Katarzyna Neubauer

Enhanced formation of advanced oxidation protein products in IBD

Background: Advanced oxidation protein products (AOPPs) are new protein markers of oxidative stress with pro‐inflammatory properties, accumulated in many pathological conditions. The issue of their enhanced formation in IBD has not been addressed yet. Methods: The concentration of relative AOPPs (rAOPP; concentration of AOPPs divided by albumin level) were measured in 68 subjects with ulcerative colitis (UC), 50 subjects with Crohns disease (CD) and 45 healthy volunteers, and related to disease phenotype, clinical and biochemical activity, and therapeutic strategy. Diagnostic utility of rAOPP was evaluated by ROC analysis. Results: In comparison with controls (1.367 &mgr;mol/g), rAOPP were increased in inactive (1.778 &mgr;mol/g, P = 0.053) and active (1.895 &mgr;mol/g, P = 0.013) UC and in active (1.847 &mgr;mol/g, P = 0.003) CD. In CD, but not UC, rAOPP correlated with disease activity (r = 0.42, P = 0.013). Significant correlations with the inflammatory/malnutrition indices‐erythrocyte sedimentation rate (ESR) (r = 0.53), leukocytes (r = 0.33), platelets (r = 0.38), IL‐6 (r = 0.36), and transferrin (r = −0.35) were demonstrated in CD. In UC, rAOPP correlated only with ESR (r = 0.35) and IL‐6 (r = 0.30). Instead, associations with antioxidant dismutase (r = 0.29) and catalase (r = 0.22) were observed. The diagnostic power of rAOPP in discriminating diseased from non‐diseased subjects was less than that of C‐reactive protein (CRP). Simultaneous determination of rAOPP and CRP did not significantly improve the power of single CRP determination. Conclusions: IBD was associated with enhanced formation of AOPP, which differed between C and UC with respect to the relationship between rAOPP and disease activity, inflammatory and antioxidant response. These differences may reflect divergent ways that oxidative stress develops in CD and UC. The diagnostic power of rAOPP was insufficient for its clinical application.

Paraoxonase-1 status in Crohn's disease and ulcerative colitis

Background: Paraoxonase 1 (PON1) is an extracellular enzyme, which in the gastrointestinal tract may act as a local detoxifier, antioxidant, immunomodulator, and/or quorum‐quenching factor. There are no data on PON1 activity in Crohns disease (CD). Methods: PON1 phenotype and activity were determined spectrophotometrically in 52 subjects with CD, 67 with ulcerative colitis (UC), and 99 healthy individuals, and related to lipid peroxidation and disease phenotype, clinical and biochemical activity, and therapeutic strategy. Diagnostic utility of PON1 was evaluated by ROC analysis and compared with C‐reactive protein (CRP). Results: In comparison with controls (166 U), PON1 was reduced only in active CD (110 U, P < 0.0001) and UC (126 U, P < 0.0001), and correlated with disease activity (r = −0.47, P = 0.001 in CD and r = −0.50, P < 0.001 in UC). PON1 significantly correlated with erythrocyte sedimentation rate (ESR) (r = −0.36), platelets (r = −0.35), interleukin‐6 (r = −0.45), hemoglobin (r = 0.29), transferrin (r = 0.46), albumin (r = 0.60) in CD, and CRP (r = −0.29), ESR (r = −0.37), platelets (r = −0.43), leukocytes (r = −0.50), interleukin‐6 (r = −0.45), hemoglobin (r = 0.34), transferrin (r = 0.54), and albumin (r = 0.50) in UC. PON1 correlated positively with lipids but not with their peroxidation markers (thiobarbituric acid‐reactive substances, lipid hydroperoxides, ox‐LDL, and ox‐LDL autoantibodies). PON1 phenotype B (protective against IBD) tended to be less frequent in IBD patients than controls, and associated with lower concentration of inflammatory indices. PON1 was a poorer indicator of CD or UC than CRP. Conclusions: PON1 was reduced in IBD, despite treatment with antioxidant 5′‐aminosalicylate derivatives. PON1 reflected disease activity, inflammation severity, and anemia but not lipid peroxidation. The diagnostic power of PON1 was insufficient for its clinical application.

