Molecular signatures and new candidates to target the pathogenesis of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic, inflammatory joint disease of unknown etiology and pronounced interpatient heterogeneity. To characterize RA at the molecular level and to uncover pathomechanisms, we performed genome-wide gene expression analysis. We identified a set of 1,054 genes significantly deregulated in pair-wise comparisons between RA and osteoarthritis (OA) patients, RA and normal donors (ND), or OA and ND. Correlation analysis revealed gene sets regulated identically in all three groups. As a prominent example secreted phosphoprotein 1 (SPP1) was identified to be significantly upregulated in RA compared with both OA and ND. SPP1 expression was found to correlate with genes expressed during an inflammatory response, T-cell activation and apoptosis, suggesting common underlying regulatory networks. A subclassification of RA patients was achieved on the basis of proteoglycan 4 (PRG4) expression, distinguishing PRG4 high and low expressors and reflecting the heterogeneity of the disease. In addition, we found that low PRG4 expression was associated with a more aggressive disease stage, which is in accordance with PRG4 loss-of-function mutations causing camptodactyly-arthropathy-coxa vara-pericarditis syndrome. Altogether we provide evidence for molecular signatures of RA and RA subclasses, sets of new candidate genes as well as for candidate gene networks, which extend our understanding of disease mechanisms and may lead to an improved diagnosis.

Gene expression in endoprosthesis loosening: chitinase activity for early diagnosis?

The aim of the study was to identify markers for the early diagnosis of endoprosthesis loosening, for the differentiation between wear particle-induced and septic loosening and to gather new insights into the pathogenesis of endoprosthesis loosening. Gene expression profiles were generated from five periprosthetic membranes of wear particle-induced and five of infectious (septic) type using Affymetrix HG U133A oligonucleotide microarrays. The results of selected differentially expressed genes were validated by RT-PCR (n = 30). The enzyme activity and the genotype of chitinase-1 were assessed in serum samples from 313 consecutive patients hospitalized for endoprosthesis loosening (n = 54) or for other reasons, serving as control subjects (n = 259). Eight hundred twenty-four genes were differentially expressed with a fold change greater than 2 (data sets on http://www.ncbi.nlm.nih.gov/geo/ GSE 7103). Among these were chitinase 1, CD52, calpain 3, apolipoprotein, CD18, lysyl oxidase, cathepsin D, E-cadherin, VE-cadherin, nidogen, angiopoietin 1, and thrombospondin 2. Their differential expression levels were validated by RT-PCR. The chitinase activity was significantly higher in the blood from patients with wear particle-induced prosthesis loosening (p = 0.001). However, chitinase activity as a marker for early diagnosis has a specificity of 83% and a sensitivity of 52%, due to a high variability both in the disease and in the control group.

Anti-TNF effects on destructive fibroblasts depend on mechanical stress.

Joint destruction in rheumatoid arthritis (RA) starts typically at sites of mechanically stressed inserts of the synovial membrane near the cartilage/bone border. In the therapy of RA, tumour necrosis factor (TNF) antagonists have rapidly emerged as a valuable class of anti-rheumatic agents that reduce joint destruction. The aim of this study was to investigate and profile genes involved in the interaction between articular movement and anti-TNF therapy in an in vitro model. Murine LS48 cells, an established substitute for invasive RA synovial fibroblasts, were cultured, stretched and/or treated with anti-TNF-alpha antibody for 24 h. RNA was isolated and gene transcript levels were determined using U74Av2 Affymetrix GeneChips to identify transcriptional events. Positive findings were verified by polymerase chain reaction (PCR). We identified 170 differentially regulated genes, including 44 of particular interest. Gene expression fell into different functional groups that can be explained by RA pathogenesis and experimental conditions. For 21 genes of the 44 of particular interest, regulation could be confirmed by real-time PCR. Remarkably, we found structural as well as functional genes differently regulated between stretched cells, anti-TNF-treated cells, and stretched cells treated with anti-TNF antibody. Additionally, we also found a large number of genes that are apparently not related to the experimental conditions. Mechanical exertion modulates gene expression and subsequently cellular response to anti-TNF therapy. Results in exerted cells correspond to current knowledge regarding RA pathogenesis and underline the relevance of our experimental approach. Finally, the central function of the interleukin-18 system in joint destruction could be confirmed by our findings.

Water induces autocrine stimulation of tumor cell killing through ATP release and P2 receptor binding.

