A reversible gene trap collection empowers haploid genetics in human cells.

Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-ß, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.

Hydrogen sulphide decreases IL-1ß-induced activation of fibroblast-like synoviocytes from patients with osteoarthritis.

Balneotherapy employing sulphurous thermal water is still applied to patients suffering from diseases of musculoskeletal system like osteoarthritis (OA) but evidence for its clinical effectiveness is scarce. Since the gasotransmitter hydrogen sulphide (H2 S) seems to affect cells involved in degenerative joint diseases, it was the objective of this study to investigate the effects of exogenous H2 S on fibroblast-like synoviocytes (FLS), which are key players in OA pathogenesis being capable of producing pro-inflammatory cytokines and matrix degrading enzymes. To address this issue primary FLS derived from OA patients were stimulated with IL-1ß and treated with the H2 S donor NaHS. Cellular responses were analysed by ELISA, quantitative real-time PCR, phospho-MAPkinase array and Western blotting. Treatment-induced effects on cellular structure and synovial architecture were investigated in three-dimensional extracellular matrix micromasses. NaHS treatment reduced both spontaneous and IL-1ß-induced secretion of IL-6, IL-8 and RANTES in different experimental settings. In addition, NaHS treatment reduced the expression of matrix metallo-proteinases MMP-2 and MMP-14. IL-1ß induced the phosphorylation of several MAPkinases. NaHS treatment partially reduced IL-1ß-induced activation of several MAPK whereas it increased phosphorylation of pro-survival factor Akt1/2. When cultured in spherical micromasses, FLS intentionally established a synovial lining layer-like structure; stimulation with IL-1ß altered the architecture of micromasses leading to hyperplasia of the lining layer which was completely inhibited by concomitant exposure to NaHS. These data suggest that H2 S partially antagonizes IL-1ß stimulation via selective manipulation of the MAPkinase and the PI3K/Akt pathways which may encourage development of novel drugs for treatment of OA.

Comparative evaluation of preclinical in vivo models for the assessment of replicating retroviral vectors for the treatment of glioblastoma.

Despite impressive improvements in neurosurgical techniques, radiation and chemotherapy during the past few years, little progress has been made in the treatment of malignant gliomas. Recently, the efficacy of suicide gene therapy based on replication-competent retroviral (RCR) vectors as delivery vehicles for the therapeutic gene has been described in the treatment of experimental cancer, including gliomas. In this study, we have thus critically evaluated a panel of human and rodent glioma/glioblastoma cell lines (U-87MG, U-118MG, LN-18, LN-229, 8-MG-BA, 42-MG-BA, A-172, T-98G, UVW, C6, 9L, G-26, GL-261, Tu-2449, Tu-9648) with respect to RCR virus vector spread, sensitivity towards the cytosine deaminase (CD)/5-flurocytosine (5-FC)/5-flurouracil (5-FU) suicide system, and orthotopic growth characteristics in mice to identify suitable preclinical animal models for the development of a glioblastoma gene therapy. Rapid virus spread was observed in eight out of nine human cell lines tested in vitro. As expected, only CD-expressing cells became sensitive to 5-FC, due to their ability to convert the prodrug in its toxic form, 5-FU. All LD(50) values were within the range of concentrations obtained in human body fluids after conventional antifungal 5-FC administration. In addition, a significant bystander effect was observed in all human glioma cell lines tested. Injection of the RCR vector into pre-established orthotopic mouse tumor xenografts revealed substantial infection and virus spread of tumor tissue from most cell types.

H2S transiently blocks IL-6 expression in rheumatoid arthritic fibroblast-like synoviocytes and deactivates p44/42 mitogen-activated protein kinase.

