Functions of Fos phosphorylation in bone homeostasis, cytokine response and tumourigenesis.

Mice lacking c-fos develop osteopetrosis due to a block in osteoclast differentiation. Carboxy-terminal phosphorylation of Fos on serine 374 by ERK1/2 and serine 362 by RSK1/2 regulates Fos stability and transactivation potential in vitro. To assess the physiological relevance of Fos phosphorylation in vivo, serine 362 and/or serine 374 was replaced by alanine (Fos362A, Fos374A and FosAA) or by phospho-mimetic aspartic acid (FosDD). Homozygous mutants were healthy and skeletogenesis was largely unaffected. Fos C-terminal phosphorylation, predominantly on serine 374, was found important for osteoclast differentiation in vitro and affected lipopolysaccharide (LPS)-induced cytokine response in vitro and in vivo. Importantly, skin papilloma development was delayed in FosAA, Fos362A and Rsk2-deficient mice, accelerated in FosDD mice and unaffected in Fos374A mutants. Furthermore, the related Fos protein and putative RSK2 target Fra1 failed to substitute for Fos in papilloma development. This indicates that phosphorylation of serines 362 and 374 exerts context-dependent roles in modulating Fos activity in vivo. Inhibition of Fos C-terminal phosphorylation on serine 362 by targeting RSK2 might be of therapeutic relevance for skin tumours.

Anti-inflammatory and cartilage-protecting effects of an intra-articularly injected anti-TNF{alpha} single-chain Fv antibody (ESBA105) designed for local therapeutic use.

OBJECTIVES: (1) To show that a single-chain Fv antibody (scFv) against tumour necrosis factor alpha (TNFalpha) (ESBA105) has efficacy comparable to a full length anti-TNFalpha IgG (infliximab); (2) to evaluate whether ESBA105 has all the properties required for the local treatment of arthritis; and (3) to investigate its discriminative tissue penetration properties. METHODS: In vivo efficacy was measured in arthritis of the knee joint induced by the intra-articular injection of recombinant human TNFalpha (rhTNFalpha) in Lewis rats. Cartilage penetration of scFv (ESBA105) and full length IgG (infliximab) were studied in bovine cartilage specimens ex vivo. Tissue penetration, biodistribution and pharmacokinetics of ESBA105 were followed and compared after intra-articular and intravenous administration. RESULTS: In cell culture, ESBA105 showed similar TNFalpha inhibitory potency to infliximab. In vivo, ESBA105 inhibited rhTNFalpha-induced synovial inflammation in rats with efficacy again comparable to infliximab. An 11-fold molar excess of ESBA105 over rhTNFalpha resulted in 90% inhibition of knee joint swelling, inflammatory infiltrates and proteoglycan loss from cartilage. In ex vivo studies of bovine cartilage, ESBA105 penetrated well into the cartilage whereas infliximab remained on the surface. In vivo, rapid penetration into the synovial tissue, cartilage and surrounding tissues was observed following intra-articular injection of [(125)I]-ESBA105 into the knee joint of rabbits. CONCLUSIONS: ESBA105 potently inhibits inflammation and prevents cartilage damage triggered by TNFalpha. In contrast to a full length IgG, ESBA105 also penetrates into cartilage and can be expected to reverse the TNFalpha-induced catabolic state of articular cartilage in arthritides.

Interleukin-1 is essential for systemic inflammatory bone loss.

OBJECTIVES: Chronic inflammation is a major risk factor for systemic bone loss leading to osteoporotic fracture and substantial morbidity and mortality. Inflammatory cytokines, particularly tumour necrosis factor (TNF) and interleukin-1 (IL1), are thought to play a key role in the pathogenesis of inflammation-induced bone loss, but their exact roles are yet to be determined. METHODS: To determine whether TNF directly triggers bone loss or requires IL1, human TNFalpha mice (hTNFtg) were crossed with mice lacking IL1alpha and IL1beta (IL1(-/-)hTNFtg). Systemic bone architecture was evaluated using CT scanning, static and dynamic bone histomorphometry and serum markers of bone metabolism. RESULTS: hTNFtg mice developed severe bone loss accompanied by a severe distortion of bone microarchitecture. Bone trabeculae were thinner and decreased in numbers, resulting in increased trabecular separation. Histomorphometric analyses revealed strongly increased bone resorption in hTNFtg mice compared with wild-type mice. In contrast, IL1(-/-)hTNFtg mice were fully protected from systemic bone loss despite still developing inflammation in their joints. Lack of IL1 completely reversed increased osteoclast formation and bone resorption in hTNFtg mice and the increased levels of RANKL in these mice. Structural parameters and osteoclast and osteoblast numbers were indistinguishable from wild-type mice. CONCLUSIONS: These data indicate that IL1 is essential for TNF-mediated bone loss. Despite TNF-mediated inflammatory arthritis, systemic bone is fully protected by the absence of IL1, which suggests that IL1 is an essential mediator of inflammatory osteopenia.

