Quality of Life and Limitations in Daily Life of Stable COPD Outpatients in a Real-World Setting in Austria - Results from the CLARA Project.

Background: COPD patients suffer from respiratory symptoms and limitations in daily life. We aimed to characterize the impact of disease on overall health, daily life, and perceived well-being in COPD outpatients. Methods: We conducted a national, cross-sectional study among pulmonologists and general practitioners (GPs). The St. George's Respiratory Questionnaire for COPD patients (SGRQ-C) was used. Inclusion criteria were a physician's diagnosis of COPD and age ≥40 years. Subjects with a history of lung surgery, lung cancer or COPD exacerbation within the last four weeks were excluded. Results: Sixty-seven pulmonologists and 6 GPs enrolled 1175 COPD patients. Two hundred forty-eight of those did not fulfill GOLD criteria for COPD (FEV1/FVC <0.7) and 77 were excluded due to missing data. Finally, 850 patients (62.8% men; mean age 66.2 ± 0.3 (SE) years; mean FEV1%pred. 51.5 ± 0.6 (SE)) were analyzed. Last year, 55.4% reported at least one exacerbation, and 12.7% were hospitalized for COPD exacerbation. Mean SGRQ-C total score was 43.1 ± 0.83 (SE) and mean component scores for symptoms, activity and impacts were 55.6, 55.4 and 30.5, respectively. Half of the patients (50.3%) reported not being able to do any sports and 78.7% stated that their respiratory symptoms did not allow them doing anything they would like to do. In patients with less severe COPD (FEV1pred ≥50% and non-frequent exacerbations), global health status was overrated, ie, estimated as better by the physician than by the patient, while it was underrated in more severe COPD. Conclusion: In Austria, the burden of disease in COPD outpatients tends to be underestimated in patients with milder airway obstruction and less exacerbations and overestimated in patients with more severe airway obstruction and frequent exacerbations. Our finding suggests that validated assessment of global health status might decrease these differences of perception.

BK virus infection activates the TNFα/TNF receptor system in Polyomavirus-associated nephropathy.

Polyomavirus-associated nephropathy due to BK virus infection (BKVAN) is recognized as an important cause of significant kidney transplant dysfunction often leading to renal graft loss. The activation of innate immune defense mechanisms during BKVAN is still poorly understood and an altered regulation of inflammatory mediators by resident kidney cells upon viral infection can be expected to contribute to the onset and progression of disease. TNFα interacting with its receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), is largely accepted to be involved in viral responses, exhibiting both proinflammatory and immunosuppressive effects. Our aim was to examine the expressions of TNFα and TNFR1 and 2 in human collecting duct epithelial cells (HCDC) after infection with BKV as well as to study the effect of TNFα and poly(I:C), a synthetic analog of viral RNA, on the expressions of TNF receptors and proinflammatory cytokines and chemokines in HCDC. Quantitative RT-PCR analyses showed a downregulation of TNFα and an upregulation of both TNFR1 and 2 upon exposure of HCDC to the BK virus. TNFα stimulation induced the expressions of IL-6, IL-8, RANTES, and TNFR2. Poly(I:C) upregulated the expressions of both TNFR1 and TNFR2, a response that could be effectively blocked by siRNA to TLR3 and RIG-I, two double-stranded (ds) RNA receptors of the innate immune system. Poly(I:C)-dependent expression of TNFR2 but not TNFR1 was enhanced by TNFα. Taken together, our results suggest an involvement of TNF/TNFR system in virus-associated nephropathy.

Hepatitis C virus induced endothelial inflammatory response depends on the functional expression of TNFα receptor subtype 2.

In hepatitis C virus (HCV) infection, morbidity and mortality often result from extrahepatic disease manifestations. We provide evidence for a role of receptors of the innate immune system in virally induced inflammation of the endothelium in vitro and in vivo. Corresponding to the in vitro finding of an HCV-dependent induction of proinflammatory mediators in endothelial cells, mice treated with poly (I:C) exhibit a significant reduction in leukocyte rolling velocity, an increase in leukocyte adhesion to the vessel wall and an increased extravasation of leukocytes. HCV directly promotes activation, adhesion and infiltration of inflammatory cells into the vessel wall by activation of endothelial viral receptors. Poly (I:C) induces the expression of TLR3 in vivo and hereby allows for amplification of all of the aforementioned responses upon viral infection. Proinflammatory effects of viral RNA are specifically mediated by TLR3 and significantly enhanced by tumor necrosis factor alpha (TNFα). HCV-RNA induces the endothelial expression of TNFα and TNFα receptor subtype 2 and we provide evidence that leucocyte adhesion and transmigration in response to activation of viral RNA receptors seem to depend on expression of functional TNFR2. Our results demonstrate that endothelial cells actively participate in immune mediated vascular inflammation caused by viral infections.