Circulating midkine in Crohn's disease: Clinical implications

Background: A noninvasive marker facilitating differential diagnosis in Crohns disease (CD) is sought after. Midkine is a heparin‐binding growth factor of angiogenic and chemotactic properties, positively evaluated as a tumor marker, and a possible association with CD has not yet been investigated. Methods: Circulating midkine was measured in 91 CD patients and 108 controls and related to disease clinical and biochemical activity, inflammation severity, and angiogenesis. Midkine diagnostic value in comparison with C‐reactive protein (CRP) was evaluated by receiver operating characteristic (ROC) analysis. Results: Circulating midkine was elevated both in quiescent and active disease compared to controls (147, 506, and 93 pg/mL, respectively), and corresponded well with disease activity (r = 0.49, P < 0.001). Midkine significantly correlated with inflammatory indices: CRP (r = 0.49), erythrocyte sedimentation rate (r = 0.31), leukocytes (r = 0.48), platelets (r = 0.52), albumin (r = −0.49), transferrin (r = −0.47), and IL‐6 (r = 0.54); hematological variables: hemoglobin (r = −0.38), hematocrit (r = −0.43), and iron (r = −0.58); angiogenic factors: vascular endothelial growth factor‐A (r = 0.42), fibroblast growth factor‐2 (r = 0.54), and platelet‐derived growth factor‐BB (r = 0.57). Midkine elevation corresponded well (r = −0.41) with the drop in paraoxonase‐1 activity—a quorum‐quenching factor. Midkine as a marker of active CD had sensitivity and specificity of 86% and 97%, respectively, whereas CRP was 83% and 92%. Conclusions: CD is associated with an elevation of midkine, which corresponds well with disease activity and reflects the severity of inflammatory response and exacerbation of pathological angiogenesis. Midkine performance as a disease marker was slightly better than that of CRP. Its high specificity and likelihood ratios for positive test results might recommend midkine as a possible “ruling in” marker in CD. Inflamm Bowel Dis 2009

Impaired Erythrocyte Antioxidant Defense in Active Inflammatory Bowel Disease: Impact of Anemia and Treatment

Background: Oxidative stress contributes to the propagation and exacerbation of inflammatory bowel disease (IBD) but the status of erythrocyte antioxidant defense remains unknown. Methods: Erythrocyte activities of superoxide dismutase‐1 (SOD1), catalase, and glutathione peroxidase‐1 (GPx1) were determined in 174 IBD patients and 105 controls and referred to IBD activity, inflammation severity, nutritional status, systemic oxidative stress, anemia, and treatment. Results: Catalase and GPx1 activities were decreased in active IBD, whereas SOD1 became upregulated by IBD‐related oxidative stress. In Crohns disease (CD) corticosteroids decreased SOD1 activity. SOD1 correlated indirectly with CD activity and erythrocyte sedimentation rate (ESR) and directly with transferrin. In ulcerative colitis (UC) anemia downregulated SOD1. Decreases in GPx activity corresponded with IBD activity, anemia, inflammation, and malnutrition. Oxidative stress in UC and corticosteroids in CD also downregulated GPx. Catalase activity was decreased by CD‐related anemia, correlating directly with hemoglobin, and indirectly with CD activity, inflammatory and protein oxidative stress markers. When co‐analyzed, anemia but not CD activity significantly contributed to catalase downregulation. In UC, catalase activity corresponded indirectly with UC endoscopic activity and inflammation and directly with hemoglobin. UC activity, anemia, and treatment with azathioprine negatively affected catalase. As indicators of active IBD, GPx1 showed a diagnostic accuracy of 73%, whereas catalase showed 63% as compared to 74% of C‐reactive protein and ESR. Conclusions: Erythrocyte antioxidant defense is impaired in active IBD. SOD1, GPx1, and CAT activities are differently affected by the disease type, activity, anemia, inflammation, oxidative stress, and treatment. As an active IBD indicator, GPx1 was comparable to C‐reactive protein and ESR. Inflamm Bowel Dis 2010

Midkine, a multifunctional cytokine, in patients with severe sepsis and septic shock: a pilot study.