Sulfur bath therapy represents the oldest form of treatment for patients with different types of rheumatic disorders. However, scientific reports about the beneficial effects of this form of therapy are controversial, rare and of poor scientific quality. Also, little is known about the role and underlying molecular mechanisms of H2S. Therefore, this topic encouraged us to investigate the influence of H2S on fibroblasts isolated from the synovial membrane of RA (rheumatoid arthritis) patients. FLSs (fibroblast-like synoviocytes) were treated with different concentrations of an exogenous H2S donor (NaHS). At defined time points, secretion of IL-6 was quantified by ELISA. Activation/deactivation of MAPKs (mitogen-activated protein kinases), p38 and p44/42 MAPK (ERK1/2) were confirmed by Western blot experiments. FLSs constitutively express and secrete large quantities of IL-6 and IL-8. Data provided prove that, in FLSs, constitutive as well as IL-1beta-induced expression of IL-6 is transiently and partially down-regulated by the short treatment of cells with low concentrations of NaHS. Another key finding is that H2S deactivates p44/42 MAPK (ERK1/2). Long-term exposure of FLSs to H2S provides stimulatory effects, leading to reinforced activation of p38 MAPK and ERK1/2 accompanied by upregulation of IL-6 expression. Presented data seem of importance for studying (patho-) physiological functions of H2S and also for re-evaluating sulfur spa therapy as one of the oldest forms of therapy for rheumatic disorders.

High concentrations of hydrogen sulphide elevate the expression of a series of pro-inflammatory genes in fibroblast-like synoviocytes derived from rheumatoid and osteoarthritis patients.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder, primarily affecting the articular structures and synovial membranes of multiple joints. Beside pharmacologically based treatments, sulphur bath therapy has long been used as a therapy for patients suffering from different rheumatic disorders. But scientific reports about the beneficial effects of H(2)S as well as about the underlying molecular mechanisms are controversial and rare. METHODS: Fibroblast-like synoviocytes (FLS) derived from RA and OA-patients were treated with the H(2)S-donor sodium hydrogen sulphide (NaHS). IL-6 release was quantified by enzyme-linked immunosorbent assay (ELISA). Gene expression of IL-6, IL-8 and COX-2 as well as of the matrix metalloproteinases (MMPs) MMP-2, MMP-3 and MMP-14 was monitored by quantitative real-time PCR (qRT-PCR). Modulation of the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2 was analysed by Western blotting. RESULTS: High concentrations of H(2)S (above 0.5mM) elevated the expression of pro-inflammatory genes in RA- and OA-FLS. This was accompanied by activation of p38 and ERK1/2 MAPK. H(2)S-induced expression of IL-6, IL-8 and COX-2 was completely blocked by specific inhibitors of p38 and ERK1/2 MAPK and NF-κB. CONCLUSION: H(2)S is a potent gaseous molecule that can upregulate the expression of a series of pro-inflammatory genes in RA and OA-FLS. Therefore, caution is advised in patients with active RA when taking sulphur bath therapy.

Dimethyl sulphoxide and dimethyl sulphone are potent inhibitors of IL-6 and IL-8 expression in the human chondrocyte cell line C-28/I2.

AIMS: Reactive oxygen species (ROS) are highly diffusable and reactive molecules which modulate gene transcription, particularly of pro-inflammatory cytokines which play a crucial role in the nascency and progression of chronic inflammatory diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA). Since thiols could be potent inhibitors of the production of cytokines, the effects of dimethyl sulphoxide (DMSO) and dimethyl sulphone (DMS) on constitutive and IL-1ß-induced IL-6 and IL-8 expression in the human chondrocyte cell line C-28/I2 were evaluated. MAIN METHODS: C-28/I2 cells were incubated for 12h with different concentrations of DMSO or DMS. The secretion of IL-6 and IL-8 was quantified by enzyme-linked immunosorbent assays (ELISAs). The impact of DMSO and DMS on the regulation of p38 and ERK1/2 mitogen-activated protein kinases (MAPKs) was confirmed by Western blot experiments. Furthermore, C-28/I2 cells were stimulated with IL-1ß in the absence or presence of DMSO and DMS and IL-6 and IL-8 expression was quantified by ELISAs and quantitative real-time polymerase chain reaction (qRT-PCR). KEY FINDINGS: C-28/I2 cells constitutively expressed large quantities of IL-6 and IL-8. Long-term exposure of cells to DMSO (1%) or DMS (100mM) led to a dramatic downregulation of IL-6 and IL-8 expression which was accompanied by the deactivation of ERK1/2. Both substances also blocked IL-1ß-induced IL-6 and IL-8 expression. SIGNIFICANCE: In this study, we demonstrate that both DMSO and DMS represent strong anti-inflammatory properties by blocking constitutive as well as IL-1ß-induced IL-6 and IL-8 expression in the human chondrocyte cell line C-28/I2.