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Prior to using a new multi-analyte platform for the detection of markers in sputum it is advisable to assess whether sputum processing, especially mucus homogenization by dithiothreitol (DTT), affects the analysis. In this study we tested a novel Human Inflammation Multi Analyte Profiling® Kit (v1.0 Luminex platform; xMAP®). Induced sputum samples of 20 patients with stable COPD (mean FEV1, 59.2% pred.) were processed in parallel using standard processing (with DTT) and a more time consuming sputum dispersion method with phosphate buffered saline (PBS) only. A panel of 47 markers was analyzed in these sputum supernatants by the xMAP®. Twenty-five of 47 analytes have been detected in COPD sputum. Interestingly, 7 markers have been detected in sputum processed with DTT only, or significantly higher levels were observed following DTT treatment (VDBP, α-2-Macroglobulin, haptoglobin, α-1-antitrypsin, VCAM-1, and fibrinogen). However, standard DTT-processing resulted in lower detectable concentrations of ferritin, TIMP-1, MCP-1, MIP-1β, ICAM-1, and complement C3. The correlation between processing methods for the different markers indicates that DTT processing does not introduce a bias by affecting individual sputum samples differently. In conclusion, our data demonstrates that the Luminex-based xMAP® panel can be used for multi-analyte profiling of COPD sputum using the routinely applied method of sputum processing with DTT. However, researchers need to be aware that the absolute concentration of selected inflammatory markers can be affected by DTT.

Actovegin(®) is a biological drug manufactured from a natural source: it is a calf blood hemodialysate. Its therapeutic benefits stem from a variety of pharmacodynamic actions that can be summarized to a common goal, i.e. the enhancement of cellular metabolism; this results from an insulin-like activity mediated by Inositol-phospho-oligosaccharides. Actovegin(®) results in beneficial effects in several pathophysiological clinical settings including malfunction of the blood circulation and trophic disturbances in the brain, impairment of peripheral blood circulation and associated diseases, dermal transplants and acute and chronic wounds. Here, we give an overview of the pharmacodynamic actions of calf-blood hemidialysate and its beneficial effects in a variety of clinical settings.

Availability of human chondrocytes is a major limiting factor regarding drug discovery projects and tissue replacement therapies. As an alternative human mesenchymal stem cells (hMSCs) from bone marrow are taken into consideration as they can differentiate along the chondrogenic lineage. However, it remains to be shown whether they could form a valid model for primary chondrocytes with regards to inflammatory mediator production, like nitric oxide (NO) and prostanoids. We therefore investigated the production of NO and prostanoids in hMSCs over the course of chondrogenic differentiation and in response to IL-1β using primary OA chondrocytes as reference. Chondrogenic differentiation was monitored over 28 days using collagen I, collagen II, and collagen X expression levels. Expression levels of inducible nitric oxide synthase (iNOS), levels of NO, and prostanoids were assessed using PCR, Griess assay, and GC/MS/MS, respectively. The hMSCs collagen expression profile during course of differentiation was consistent with a chondrocytic phenotype. Contrary to undifferentiated cells, differentiated hMSCs expressed iNOS and produced NO following stimulation with IL-1β. Moreover, this induction of iNOS expression was corticosteroid insensitive. The spectrum of prostanoid production in differentiated hMSCs showed similarities to that of OA chondrocytes, with PGE2 as predominant product. We provide the first detailed characterization of NO and prostanoid production in hMSCs in the course of chondrogenic differentiation. Our results suggest that differentiated hMSCs form a valid model for chondrocytes concerning inflammatory mediator production. Furthermore, we propose that IL-1β stimulation, leading to corticosteroid-insensitive NO synthesis, can be used as a sensitive marker of chondrogenesis. J. Cell. Biochem. 98: 798–809, 2006. © 2006 Wiley-Liss, Inc.