The objective of the study was to evaluate whether severe sepsis and septic shock are related to alterations in midkine concentrations, to identify disease-related factors associated with these alterations, and to initially appraise whether midkine might serve as a biomarker in sepsis. Prospective observational cross-sectional study with 5-day follow-up. Circulating midkine was measured (enzyme-linked immunosorbent assay) in 38 septic (13 with severe sepsis, 25 with septic shock), 82 active inflammatory bowel disease (IBD) (26 with systemic inflammatory response syndrome [SIRS]) patients, and 87 healthy subjects. Midkine significantly increased along with a sequence: health-inflammation (IBD)-systemic inflammation (IBD-SIRS)-severe sepsis/septic shock. High midkine levels (>1,000 ng/L) were found in 63% of septic and in 19% of IBD-SIRS patients, whereas extremely high concentrations (>5,000 ng/L) were found in 16% vs. 4%. Although not different at admission, midkine gradually decreased in severe sepsis and remained high in shock. Similarly, persistently high midkine was observed in patients with cardiovascular insufficiency (CVI) and in mechanically ventilated as compared with normalizing levels in patients without CVI and not requiring ventilation. The differences in devised simple rates (&Dgr;5th-1st) were significant in all these cases. Accordingly, admission midkine was higher in patients with metabolic acidosis. Concerning pathogen, gram-positive infections were associated with the highest midkine levels. In conclusion, sepsis and septic shock are associated with midkine elevation, substantially more pronounced than in inflammation, even systemic, revealing a new potential mediator of deregulation of neutrophil migration. Sepsis-related global hypoxia seems to contribute to midkine elevation. Our results substantiate further research on possible midkine application as a sepsis biomarker: in differentiating SIRS from sepsis and identifying gram-positive sepsis and septic patients at risk of CVI and shock.

Matrix metalloproteinase-9: its interplay with angiogenic factors in inflammatory bowel diseases.

Matrix metalloproteinase- (MMP-) 9 is one of the main metalloproteinases reported to be involved in extracellular matrix degradation and recently also in triggering of angiogenic switch in the course of inflammatory bowel diseases (IBD). The goal of our studies was to estimate in one experimental setting the levels of MMP-9 in sera of Crohns Disease (CD) and ulcerative colitis (UC) patients and to evaluate its possible diagnostic potential in comparison with other biochemical markers and selected proinflammatory and angiogenic factors. The study group included 176 subjects (CD = 64, UC = 85, control = 27). Concentrations of serum MMP-9 were significantly higher in active than inactive forms of IBD, being higher in active UC than in active CD. Both in the case of CD and UC serum MMP-9 positively correlated with disease activity, IL-6 levels, platelet and leukocyte count, midkine, and PDGF-BB, as well as in UC with ESR and in CD with CRP, IL-1, and VEGF-A. Diagnostic accuracy of MMP-9 in distinguishing active UC from active CD was 66%, and displayed higher specificity than CRP (79.0% versus 61.6%, resp.). Evaluation of serum MMP-9 concentrations could aid in differentiation of active UC from active CD. MMP-9 correlated better with inflammatory and angiogenic parameters in CD than in UC.

Platelet-derived growth factor-BB reflects clinical, inflammatory and angiogenic disease activity and oxidative stress in inflammatory bowel disease.

OBJECTIVES The goal of the studies was the evaluation of platelet-stored (serum) and circulating (plasma) pools of platelet-derived growth factor (PDGF)-BB in inflammatory bowel disease (IBD) and the assessment of a possible application of PDGF as the disease marker. DESIGN AND METHODS Serum and plasma PDGF-BB were measured in 134 IBD patients and 81 controls and evaluated with respect to the disease status, endoscopic, inflammatory, and angiogenic activity. The diagnostic utility was evaluated using ROC analysis. RESULTS PDGF was increased exclusively in active IBD regardless the disease type and associated with its clinical and endoscopic activity. Serum- and plasma-PDGF were poorly interrelated. Plasma-PDGF better reflected oxidative stress whereas serum-PDGF reflected inflammation and angiogenesis. In multivariate analysis, platelets alone explained about 30% in the PDGF variability and seemed to mediate most of the observed relationships. CONCLUSIONS IBD is associated with the increases in platelet-stored and circulating PDGF, which correspond with the disease clinical, endoscopic, inflammatory, and angiogenic activity and IBD-associated oxidative stress. However, PDGF as an active-IBD marker was not better than currently applied C-reactive protein, erythrocyte sedimentation rate and platelets.