Prothrombotic effects of tumor necrosis factor alpha in vivo are amplified by the absence of TNF-alpha receptor subtype 1 and require TNF-alpha receptor subtype 2.

INTRODUCTION: Elevated serum levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) correlate with an increased risk for atherothrombotic events and TNFα is known to induce prothrombotic molecules in endothelial cells. Based on the preexisting evidence for the impact of TNFα in the pathogenesis of autoimmune disorders and their known association with an acquired hypercoagulability, we investigated the effects of TNFα and the role of the TNF receptor subtypes TNFR1 and TNFR2 for arteriolar thrombosis in vivo. METHODS: Arteriolar thrombosis and platelet-rolling in vivo were investigated in wildtype, TNFR1-/-, TNFR2-/- and TNFR1-/R2-/- C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. In vitro, expression of prothrombotic molecules was assessed in human endothelial cells by real-time PCR and flow cytometry. RESULTS: In wildtype mice, stimulation with TNFα significantly accelerated thrombotic vessel occlusion in vivo upon ferric chloride injury. Arteriolar thrombosis was much more pronounced in TNFR1-/- animals, where TNFα additionally led to increased platelet-endothelium-interaction. TNFα dependent prothrombotic effects were not observed in TNFR2-/- and TNFR1-/R2- mice. In vitro, stimulation of human platelet rich plasma with TNFα did not influence aggregation properties. In human endothelial cells, TNFα induced superoxide production, p-selectin, tissue factor and PAI-1, and suppressed thrombomodulin, resulting in an accelerated endothelial dependent blood clotting in vitro. Additionally, TNFα caused the release of soluble mediators by endothelial cells which induced prothrombotic and suppressed anticoagulant genes comparable to direct TNFα effects. CONCLUSIONS: TNFα accelerates thrombus formation in an in vivo model of arteriolar thrombosis. Its prothrombotic effects in vivo require TNFR2 and are partly compensated by TNFR1. In vitro studies indicate endothelial mechanisms to be responsible for prothrombotic TNFα effects. Our results support a more selective therapeutic approach in anticytokine therapy favouring TNFR2 specific antagonists.

TNFα enhances TLR3-dependent effects on MMP-9 expression in human mesangial cells.

The role of MMPs (matrix metalloproteinases) in kidney diseases has been widely accepted, where they can regulate inflammatory response because of their effects on both recruitment and survival of inflammatory cells. TNFα (tumour necrosis factor α) has also been implicated in the pathogenesis of inflammatory kidney diseases, including forms of glomerulonephritis associated with viral diseases. Previously, we established the functional linkage between viral receptors of the innate immune system, the TLRs (Toll-like receptors) and control of MMP activity in human MC (mesangial cells). Expression levels of MMP-2, MMP-7, MMP-9, TIMP-1 (tissue inhibitor of metalloproteinase 1) and TIMP-2 in human MC in culture were analysed by RT-PCR (reverse transcription-PCR). TNFα significantly enhanced the TLR3-dependent induction of MMP-9 in human MC. Expression levels of MMP-2, TIMP-1 and TIMP-2 were not significantly affected by the activation of TLR3 or TNFα stimulation. No significant MMP-7 expression was found. We conclude that the role of MMP-9 in chemotaxis, activation and proliferation of inflammatory cells is amplified by TNFα originating from infiltrating cells, especially monocytes, producing a regulatory loop that potentially leads to a self-propagating inflammation.

Response of VEGF to activation of viral receptors and TNFα in human mesangial cells.

