Iwona Bednarz-Misa

Visfatin in juvenile obesity – the effect of obesity intervention and sex

Eur J Clin Invest 2011; 41 (12): 1284–1291

Localization of Enolase in the Subfractions of a Breast Cancer Cell Line

Enolase detected on the cell surface may be a receptor for certain ligands, especially for plasminogen. It is important for the pathogen invasiveness and in the development of a tumour. Therefore, we sought to preliminarily determine the enolase location and catalytic activity in the subfractions of MCF-7 cells. The latter was done on intact cells and in subfractions of MCF-7 cells. We identified enolase by immunoblotting. The binding of human plasminogen to enolase was performed by immunoblotting using monoclonal antibodies against plasminogen. The intact MCF-7 cells demonstrated activity of enolase. Enolase in postnuclear and perinuclear fractions is catalyticly active too. We identified the enolase protein in immunoblots of these fractions, except for the nuclear subfraction. These results provide evidence that enolase is present on the intact surface of MCF-7 cells and in post- and perinuclear fractions. The surface protein maintained catalytic activity, which suggests that its location in the plasma membrane didn’t change the active centre of the enzyme

Circulating adipocyte fatty acid-binding protein, juvenile obesity, and metabolic syndrome.

Abstract Adipocyte fatty acid-binding protein (A-FABP) links obesity and metabolic syndrome (MetS) and might be targeted in future therapies. Its utility as a MetS biomarker has been suggested in adults but has not been examined in children/adolescents. Our objectives were to identify metabolic parameters associated with A-FABP elevation in children and adolescents and to evaluate the effect of obesity intervention and A-FABP diagnostic utility. A-FABP and anthropometric, metabolic, and inflammatory indices were measured in 31 lean and 114 overweight/obese children and adolescents and reassessed after obesity intervention (1 year; diet and enhanced physical activity, with or without metformin). A-FABP was significantly higher in overweight/obese than lean individuals, where it correlated with insulin, waist circumference (WC), and 2-h glucose independent of body mass index (BMI), age, gender, and developmental stage. The pattern of A-FABP associations differed between sexes. As a MetS indicator, A-FABP had 68% accuracy. The weight reduction program was effective in reducing A-FABP, BMI%, WC, triglycerides, and cholesterol. In conclusion, elevation in A-FABP is associated with MetS components independent of BMI status and can be reduced by diet and enhanced physical activity. A-FABP as a single MetS biomarker has a moderate accuracy.

Elevated systemic interleukin-7 in patients with colorectal cancer and individuals at high risk of cancer: association with lymph node involvement and tumor location in the right colon

Interleukin (IL)-7 is a cytokine essential for protective immunity, and it is considered as a promising agent for cancer immunotherapy. Recent studies, however, appear to associate IL-7 with aggressiveness of solid tumors. The IL-7 has been less studied in colorectal cancer (CRC) and conditions associated with increased risk of CRC development. To explore IL-7 status in bowel diseases, it was measured immunofluorometrically in 431 individuals (110 with CRC) by using Luminex platform. A level of IL-7 in CRC patients was significantly higher than in controls, did not differ from those with adenomas, but was lower than in both active and inactive inflammatory bowel disease (IBD) cases. In CRC, IL-7 was higher in patients with lymph node and distant metastases and with tumors located in right colon. In adenomas, IL-7 elevation was associated exclusively with villous growth pattern, while in IBD, circulating IL-7 reflected clinical activity of Crohn’s disease and ulcerative colitis. Systemic TNFα, IL-10, and PDGF-BB were independent predictors of circulating IL-7. In summary, our study is the first to demonstrate IL-7 elevation in CRC in association with metastatic disease and tumor location. Both associations should be considered when designing IL-7-based immunotherapies for CRC. Further studies on IL-7 functionality in CRC are necessary.

Distribution of beta-enolase in normal and tumor rat cells.

Enolase - a glycolytic enzyme is also expressed on the surface of eukaryotic cells such as macrophages, neutrophils, endothelial, neuronal, tumor cells. Surface enolase as plasminogen receptor plays an important role in myogenesis, tumorgenesis and angiogenesis. Determination of enolase localization in the cell lines may give rise to the elucidation of its receptor function in tumor cells. The cellular localization of the muscle-specific isoform of the enolase in normal rat cardiomyocytes (H9c2, an embryonic rat heart-derived cell line) and a rat sarcoma (R1) cell line is reported here. Immunocytochemical assays showed that this enolase isoform is freely diffused in the sarcoplasm of rat cells. The evident location of enolase molecules on the perinuclear surface is observed in immunofluorescence assays. Enolase localization on the surface of some intact normal rat cardiomyocytes was also observed. This surface protein maintains enolase catalytic activity.

