Terje Haug

In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability.

Human bone marrow mesenchymal stem cells (hMSCs) represent an appealing source of adult stem cells for cell therapy and tissue engineering, as they are easily obtained and expanded while maintaining their multilineage differentiation potential. All current protocols for in vitro culture of hMSCs include fetal bovine serum (FBS) as nutritional supplement. FBS is an undesirable additive to cells that are expanded for therapeutic purposes in humans because the use of FBS carries the risk of transmitting viral and prion diseases and proteins that may initiate xenogeneic immune responses. In the present study, we have therefore investigated if autologous serum (AS) or allogeneic human serum (alloHS) could replace FBS for the expansion of hMSCs in vitro. We discovered that the choice of serum affected hMSCs at several different levels. First, hMSCs in AS proliferated markedly faster than hMSCs in FBS, whereas use of alloHS resulted in hMSC growth arrest and death. Second, hMSCs in FBS differentiated more rapidly toward mesenchymal lineages compared with hMSCs in AS. Interestingly, genome‐wide microarray analysis identified several transcripts involved in cell cycle and differentiation that were differentially regulated between hMSCs in FBS and AS. Finally, several transcripts, including some involved in cell cycle inhibition, were upregulated in hMSCs in FBS at a late passage, whereas the hMSC transcriptome in AS was remarkably stable. Thus, hMSCs may be expanded rapidly and with stable gene expression in AS in the absence of growth factors, whereas FBS induces a more differentiated and less stable transcriptional profile.

Comparison of gene expression in HCT116 treatment derivatives generated by two different 5-fluorouracil exposure protocols

BackgroundEstablished colorectal cancer cell lines subjected to different 5-fluorouracil (5-FU) treatment protocols are often used as in vitro model systems for investigations of downstream cellular responses to 5-FU and to generate 5-FU-resistant derivatives for the investigation of biological mechanisms involved in drug resistance. We subjected HCT116 colon cancer cells to two different 5-FU treatment protocols in an attempt to generate resistant derivatives: one that simulated the clinical bolus regimens using clinically-achievable 5-FU levels, the other that utilized serial passage in the presence of increasing 5-FU concentrations (continuous exposure). HCT116 Bolus3, ContinB, and ContinD, corresponding to independently-derived cell lines generated either by bolus exposure or continuous exposure, respectively, were characterized for growth- and apoptosis-associated phenotypes, and gene expression using 8.5 K oligonucleotide microarrays. Comparative gene expression analyses were done in order to determine if transcriptional profiles for the respective treatment derivatives were similar or substantially different, and to identify the signaling and regulatory pathways involved in mediating the downstream response to 5-FU exposure and possibly involved in development of resistance.ResultsHCT116 ContinB and ContinD cells were respectively 27-fold and >100-fold more resistant to 5-FU and had reduced apoptotic fractions in response to transient 5-FU challenge compared to the parental cell line, whereas HCT116 Bolus3 cells were not resistant to 5-FU after 3 cycles of bolus 5-FU treatment and had the same apoptotic response to transient 5-FU challenge as the parental cell line. However, gene expression levels and expression level changes for all detected genes in Bolus3 cells were similar to those seen in both the ContinB (strongest correlation) and ContinD derivatives, as demonstrated by correlation and cluster analyses. Regulatory pathways having to do with 5-FU metabolism, apoptosis, and DNA repair were among those that were affected by 5-FU treatment.ConclusionAll HCT116 derivative cell lines demonstrated similar transcriptional profiles, despite the facts that they were generated by two different 5-FU exposure protocols and that the bolus exposure derivative had not become resistant to 5-FU. Selection pressures on HCT116 cells as a result of 5-FU challenge are thus similar for both treatment protocols.

Increased Expression of Interleukin-1 in Coronary Artery Disease With Downregulatory Effects of HMG-CoA Reductase Inhibitors

Background—Inflammation is important in atherogenesis. Interleukin (IL)-1 is the prototypic inflammatory cytokine. We hypothesized a dysbalance between inflammatory and anti-inflammatory mediators in the IL-1 family in coronary artery disease (CAD) and a possible modulation of these mediators by HMG-CoA inhibitors (statins). Methods and Results—In a microarray screening experiment examining peripheral blood mononuclear cells (PBMCs) from 6 CAD patients and 4 healthy control subjects, IL-1β was identified as 1 of 25 genes whose expression were upregulated in CAD and downregulated by statins. In the following, we studied the role of IL-1β and related mediators in CAD. Our major findings were as follows. (1) Although mRNA levels of IL-1&agr; and IL-1β were markedly reduced in PBMCs from CAD patients after 6 months of simvastatin (20 mg/d, n=15) and atorvastatin (80 mg/d, n=15) therapy, the reduction in IL-1 receptor antagonist (IL-1Ra) was more modest. Statins also reduced the spontaneous release of IL-1β and IL-1Ra from PBMCs in CAD patients. (2) mRNA levels of IL-1&agr;, IL-1β, and IL-1Ra were increased in PBMCs from patients with stable (n=20) and unstable (n=20) angina compared with healthy control subjects (n=15). Although the unstable patients had particularly high levels of IL-1β and IL-1&agr;, IL-1Ra was not correspondingly increased. (3) IL-1β induced release of proatherogenic cytokines from PBMCs, whereas atorvastatin partly abolished this effect. Conclusions—Our findings suggest that cytokines in the IL-1 family may represent therapeutic targets in CAD. The ability of statins to modulate these cytokines in an anti-inflammatory direction underscores their immunomodulatory potential.

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.