Vascular endothelial growth factor (VEGF) plays an important role in glomerular homeostasis as well as in the pathogenesis of kidney diseases as glomerulonephritis (GN) and diabetic nephropathy. Mesangial cells (MC), which are an integral part of the functional glomerular filtration barrier in that providing structural support, can behave like inflammatory cells and produce mediators as chemokines and growth factors; they are known to express viral receptors, with TLR3 having been attributed relevance in viral disease-associated GN. Experiments were performed on human MC in cell culture. Stimulation experiments were performed with poly (I:C) and hepatitis C RNA from patients with hepatitis C infection. We hereby show a TLR3-mediated upregulation of VEGF and its receptor subtype 2 (VEGF-R2) in human MC upon activation of viral receptors by poly (I:C) and hepatitis C virus. The increase in VEGF expression levels is further enhanced by tumor necrosis factor alpha (TNFα) which also induces the cytokines IL-6 and IL-8 as well as the chemokines MCP-1 and RANTES. These effects are potentiated by preincubation of MC with poly (I:C), just as the induction of the viral receptors TLR3, RIG-1, and MDA5 themselves. Moreover, MCP-1 itself is able to significantly increase mesangial VEGF expression. Therefore, with VEGF and VEGF-R2 being induced upon viral receptor activation in human MC, a novel role of TLR3 in mediating glomerular damage in virally induced or aggravated GN is inferred. TNFα and MCP-1 are seemingly important in amplifying VEGF effects in the setting of virally induced inflammation, with TNFα being also able to induce other mediators of glomerular pathology in GN.

TLR3-dependent immune regulatory functions of human mesangial cells.

In glomerulonephritis, the migration of inflammatory cells into the glomerulus is an important step in disease initiation and progression. The viral receptor Toll-like receptor 3 (TLR3) is known to play a role in virus-associated glomerulonephritis. Based on this knowledge, this study aimed to define the effects of the TLR3 ligand polyriboinosinic:polyribocytidylic acid (poly(I:C)) on the expression of adhesion molecules and macrophage colony-stimulating factor (M-CSF) on resident glomerular cells. Experiments in MCs demonstrated that the activation of viral receptors by poly(I:C) leads to a time- and dose-dependent induction of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and M-CSF at both the mRNA and protein levels; these results were confirmed by incubating MCs with HCV RNA. As shown in knockdown experiments, this effect is specifically mediated by TLR3. The prestimulation of MCs with proinflammatory cytokines increases the effects of poly(I:C), except for its induction of VCAM-1. Tumor-necrosis factor (TNF)-α, likewise, induces ICAM-1, VCAM-1 and M-CSF, and amplifies the mesangial response to poly(I:C). These results were confirmed by incubating MCs with HCV RNA. We thus provide evidence that human MCs represent a potential target of the leukocytes and monocytes that infiltrate the glomerulus in viral disease-associated GN, highlighting the possibility that MCs may act as resident antigen-presenting cells.

Inhibition of TLR3-mediated proinflammatory effects by Alkylphosphocholines in human retinal pigment epithelial cells.

PURPOSE. To elucidate the role of Toll-like receptor 3 (TLR3) in the pathogenesis of age-related macular degeneration (AMD) and to investigate the effect of alkylphosphocholines (APCs) on the TLR3-mediated expression of cytokines and growth factors in human retinal pigment epithelial (RPE) cells. METHODS. Confluent cultures of human RPE cells (ARPE-19) were stimulated with poly (I:C) RNA as a well-established ligand for TLR3. Cytokine profiles were determined by RT-PCR on the activation of TLR3. RPE cells were transfected with siRNA specific for TLR3 and RIG-1 to determine the receptors involved. The effect of preincubation of RPE cells with APCs on the expression level of target genes was assessed. RESULTS. Poly (I:C) RNA stimulation led to a dose-dependent increase in the expression of TLR3 and RIG-I. A significant increase in expression levels of IL-6, TNF-α, IL-8, MCP-1, ICAM-1, and BFGF was observed after poly (I:C) RNA stimulation (P < 0.05). This effect was time and dose dependent. No effect on PEDG or VEGF expression was seen. Transfection of RPE cells with siRNA specific for TLR3 reduced poly (I:C) RNA-induced mRNA expression of the genes (P < 0.05). Preincubation of RPE cells with APCs significantly reduced the poly (I:C) RNA-induced expression of the target genes (P < 0.05). CONCLUSIONS. The authors demonstrate that the expression of proinflammatory cytokines and chemokines in RPE cells depends on the activation of TLR3. The induction of downstream gene expression is blocked by siRNA specific for TLR3 and alkylphosphocholines. Therefore, TLR3 should be considered a novel target in AMD therapy.

TLR3-dependent regulation of cytokines in human mesangial cells: a novel role for IP-10 and TNF-alpha in hepatitis C-associated glomerulonephritis.