Systemic interleukin-9 in inflammatory bowel disease: Association with mucosal healing in ulcerative colitis

AIM To evaluate circulating IL9 in inflammatory bowel disease and disease-associated anemia/cachexia and assess its potential as a mucosal healing marker. METHODS Serum IL9 as well as other cytokines (IL1β, IL6, IL13, IFNγ, TNFα, and VEGF-A) were determined in 293 individuals: 97 patients with Crohn’s disease (CD) and 74 with ulcerative colitis (UC) and in 122 apparently healthy controls. The clinical activity of CD and UC was expressed in terms of the Crohn’s Disease Activity Index (CDAI) and the Mayo Scoring System (MDAI), respectively, and the severity of bowel inflammation in UC patients was assessed using Mayo endoscopic score. Cytokine concentrations were measured by a flow cytometry-based method using Luminex xMAP® technology. High-sensitive C-reactive protein concentrations (hsCRP) were determined in CD and UC patients using the enhanced immunoturbidimetric method. RESULTS Systemic IL9 was significantly lower in healthy individuals [9 pg/mL (95%CI: 8.2-10)] than in patients with inflammatory bowel disease (IBD): both inactive [14.3 pg/mL (11.9-19.9)] and active [27.6 pg/mL (24.5-32), P < 0.0001]. Cytokine concentrations were significantly higher in active CD [27.4 pg/mL (23.4-32.2)] and in active UC [32.7 pg/mL (27-38.9)] compared to inactive diseases [15.9 pg/mL (10.8-23.4) in CD and 19.4 pg/mL (13.9-27.1) in UC, P = 0.001]. IL9 correlated weakly with CDAI (ρ = 0.32, P = 0.003) and MDAI (ρ = 0.35, P = 0.002) and strongly with endoscopic inflammation in UC (ρ = 0.74, P < 0.0001). As a negative marker of mucosal healing (MH), IL9 had an accuracy superior to hsCRP and IL6 [97% (P < 0.0001), 67% (P = 0.071), and 55% (P = 0.525), respectively]. IL9 was significantly higher in cachectic IBD patients [30.25 pg/mL (24.4-37.5) vs 21.88 pg/mL (18-26.5), P = 0.026] and negatively correlated with hemoglobin concentrations (ρ = -0.27, P < 0.001). Multiple regression showed IL1β and IL13 to be the independent predictors of circulating IL9 in healthy individuals, IFNγ or IL6 in active and inactive UC, respectively, and IL13 and VEGF-A in both active and inactive CD. CONCLUSION The systemic IL9 level is higher in IBD and corresponds with endoscopic inflammation, suggesting its possible application as a negative marker of mucosal healing in UC.

Midkine is up-regulated in both cancerous and inflamed bowel, reflecting lymph node metastasis in colorectal cancer and clinical activity of ulcerative colitis.

HighlightsMidkine overexpression in colon and leukocytes accompanies regional metastasis in CRC.Midkine is overexpressed in inflamed bowel only in ulcerative colitis.Midkine expression in tumor‐adjacent tissue is upregulated and depends on CRC stage.Serum midkine drops directly after surgery but its levels are restored on 3rd day. ABSTRACT Midkine is a multifunctional cytokine and growth factor displaying proinflammatory and pro‐tumorigenic activity. Its association with bowel diseases has not been fully elucidated. Our purpose was to delineate midkine expression pattern by RT‐qPCR in inflamed/cancerous bowel (n = 208) and whole blood (n = 150) in colorectal cancer (CRC), Crohns disease (CD), and ulcerative colitis (UC) and to evaluate midkine dynamics in early postoperative period following colorectal surgery. The expression of midkine was significantly up‐regulated in stage III CRC and independently associated with lymph node metastasis. The expression of midkine in whole blood was up‐regulated solely in N1 CRC. Midkine expression in cancer‐free tissue (CRC) was also elevated and dependent on CRC advancement. In IBD, inflammation increased the bowel expression of midkine solely in UC, in a manner proportional to the disease clinical activity. Large and small bowel differed with respect to the expression of midkine in quiescent tissue (higher in small bowel) and to its correlation pattern with chemokines (in a large bowel) and angiogenic factors and cell cycle regulators (in a small bowel). Circulating midkine and its expression in whole blood dropped directly following colorectal surgery; however, the concentration of midkine in serum was restored on postoperative day three. Midkine is involved in bowel inflammation in UC and lymph node metastasis in CRC, rendering midkine an attractive target for their treatment. Owing to midkine elevation in early postoperative period and its overexpression in tumor‐adjacent tissue, targeting midkine might be considered also as a prevention of CRC recurrence following curative tumor resection.