Objective : The present cross sectional study was performed to test the hypothesis that in osteoarthritis (OA) of the knee severity of this disease is related to local levels of inflammatory metabolites and their corresponding enzymes. Methods : From 41 patients with OA of the knee (age range 45–79 years) undergoing arthroscopy blood, synovial fluid (SF) and synovial membrane (SM) were collected. Clinical conditions were primarily assessed by the WOMAC-index and radiographic grading (K&L-grade). Concentrations of PGE 2 , TxB 2 and NO 2/3 and that of IL-6, IL-1α, IL-1β, TNFα, COX-2 and iNOS were determined in SF and SM, respectively. Results : With advancing age K&L-grade and COX-2 in SM increased significantly ( P =0.005 and P =0.01, respectively). TNFα and IL-1α were not detectable in SM samples. Apart from a correlation between PGE 2 and WOMAC-index (r=0.36, P =0.035) no significant relationships could be found between the various inflammatory parameters and any of the assessed clinical signs. Conclusions : Apparently no direct relationships exist between the measured markers of inflammation (e.g. PGE 2 , NO 2/3 ) or the involved enzymes (e.g. COX-2, iNOS) and the severity of OA of the knee. The degenerative condition of this disease might be due to the more local, mainly mechanical injury with little systemic upset. However, further longitudinal studies are needed to clarify whether the assessed biochemical markers could serve as predictors for the progression of OA.

Rationale: COPD is a chronic inflammatory disease. It appears, that a single parameter or the cellular composition within the airways alone is not sufficient to characterize the degree of airway inflammation. Aim: Definition of a robust inflammatory phenotype using multiple sputum markers with a better reproducibility as compared to sputum neutrophils alone. Methods: 24 COPD patients (GOLD II) and 23 age and gender matched healthy controls were included (current smokers with 10 pack-years). Blood, bronchial biopsies, bronchoalveolar lavage (BAL), and induced sputum were collected on two occasions within 6 weeks. Cell composition and a broad panel of proteins were analyzed. Results: The top 4 most reproducible (r>0.7) sputum inflammatory markers (A1AT, IL6, MMP7, HSA) and the percentage of sputum neutrophils (r=0.52) were combined to define the inflammatory phenotype (IP) independently for both visits. The IP showed a better reproducibility (r=0.70; p

Background Pulmonary surfactant protein D (SP-D) is considered as a candidate biomarker for the functional integrity of the lung and for disease progression, which can be detected in serum. The origin of SP-D in serum and how serum concentrations are related to pulmonary concentrations under inflammatory conditions is still unclear. Methods In a cross-sectional study comprising non-smokers (n = 10), young - (n = 10), elderly smokers (n = 20), and smokers with COPD (n = 20) we simultaneously analysed pulmonary and serum SP-D levels with regard to pulmonary function, exercise, repeatability and its quaternary structure by native gel electrophoresis. Statistical comparisons were conducted by ANOVA and post-hoc testing for multiple comparisons; repeatability was assessed by Bland-Altman analysis. Results In COPD, median (IQR) pulmonary SP-D levels were lower (129(68) ng/ml) compared to smokers (young: 299(190), elderly: 296(158) ng/ml; p < 0.01) and non-smokers (967(708) ng/ml; p < 0.001). The opposite was observed in serum, with higher concentrations in COPD (140(89) ng/ml) as compared to non-smokers (76(47) ng/ml; p < 0.01). SP-D levels were reproducible and correlated with the degree of airway obstruction in all smokers. In addition, smoking lead to disruption of the quaternary structure. Conclusions Pulmonary and serum SP-D levels are stable markers influenced by smoking and related to airflow obstruction and disease state. Smaller subunits of pulmonary SP-D and the rapid increase of serum SP-D levels in COPD due to exercise support the translocation hypothesis and its use as a COPD biomarker. Trial registration no interventional trial

The present cross sectional study was performed to test the hypothesis that in osteoarthritis (OA) of the knee severity of this disease is related to local levels of inflammatory metabolites and their corresponding enzymes.From 41 patients with OA of the knee (age range 45-79 years) undergoing arthroscopy blood, synovial fluid (SF) and synovial membrane (SM) were collected. Clinical conditions were primarily assessed by the WOMAC-index and radiographic grading (KL-grade). Concentrations of PGE(2), TxB(2)and NO(2/3)and that of IL-6, IL-1 alpha, IL-1 beta, TNF alpha, COX-2 and iNOS were determined in SF and SM, respectively.With advancing age KL-grade and COX-2 in SM increased significantly (P=0.005 and P=0.01, respectively). TNF alpha and IL-1 alpha were not detectable in SM samples. Apart from a correlation between PGE(2)and WOMAC-index (r=0.36, P=0.035) no significant relationships could be found between the various inflammatory parameters and any of the assessed clinical signs.Apparently no direct relationships exist between the measured markers of inflammation (e.g. PGE(2), NO(2/3)) or the involved enzymes (e.g. COX-2, iNOS) and the severity of OA of the knee. The degenerative condition of this disease might be due to the more local, mainly mechanical injury with little systemic upset. However, further longitudinal studies are needed to clarify whether the assessed biochemical markers could serve as predictors for the progression of OA.