Eotaxin-3 is involved in Churg-Strauss syndrome--a serum marker closely correlating with disease activity.

OBJECTIVE: Churg-Strauss Syndrome (CSS) is characterized by excessive eosinophil accumulation in peripheral blood and affected tissues with development of granulomatous vasculitic organ damage. The contribution of eosinophil-chemotactic cytokines (eotaxin family) to eosinophilia and disease activity in CSS is unknown. Thus, we compared serum levels of the eotaxin family members in CSS patients with healthy and disease controls. METHODS: Forty patients with CSS diagnosed according to ACR 1990 criteria, 30 healthy controls (HC) and 57 disease controls (28 asthma, 20 small vessel vasculitis, 9 hypereosinophilic syndrome) were studied. Clinical data were collected and serum levels of eotaxin-1, -2 and -3 were determined by ELISA. Further, immunohistochemistry was applied to identify eotaxin-3 expression in tissue biopsies from patients with CSS. RESULTS: In contrast to eotaxin-1 and -2, eotaxin-3 was highly elevated in serum samples of active CSS patients and correlated highly significantly with eosinophil counts, total immunoglobulin E (IgE) levels and acute-phase parameters. Moreover, eotaxin-3 was not elevated in other eosinophilic and vasculitic diseases. Immunohistochemical analysis revealed strong expression of eotaxin-3 in endothelial and inflammatory cells in affected tissues of active CSS patients. CONCLUSIONS: This study reveals the specific association of elevated eotaxin-3 expression with high disease activity and eosinophilia in CSS patients. Eotaxin-3 might thus be a pathogenic player, biomarker and potential therapeutic target in CSS.

Tumour necrosis factor blockade increases lymphangiogenesis in murine and human arthritic joints.

OBJECTIVE: To investigate the presence and regulation of lymphatic vessels in inflamed joints of mice with experimental arthritis as well as patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA). METHODS: Lymphatic vessels and blood vessels were assessed in synovial tissue of human tumour necrosis factor transgenic (TNFtg) mice and synovial biopsies from patients with RA and SpA by immunohistochemistry for podoplanin and CD31, respectively. Assessments were performed before and after TNF blockade in all biopsies. RESULTS: Lymphatic vessels were abundantly present in the synovial tissue of hTNFtg mice as well as patients with RA and SpA. The number of lymphatic vessels was positively related to the severity of synovial inflammation. Treatment with infliximab led to an increase in the formation of lymphatic vessels in murine and human inflammatory tissue. CONCLUSIONS: This study shows that TNF blockade promotes the proliferation of lymphatic vessels in the inflamed synovium of RA and SpA. This finding leads to the assumption that promotion of lymphangiogenesis may play an important part in efflux of cells and fluid out of the inflamed tissue.

CTLA-4 directly inhibits osteoclast formation.

CTLA-4 is a regulator of co-stimulation and inhibits the activation of T cells through interfering with the interaction of CD80/86 on antigen-presenting cells with CD28 on T cells. CTLA-4 binds to the surface of antigen-presenting cells, such as dendritic cells and monocytes through CD80/86. Monocytes can differentiate in osteoclasts, the primary bone resorbing cells. Herein, we investigated whether the binding of CTLA-4 affects the differentiation of monocytes into osteoclasts in vitro and vivo. We show that CTLA-4 dose-dependently inhibits RANKL- as well as tumour necrosis factor (TNF)-mediated osteoclastogenesis in vitro without the presence of T cells. Furthermore, CTLA-4 was effective in inhibiting TNF-induced osteoclast formation in a non-T cell dependent TNF-induced model of arthritis as well as the formation of inflammatory bone erosion in vivo. These data suggest that CTLA-4 is an anti-osteoclastogenic molecule that directly binds osteoclast precursor cells and inhibits their differentiation. These findings are an attractive explanation for the anti-erosive effect of abatacept, a CTLA-4 immunoglobulin fusion protein used for the treatment of rheumatoid arthritis.

Selective p38MAPK isoform expression and activation in antineutrophil cytoplasmatic antibody-associated crescentic glomerulonephritis: role of p38MAPKalpha.