Clinical relevance of circulating midkine in ulcerative colitis

Abstract Background: Non-invasive biochemical markers are needed to support the diagnosis of ulcerative colitis (UC), an incurable disease of unknown pathology. Midkine is an angiogenic cytokine, chemotactic towards neutrophils and macrophages, and a T-regulatory cell suppressor. Methods: Serum midkine was measured immunoenzymatically in 93 UC patients and 108 healthy subjects, and evaluated with respect to disease status, endoscopic, inflammatory and angiogenic activity. The diagnostic value of midkine was compared to C-reactive protein (CRP) using receiver operating characteristics (ROC) analysis. Results: Midkine was higher (p<0.0001) in inactive (199 ng/L) and active UC (351 ng/L) compared with controls (93 ng/L), and reflected disease activity (r=0.427, p<0.001). Midkine was correlated with CRP, erythrocyte sedimentation rate (ESR), leukocytes, platelets, interleukin-6, paraoxonase-1, albumin, transferrin, iron, hemoglobin, and hematocrit. Midkine correlated with angiogenic factors: vascular endothelial growth factor-A and platelet-derived growth factor-BB. As a marker of UC, midkine showed a diagnostic accuracy of 85%, sensitivity of 72%, specificity of 82%, whereas CRP showed 83%, 65% and 91%, respectively. As a marker of active UC, midkine showed a diagnostic accuracy of 87%, sensitivity of 84%, specificity of 75%, whereas CRP showed 75%, 63% and 83%, respectively. Combined assessment of midkine and CRP improved sensitivity but substantially decreased specificity. Conclusions: UC is associated with increased circulating midkine, which corresponds with clinical, endoscopic, inflammatory and angiogenic activity, and anemia. Performance of midkine as a marker of UC or active UC was comparable to that of CRP. Clin Chem Lab Med 2009;47:1085–90.

Nampt/PBEF/Visfatin Upregulation in Colorectal Tumors, Mirrored in Normal Tissue and Whole Blood of Colorectal Cancer Patients, Is Associated with Metastasis, Hypoxia, IL1β, and Anemia

Targeting Nampt/PBEF/visfatin is considered a promising anticancer strategy, yet little is known about its association with colorectal cancer (CRC). We quantified Nampt/PBEF/visfatin expression in bowel and blood (mRNA and protein), referring it to CRC advancement and inflammatory, angiogenic, hypoxia, and proliferation indices. Tumor Nampt/PBEF/visfatin upregulation was associated with metastasis, anemia, tumor location, HIF1α, and inflammatory and angiogenic indices, of which HIF1α, IL1β, and anemia explained 70% in Nampt/PBEF/visfatin variability. Nampt/PBEF/visfatin expression in nontumor tissue, both mRNA and protein, increased in patients with metastatic disease and mild anemia, and, on transcriptional level, correlated with HIF1α, IL1β, IL8, CCL2, and CCL4 expression. Whole blood Nampt/PBEF/visfatin tended to be elevated in patients with metastatic cancer or anemia and correlated with inflammatory indices, of which IL1β, IL8, and hematocrit explained 60% of its variability. Circulating visfatin was associated with lymph node metastasis and inflammatory and angiogenic indices. In vitro experiments on SW620 cells demonstrated Nampt/PBEF/visfatin downregulation in response to serum withdrawal but its upregulation in response to serum induction and hypoxia. Stimulation with recombinant visfatin did not provide growth advantage. Summarizing, our results link Nampt/PBEF/visfatin with tumor metastatic potential and point at inflammation and hypoxia as key inducers of its upregulation in CRC.

Circulating midkine in malignant and non-malignant colorectal diseases

Midkine is a multifunctional cytokine found to be a promising cancer biomarker, however, its suitability in colorectal cancer (CRC) has not been evaluated yet. We assessed midkine circulating levels immunoenzymatically in 105 CRC patients, 86 individuals with increased risk for CRC (56 with inflammatory bowel disease (IBD) and 30 with adenomas), and 70 healthy controls and compared its performance as CRC biomarker to carcinoembryonic antigen (CEA). Midkine was higher in CRC (807 ng/L) than in IBD (477 ng/L; 633 ng/L in active and 335 ng/L in inactive), adenomas (418 ng/L) or controls (245 ng/L). Its levels increased along with advancing CRC stage, being significantly higher compared to controls already in stage I, and dedifferentiation (higher in grade 3 than 1 and 2). Lymph node or distant metastases were associated with significant midkine elevation as well. Midkine positively correlated with IL-1β, IL-6, IL-8, TNF-α, MCP-1, MIP-1α, G-CSF, GM-CSF, VEGF-A, and PDGF-BB with IL-1β and PDGF-BB explaining 40% in its variability. Midkine was better marker of CRC than CEA with 80% accuracy, 83% sensitivity and 68% specificity as compared to 60%, 37%, and 88% of CEA, also in its early stages (74% vs. 52% accuracy). Midkine better differentiated CRC from inactive while CEA from active IBD. Midkine was included in the multimarker panel and significantly contributed to efficient (Λ=0.16) differentiation of healthy controls, adenomas and CRC. Concluding, midkine may lack sufficient specificity to be a sole CRC marker but seems to constitute a valuable addition to multimarker panels devised for CRC screening and/or surveillance.