Although exposure of cells to extreme hypotonic stress appears to be a purely experimental set up, it has found an application in clinical routine. For years, surgeons have washed the abdominal cavity with distilled water to lyse isolated cancer cells left after surgery. No data are available supporting this practice or evaluating the potential mechanisms of cell injury under these circumstances. Recent evidence indicates that increases in cell volume stimulate release of adenosine triphosphate and autocrine stimulation of purinergic (P2) receptors in the plasma membrane of certain epithelial cell types. Under physiological conditions, purigenic stimulation can contribute to cell volume recovery through activation of solute efflux. In addition, adenosine triphosphate-P2 receptor binding might trigger other mechanisms affecting cell viability after profound hypotonic stress. This study demonstrates a novel pathway of cell death by apoptosis in human colon cancer cells following a short hypotonic stress. This pathway is induced by transitory cell swelling which leads to extracellular release of adenosine triphosphate (ATP) and specific binding of ATP to P2 receptors (probably P2X7). Extracellular ATP induced activation of caspases 3 and 8, annexin V, release of cytochrome c, and eventually cell death. The effect of ATP can be blocked by addition of (i) apyrase to hydrolyse extracellular ATP and (ii) suramin, a P2 receptor antagonist. Finally, (iii) gadolinium pretreatment, a blocker of ATP release, reduces sensitivity of the cells to hypotonic stress. The adenosine triphosphate-P2 receptor cell death pathway suggests that autocrine/paracrine signaling may contribute to regulation of viability in certain cancer cells disclosed with this pathway.

Relationship of interleukin-10 plasma levels to severity of injury and clinical outcome in injured patients.

Interleukin-10 (IL-10) markedly inhibits lymphocyte and phagocytic functions, which are essential for an adequate immune response to invading microbes. Although various animal and clinical studies revealed an increased release of IL-10 during sepsis, alterations of circulating IL-10 after injury and potential relationships to severity of injury and clinical outcome are unknown. Injured patients (n = 417) showed elevated (p < 0.001) IL-10 levels throughout the observation period of 21 days compared with healthy volunteers (n = 137). Patients with severe injury (Injury Severity Score > or = 25 points) demonstrated significantly increased IL-10 levels compared with patients with minor trauma (Injury Severity Score < 25 points). Patients who died from injury or developed posttraumatic complications (sepsis, multiple organ dysfunction syndrome) revealed elevated IL-10 levels in comparison with injured patients with uneventful posttraumatic course. Thus, trauma causes an enhanced release of IL-10 dependent on the severity of injury. Because increased IL-10 levels are significantly related to posttraumatic complications, IL-10 may be involved in their pathogenesis.

Inhibition of the defense system stimulating interleukin-12 interferon-gamma pathway during critical Illness.

Interleukin-12 (IL-12) and interferon-gamma (IFN-gamma) exert protective effects during experimental endotoxemia through upregulation of cellular immunity and phagocytic functions. They are part of a positive regulatory feedback loop that enhances the production of the other. Because critically ill patients show a marked suppression of T-cell and macrophage functions with a high susceptibility to infection, potential defects in the immunity/inflammation upregulating IL-12 IFN-gamma pathway were studied. As an ex vivo model of endotoxemia, lipopolysaccharide (LPS) stimulated whole blood from 25 critically ill patients and 12 healthy individuals was incubated with either recombinant human (rh) IL-12 or rhIFN-gamma, respectively. IFN-gamma dose-dependently (P < .05) increased the release of IL-12 p40 and p70 into LPS-stimulated whole blood from healthy humans without effect in whole blood from critically ill patients. RhIL-12 p70 enhanced (P < .05) the secretion of IFN-gamma in controls, while it was ineffective in LPS-stimulated whole blood from critically ill patients. The observed inhibition of the IL-12 IFN-gamma pathway is not specific to LPS, since Staphylococcus aureus Cowan strain I (SAC)-stimulated whole blood from critically ill patients showed similar suppression. The secretion of IL-12 and IFN-gamma was less reduced in critically ill patients when using isolated cultures of adherent cells or lymphocytes. Although preculture of whole blood from healthy humans with IL-10, but not with IL-4, mimicked suppression of the IL-12 IFN-gamma pathway similar to that observed during critical illness, the release of antiinflammatory reacting cytokines (IL-4, IL-10, transforming growth factor [TGF]-beta 1) was decreased into LPS-stimulated whole blood from critically ill patients. These results indicate at least two mechanisms responsible for dramatic disturbances of the IL-12 IFN-gamma pathway during critical illness: (1) deactivation of IL-12 and IFN-gamma producing leukocytes in vivo early after the primary insult, and (2) presence of serum suppressive factors different from IL-4, IL-10, or TGF-beta 1. Because IL-12 and IFN-gamma upregulate essential immune functions, the marked inhibition of IL-12 and IFN-gamma release may be pivotal for high susceptibility of critically ill patients to infection.