Degradation of angiogenic mediators might be an underlying cause of chronic wounds. To test this hypothesis, we evaluated the expression and integrity of vascular endothelial growth factor, a potent angiogenic mediator, and its receptors, Flt-1 and KDR, in chronic venous leg ulcerations. Immunohisto- chemical, in situ hybridization, and semiquantitative reverse transcriptase polymerase chain reaction analyses all indicate that expression of vascular endothelial growth factor is elevated in ulcerative tissue, with vascular endothelial growth factor mRNA being especially pronounced in the hyperplastic epithelium of the wound margin. Flt-1 and KDR protein and mRNA were detected in the papillary vessels in close vicinity to the lesional epithelium of chronic wounds. Although increased expression of vascular endothelial growth factor protein was detected in the epidermis, the intensity of this staining was weak compared with the epidermal staining in psoriatic lesions and compared with the strong vascular endothelial growth factor mRNA signal in chronic wounds and psoriasis. To analyze whether this apparent decrease in immunoreactivity could be the result of degradation of vascular endothelial growth factor by proteolytic activities from the wound environment, we examined the stability of recombinant vascular endothelial growth factor in wound fluid from chronic leg ulcers. As demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, incubation of rVEGF165 with chronic, but not acute, wound fluid resulted in rapid proteolytic degradation of rVEGF165. Protease inhibitor studies indicate that serine proteases, such as plasmin, are involved in this degradation. Together, our data show that, although vascular endothelial growth factor expression is elevated in chronic wounds, increased proteolytic activity in this environment results in its degradation, which may contribute to an impaired wound healing response.

Vascular endothelial growth factor (VEGF) is a potent angiogenic mediator in tissue repair. In non-healing human wounds plasmin cleaves and inactivates VEGF165. In the present study, we generated recombinant VEGF165 mutants resistant to plasmin proteolysis. Substitution of Arg110 with Ala110 or Gln110, and Ala111 with Pro111 yielded plasmin-resistant and biologically active VEGF165 mutants. In addition, substitution of Ala111 with Pro111 resulted in a substantial degree of stabilization when incubated in wound fluid obtained from non-healing wounds. These results suggest that the plasmin cleavage site Arg110/Ala111 and the carboxyl-terminal domain play an important role in the mitogenic activity of VEGF165.

Background The aim of this study was to test whether repeatable biomarkers collected from serum, bronchoalveolar lavage (BAL) and sputum of healthy smokers and smokers with COPD would have a prognostic value with respect to the decline in lung function over a 5 year period. Methods In 2006/2007 we had repeatedly collected serum, BAL and sputum of 23 healthy smokers and 24 smokers with COPD (GOLD II) and analysed a panel of more than 100 different parameters. In 2012 we reinvited these subjects to assess the change in lung function to enable the investigation of the potential prognostic value of the 2006/2007 markers and to determine the long-term repeatability of selected blood and serum markers. In this follow-up study we performed body-plethysmography, a blood gas analysis and collected blood and urine samples. The change in lung function was compared with 67 markers from BAL, sputum, serum and whole blood that were shown in the 2006/2007 assessment to be repeatable over a 6 week period. Results We were able to recruit 13 (54%) smokers with COPD and 11 (48%) former healthy smokers that participated in the 2006/2007 study. The decline in lung function was larger in COPD smokers; five of them changed to GOLD III, one to GOLD IV. Two healthy smokers changed to GOLD I. Blood cells, serum von Willebrand factor and alpha-1-antitrypsin showed a good repeatability over 5 years. In COPD smokers a weak correlation between 2006/2007 sputum markers of neutrophilic inflammation and the 5 year change in FEV1/FVC was found. Conclusions Our data suggests that inter-individual and group differences are maintained over a five year period. Despite the large panel of markers available for this analysis, a potential prognostic value appears to exist only for some sputum inflammatory markers. If these data can be confirmed in larger COPD cohorts, it would emphasize the value of sputum markers in clinical trials and support the assumption that an anti-inflammatory treatment can have long term benefits in COPD.