Serum availability affects expression of common house-keeping genes in colon adenocarcinoma cell lines: implications for quantitative real-time PCR studies

Careful selection of housekeeping genes (HKG) is prerequisite to yield sound qPCR results. HKG expression varies in response to hypoxia but the effect of manipulations of serum availability, a common experimental procedure, remains unknown. Also, no data on HKG expression stability across colon adenocarcinoma lines that would aid selection of normalizers suitable for studies involving several lines are available. Thus, we evaluated the effect of serum availability on the expression of commonly used HKG (ACTB, B2M, GAPDH, GUSB, HPRT1, IPO8, MRPL19, PGK1, PPIA, RPLP0, RPS23, SDHA, TBP, UBC, and YWHAZ) in seven colon adenocarcinoma cell lines (Caco-2, DLD-1, HCT116, HT29, Lovo, SW480, and SW620). Sets of stably expressed line-specific and pan-line HKG were validated against absolutely quantified CDKN1A, TP53, and MDK transcripts. Both serum availability and line type affected HKG expression. UBC was fourfold down-regulated and HPRT1 1.75-fold up-regulated in re-fed HT29 cultures. Line-to-line variability in HKG expression was more pronounced than that caused by altering serum availability and could be found even between isogenic cell lines. PPIA, RPLP0, YWHAZ, and IPO8 were repeatedly highly ranked while ACTB, B2M, UBC, and PGK1 were ranked poorly. Normalization against PPIA/RPLP0/SDHA was found optimal for studies involving various colon adenocarcinoma cell lines subjected to manipulations of serum availability. We found HKG expression to vary, more pronouncedly by line type than growth conditions with significant differences also between isogenic cell lines. Although using line-specific normalizers remains optimal, a set of pan-line HKG that yields good estimation of relative expression of target genes was proposed.

Enolase-like protein present on the outer membrane of Pseudomonas aeruginosa binds plasminogen.

Pseudomonas aeruginosa is one of the pathogenic bacteria which utilize binding of the host plasminogen (Plg) to promote their invasion throughout the host tissues. In the present study, we confirmed that P. aeruginosa exhibits binding affinity for human plasminogen. Furthermore, we showed that the protein detected on the cell wall of P. aeruginosa and binding human plasminogen is an enolase-like protein. The hypothesis that alpha-enolase, a cytoplasmatic glycolytic enzyme, resides also on the cell surface of the bacterium was supported by electron microscopy analysis. The plasminogen-binding activity of bacterial cell wall outer membrane enolase-like protein was examined by immunoblotting assay.

The perioperative dynamics of IL-7 following robot-assisted and open colorectal surgery

Interleukin-7 is critical for T-cell development and displays antimicrobial and antitumor properties. It is referred to as a “critical enhancer of protective immunity”. However, there is no information on interleukin-7 dynamics following colorectal surgery. Moreover, although robot-assisted surgery is gaining popularity, data on the immune response to it is almost non-existent. In this prospective non-randomized case-control study we found interleukin-7 dynamics to differ following robot-assisted and open approach and to affect postoperative immunity. Linear increases were seen in the robotic group while a cubic pattern with a maximum at 8 h in the open one. Low preoperative interleukin-7 was associated with developing surgical site infection. In turn, higher preoperative interleukin-7 was associated with preserved immune function: less pronounced drop in lymphocyte count and higher Δlymphocyte/Δneutrophil ratio in patients undergoing robotic surgery. The changes in other cytokines, namely, interleukin-12(p70), TNFα, interferon-γ, and interleukin-10 were independently associated with interleukin-7 dynamics. In turn, relative changes in interleukin-7 were independent predictors of changes in interferon-γ, key cytokine of favourable Th1 immune response. Taken together, we demonstrated different perioperative dynamics of interleukin-7, which may contribute to favourable outcomes following robotic colorectal surgery including lower incidence of surgical site infections, milder surgery-induced lymphopenia, and beneficial interferon-γ dynamics.