In viral infections, disease manifestations and tissue damage often result primarily from immune cells infiltrating target organs on the basis of an ineffectual viral clearance with persistent antigenemia or an inappropriate immune response. Cell types and mediators defining these inflammatory processes are still inadequately understood. In hepatitis C virus-associated glomerulonephritis, analysis of interferon-γ-inducible protein (IP-10) as a chemokine centrally involved in early antiviral response and TNF-α known to balance proinflammatory and immunosuppressive effects in inflammation shows a significant upregulation of both IP-10 and TNF-α mediated specifically by the viral receptor Toll-like receptor 3 expressed on mesangial cells. IP-10 induction is further potentiated by TNF-α signaling, preferentially via the TNF-α receptor subtype 2 selectively increased upon stimulation of viral receptors in the proinflammatory milieu.

Synthetic double-stranded RNA stimulates the expression of interferon-inducible protein 10 in human mesothelial cells.

Interferon-inducible protein 10 (IP-10) is a chemokine playing an important role in the restriction of viral spread. A time- and dose-dependent increase in IP-10 is found upon activation of viral receptors expressed on mesothelial cells, which provides novel evidence for a link between viral infections and inflammation of serous membranes.

Cystatin C and creatinine as markers for radiocontrast-induced nephropathy in patients treated with N-acetylcysteine.

The beneficial effect of N-acetylcysteine (NAC) in the prevention of radiocontrast-induced nephropathy (RCIN) as well as the definition of an adequate surrogate parameter for the evaluation of the incidence of RCIN remain points of controversial discussion. Nearly all clinical studies used an increase in serum creatinine to define renal injury, although cystatin C is suggested to be superior to creatinine in estimating glomerular filtration rate (GFR). Furthermore, a recent study showed that in healthy volunteers, NAC leads to a decrease in serum creatinine without influencing serum cystatin C concentrations, implicating a possible overestimation of the protective effect of NAC on the incidence of RCIN. We compared serum creatinine and cystatin C levels in patients with chronic kidney disease undergoing coronary angiography, as these patients are to be considered at highest risk for the development of RCIN. A total of three doses of NAC was given orally, and patients received isotonic saline intravenously. Serum levels at baseline and 24 hours after angiography were not significantly different for serum creatinine (1.72 +/- 0.08 mg/dl and 1.72 +/- 0.08 mg/dl) and for cystatin C (1.72 +/- 0.09 mg/dl and 1.76 +/-0.10 mg/dl). There was a significant positive correlation between creatinine and cystatin C serum levels before and after exposure to radiocontrast medium (p < 0.05) in all patients, including subgroup analyses. We conclude that serum creatinine and cystatin C are equivalent surrogate parameters for the evaluation of NAC in the prevention of RCIN. Furthermore, we present a prophylactic treatment regime easily applicable even in an outpatient setting, which seems to protect very effectively against RCIN in a high-risk group of patients.

Effect of activation of viral receptors on the gelatinases MMP-2 and MMP-9 in human mesothelial cells.

BACKGROUND: Extracellular matrix (ECM) not only provides molecular and spatial information that influence cell proliferation, differentiation and apoptosis but also has the potential to bind and present or release cytokines and cytotactic factors. Synthesis and degradation of extracellular matrix components are balanced by matrix metalloproteinases (MMP) and their inhibitors. In the pericardium as well as in the pleural and peritoneal cavities a multitude of clinically relevant disease states ranging from inflammation to fibrosis and tumor invasion result from altered regulation of MMP activity and are known to be associated with viral disease. METHODS: Therefore, the functional linkage between viral receptors of the innate immune system, the toll-like receptors (TLR), and control of MMP activity was exemplarily analyzed by stimulating human mesothelial cells with poly (I:C) RNA. RESULTS: We hereby show that human mesothelial cells (MC) express TLR3. After stimulation of MC with the cytokines TNF-alpha, IL-1beta and IFN-gamma alone or in combination to simulate a proinflammatory milieu as would occur during immune-mediated inflammatory disease, an upregulation of TLR3 is seen. Furthermore, a selectively TLR3 mediated, time- and dose-dependent upregulation of MMP-9 and TIMP-1 is found, whereas MMP-2 expression is not significantly affected by TLR3 stimulation. CONCLUSIONS: With these results we provide evidence for a mechanism by which infectious agents can mediate processes of the final common path of inflammation as fibrosis via regulation of MMP and TIMP.

Novel role of toll-like receptor 3 in hepatitis C-associated glomerulonephritis.