Gene expression analysis of peripheral T cells in a subgroup of common variable immunodeficiency shows predominance of CCR7– effector‐memory T cells

Common variable immunodeficiency (CVID) represents a heterogeneous group of antibody deficiency syndromes, characterized by defective antibody production in which T cell deficiency may play a pathogenic role. A subgroup of CVID patients has impaired in vitro T cell proliferation. Using microarray analyses of T cells from these patients, we found a gene expression pattern different from healthy controls and patients with X‐linked agammaglobulinaemia. The profile of the differentially expressed genes suggests enhanced cytotoxic effector functions, antigen experienced or chronically activated T cells and a predominance of CCR7– T cells. Further experiments using flow cytometry revealed a striking predominance of CCR7– T cells in a subgroup of CVID patients, and an association with impaired T cell proliferation. Our observations indicate that a predominance of CCR7– T cells with effector‐memory cell features and with reduced proliferative capacity may characterize a subgroup of CVID.

DNA transfection of mononuclear cells in muscle tissue

Genes encoding non‐self proteins may be injected into skeletal muscles in vivo to obtain induction of cellular and humoral immune responses against the encoded antigens (DNA vaccination). Bone marrow derived professional antigen‐presenting cells (APCs) play a key role in the induction of immunity by DNA vaccination. In the present work we have investigated whether the APCs are transfected by DNA injection into muscle.

Effects of interferon-α on gene expression of chemokines and members of the tumour necrosis factor superfamily in HIV-infected patients

We examined the effect of interferon (IFN)‐α on the expression of 375 genes relevant to inflammatory and immunological reactions in peripheral blood mononuclear cells (PBMC) from HIV‐infected patients by cDNA expression array and real‐time quantitative RT‐PCR. Our main findings were: (i) IFN‐α induced up‐regulation of several genes in the tumour necrosis factor (TNF) superfamily including the ligands APRIL, FasL, TNF‐α and TRAIL, with particularly enhancing effects on the latter in HIV‐infected patients. (ii) While IFN‐α markedly up‐regulated the expression of anti‐angionetic ELR– CXC‐chemokines (e.g. MIG and IP‐10), it suppressed the expression of angiogenic ELR+ CXC‐chemokines (e.g. GRO‐α, IL‐8 and ENA‐78), with similar patterns in both patients and controls. (iii) IFN‐α induced a marked increase in gene expression of the HIV co‐receptor CCR5 in both patients and controls. We suggest that these effects may contribute to both the therapeutic and toxic effects of IFN‐α. Moreover, our findings underscore that the biological effects of IFN‐α in HIV infection are complex and that the clinical net effects of IFN‐α treatment may be difficult to predict. However, the potent enhancing effect of IFN‐α on several pro‐apoptotic genes in the TNF superfamily and the enhancing effect on CCR5 expression suggest a possible pathogenic role of IFN‐α in the progression of HIV‐related immunodeficiency and suggests caution in the therapeutic use of IFN‐α in HIV‐infected individuals.

Myocardial gene expression of inflammatory cytokines after heart transplantation in relation to the development of transplant coronary artery disease

Long-term success of cardiac transplantation is mainly limited by the development of transplant coronary artery disease (CAD); it is generally accepted that it is immune mediated, involving cytokines and growth factors. We show that development of transplant CAD is associated with a particular cytokine profile in myocardial biopsies characterized by a late (i.e., 1 year) increase in tumor necrosis factor-alpha and interferon-gamma gene expression, which precede and potentially contribute to the development of allograft vasculopathy, further supporting a role for inflammation and the pathogenesis of transplant CAD in humans.

Macrophage enrichment from induced sputum

Since induced sputum has become a widely used non-invasive method of recovering cells from the surfaces of the bronchial airways, isolating specific cell populations will be necessary in order to learn more about their specific role in innate immunity and inflammation in the airways. Several studies have demonstrated the ability to conduct ex vivo analyses on sputum cells such as phagocytosis and surface marker measurements, but these have not been performed on isolated cell types.1–3 This study demonstrates the capability to isolate sputum macrophages from human volunteers in order to advance our understanding of macrophage biology in the airways. To this end, techniques that can enrich and isolate cells without significant activation would prove extremely useful. We compared two common methods for isolating and enriching macrophages in sputum: (1) magnetic bead separation; and (2) Percoll gel density gradient centrifugation. Cell purity and markers of cell activation (mRNA tumour necrosis factor α (TNFα)) …

Airway inflammation in paper mill workers.

Paper mill workers are exposed to culturable microorganisms (MOs). We hypothesized that inflammatory airway response could be detected in sputum of nonsymptomatic workers. From four paper mills, we included 29 healthy nonsmoking men. Workers exposed to high levels of MOs (HMOE, n = 17) were compared with workers exposed to low levels of MO (LMOE, n = 12). A reference group of 22 healthy, nonsmoking, nonexposed (NE) men were also included. We performed differential cell counts of induced sputum, studied gene expressions of isolated sputum macrophages and analyzed inflammatory parameters, including matrix metalloproteinases. Sputum from HMOE workers had a significantly higher percentage of neutrophils than that from LMOE workers (P < 0.05) and NE controls (P < 0.001). There was also an increased gene expression of different pro-inflammatory cytokines, interleukin-6, tumor necrosis factor-&agr;, and macrophage inflammatory protein-1&bgr; in isolated airway macrophages and increased levels of total matrix metalloprotease-9 activity in induced sputum from the HMOE group. Our findings indicate that paper industry workers exposed to MOs develop subclinical airway inflammation.