OBJECTIVE: Crescentic glomerulonephritis (crGN) is a frequent and life-threatening manifestation of antineutrophil cytoplasmatic antibody-associated vasculitis. Up-regulation of proinflammatory cytokines contributes to renal damage by activation of p38 mitogen-activated protein kinases (MAPKs). However, it is unclear which of the four p38MAPK isoforms are expressed, activated and hence of major importance in crGN. METHODS: Kidney biopsies of patients with antineutrophil cytoplasmatic antibody-positive crGN and control samples were investigated for the expression and phosphorylation of p38MAPK isoforms and downstream target kinase MAPKAP2 by immunohistochemistry. Expression and functional activation of p38MAPK isoforms by TNF was also assessed in a human podocyte cell line by reverse transcription-polymerase chain reaction, immunoblotting and kinase array. RESULTS: Strong expression of p38MAPKalpha, beta and gamma isoforms was found in glomerular podocytes and crescents. Infiltrating leucocytes showed predominant p38MAPKalpha expression. Activation of p38MAPK and its downstream mediator MAPKAP2 was found in crGN confined to glomerular podocytes, crescents and inflammatory infiltrates. Interestingly, corticosteroid treatment before kidney biopsy diminished p38MAPK activation in crGN. Activated p38MAPK co-localised with alpha, beta and gamma isoforms in podocytes and crescents, while leucocytes showed mainly p38MAPKalpha activation. In a human podocyte cell line mRNA and protein of all four p38MAPK isoforms was expressed but only p38MAPKalpha was activated upon challenge with TNF. CONCLUSIONS: This study shows selective p38MAPK isoform expression and activation in crGN. Podocytes and podocyte-induce crescent formation is the main source of p38MAPK activation in crGN. TNF is a potent and selective activator of the alpha-isoform in podocytes, which therefore appears as a main contributor to proinflammatory signalling in the glomerulum of crGN.

The role of risk adjustment in national health reform.

Efforts to contain health insurance costs through competitive strategies are undermined by the economic incentive facing buyers and sellers to avoid high-risk individuals. To deal with this problem, proponents of competitive strategies, in which cost containment would be achieved by having consumers move to the most efficient health plans, suggest developing risk-assessment methods and using them to make transfer payments from plans enrolling relatively healthier people to plans with relatively sicker ones. Effective risk adjustment is also of interest to payers such as Medicare, large employers offering multiple-choice programs, and risk-bearing providers seeking fair compensation. So far, however, the ability to predict the variability of future medical costs on an individual basis is very limited. In a market in which individuals are free to change plans annually, the potency of current risk-adjustment technology would leave plans with ample incentive to attract healthier people and to avoid sicker people. The state of current risk-assessment methods leads some analysts to advocate a mixed payment system, partly based on a risk-adjusted prospective payment and partly based on retrospective adjustments made once competing plans' actual experiences are known. New York State is trying such an approach. Many analysts emphasize the importance of other insurance reforms and the institutional framework in which risk adjustments might be made as key factors in helping such a process succeed.(ABSTRACT TRUNCATED AT 250 WORDS)

[Intraoperative changes in extravascular lung water. Study on heart surgery patients]. / Intraoperative Veränderungen des extravaskulären Lungenwassers. Untersuchung an kardiochirurgischen Patienten.

The intraoperative changes in extravascular lung water (EVLW) were studied in 40 patients undergoing aortic-coronary bypass grafting. The patients were divided into two groups on the basis of preoperative ejection fraction (EF) values (group I: EF greater than 45%; group II: EF less than 45%). EVLW was measured using the double-indicator dilution method (thermo/dye). In a control study, changes in transthoracic impedance (ZoTh) were recorded. The initial EVLW value in group I was 4.3 +/- 0.4 ml/kg body wt. and in group II, 4.4 +/- 0.3 ml/kg body wt. After extracorporeal circulation, significant changes in EVLW could be observed (group I: from 4.5 +/- 0.5 ml/kg body wt. to 7.0 +/- 0.2 ml/kg body wt.; group II: from 5.1 +/- 0.8 ml/kg body wt. to 7.8 +/- 0.9 ml/kg body wt. (p less than 0.001). At the end of the operation, no changes in EVLW were observed in group I. However, in group II EVLW was significantly different to initial values (6.3 +/- 1.0 ml/kg body wt., p less than 0.01). The results obtained using the double-indicator method were identical with those obtained using the transthoracic impedance method. A marked correlation could be seen between length of ECC recording and EVLW values at the end of the operation, especially when the ECC time was 90 min or more (r = 0.84). Based on our results, it must be assumed that intraoperative damage to capillary membranes occurs if the ECC time is above 90 min.