Hepatitis C virus (HCV) infection is frequently complicated by glomerulonephritis with immune complexes containing viral RNA. We examined the potential influence of Toll-like receptors (TLRs), specifically TLR3 recognition of viral dsRNA exemplified by polyriboinosinic:polyribocytidylic acid [poly(I:C) RNA]. Normal human kidney stained positive for TLR3 on mesangial cells (MCs), vascular smooth muscle cells, and collecting duct epithelium. Cultured MCs have low TLR3 mRNA levels with predominant intracellular protein localization, which was increased by tumor necrosis factor-alpha, interleukin (IL)-1beta, interferon (IFN)-gamma, and the TLR3 ligand poly(I:C) RNA. Poly(I:C) RNA stimulation of MCs increased mRNA and protein synthesis of IL-6, IL-1beta, M-CSF, IL-8/CXCL8, RANTES/CCL5, MCP-1/CCL2, and ICAM-I; it also increased anti-proliferative and proapoptotic effects, the latter of which was decreased by inhibiting caspase-8. In microdissected glomeruli of normal and non-HCV membranoproliferative glomerulonephritis biopsies, TLR3 mRNA expression was low. In contrast TLR3 mRNA expression was significantly increased in hepatitis C-positive glomerulonephritis and was associated with enhanced mRNA for RANTES/CCL5 and MCP-1/CCL2. We hypothesize that immune complexes containing viral RNA activate mesangial TLR3 during HCV infection, thereby contributing to chemokine/cytokine release and effecting proliferation and apoptosis. Thus, TLR3 expression on renal cells, and especially MCs, may establish a link between viral infections and glomerular diseases.

Sam68-like mammalian protein 2, identified by digital differential display as expressed by podocytes, is induced in proteinuria and involved in splice site selection of vascular endothelial growth factor.

Podocytes, the glomerular epithelial cells of the kidney, share important features with neuronal cells. In addition to phenotypical and functional similarities, a number of gene products have been found to be expressed exclusively or predominantly by both cell types. With the hypothesis of a common transcriptome shared by podocytes and neurons, digital differential display was used to identify novel podocyte-expressed gene products. Comparison of brain and kidney cDNA libraries with those of other organs identified Sam68-like mammalian protein 2 (SLM-2), a member of the STAR family of RNA processing proteins, as expressed by podocytes. SLM-2 expression was found to be restricted in the kidney to podocytes. In proteinuric diseases, SLM-2, a known regulator of neuronal mRNA splice site selection, was found significantly upregulated on mRNA and protein levels. Knockdown of SLM-2 by short interfering RNA in podocytes was performed to evaluate its biologic role. RNA splicing of vascular endothelial growth factor (VEGF), a key regulator of the filtration barrier and expressed as functionally distinct splice isoforms, was evaluated. VEGF(165) expression was found to be reduced by 25% after SLM-2 knockdown. In vivo, the glomerular expression of SLM-2 correlated with the mRNA levels of VEGF(165). This study demonstrates the power of digital differential display to predict cell type-specific gene expression by hypothesis-driven analysis of tissue cDNA libraries. SLM-2-dependent VEGF splicing indicates the importance of mRNA splice site selection for glomerular filtration barrier function.

Effects of chemokines on proliferation and apoptosis of human mesangial cells.

BACKGROUND: Proliferation and apoptosis of mesangial cells (MC) are important mechanisms during nephrogenesis, for the maintenance of glomerular homeostasis as well as in renal disease and glomerular regeneration. Expression of chemokines and chemokine receptors by intrinsic renal cells, e.g. SLC/CCL21 on podocytes and CCR7 on MC is suggested to play a pivotal role during these processes. Therefore the effect of selected chemokines on MC proliferation and apoptosis was studied. METHODS: Proliferation assays, cell death assays including cell cycle analysis, hoechst stain and measurement of caspase-3 activity were performed. RESULTS: A dose-dependent, mesangioproliferative effect of the chemokine SLC/CCL21, which is constitutively expressed on human podocytes was seen via activation of the chemokine receptor CCR7, which is constitutively expressed on MC. In addition, in cultured MC SLC/CCL21 had a protective effect on cell survival in Fas-mediated apoptosis. The CXCR3 ligands IP-10/CXCL10 and Mig/CXCL9 revealed a proproliferative effect but did not influence apoptosis of MC. Both the CCR1 ligand RANTES/CCL5 and the amino-terminally modified RANTES analogue Met-RANTES which blocks CCR1 signalling had no effect on proliferation and apoptosis. CONCLUSIONS: The different effects of chemokines and their respective receptors on proliferation and apoptosis of MC suggest highly regulated, novel biological functions of chemokine/chemokine receptor pairs in processes involved in renal inflammation, regeneration and glomerular homeostasis.