Levels and determinants of inflammatory biomarkers in a Swiss population-based sample (CoLaus study)

Objective to assess the levels and determinants of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and C-reactive protein (CRP) in a healthy Caucasian population. Methods population sample of 2884 men and 3201 women aged 35 to 75. IL-1β, IL-6 and TNF-α were assessed by a multiplexed particle-based flow cytometric assay and CRP by an immunometric assay. Results Spearman rank correlations between duplicate cytokine measurements (N = 80) ranged between 0.89 and 0.96; intra-class correlation coefficients ranged between 0.94 and 0.97, indicating good reproducibility. Among the 6085 participants, 2289 (37.6%), 451 (7.4%) and 43 (0.7%) had IL-1β, IL-6 and TNF-α levels below detection limits, respectively. Median (interquartile range) for participants with detectable values were 1.17 (0.48–3.90) pg/ml for IL-1β; 1.47 (0.71–3.53) pg/ml for IL-6; 2.89 (1.82–4.53) pg/ml for TNF-α and 1.3 (0.6–2.7) ng/ml for CRP. On multivariate analysis, greater age was the only factor inversely associated with IL-1β levels. Male sex, increased BMI and smoking were associated with greater IL-6 levels, while no relationship was found for age and leisure-time PA. Male sex, greater age, increased BMI and current smoking were associated with greater TNF-α levels, while no relationship was found with leisure-time PA. CRP levels were positively related to age, BMI and smoking, and inversely to male sex and physical activity. Conclusion Population-based levels of several cytokines were established. Increased age and BMI, and to a lesser degree sex and smoking, significantly and differentially impact cytokine levels, while leisure-time physical activity has little effect.

Flow cytometry and FISH to measure the average length of telomeres (flow FISH)

Telomeres have emerged as crucial cellular elements in aging and various diseases including cancer. To measure the average length of telomere repeats in cells, we describe our protocols that use fluorescent in situ hybridization (FISH) with labeled peptide nucleic acid (PNA) probes specific for telomere repeats in combination with fluorescence measurements by flow cytometry (flow FISH). Flow FISH analysis can be performed using commercially available flow cytometers, and has the unique advantage over other methods for measuring telomere length of providing multi-parameter information on the length of telomere repeats in thousands of individual cells. The accuracy and reproducibility of the measurements is augmented by the automation of most pipetting (aspiration and dispensing) steps, and by including an internal standard (control cells) with a known telomere length in every tube. The basic protocol for the analysis of nucleated blood cells from 22 different individuals takes about 12 h spread over 2-3 days.

OCT4 expression in human non-small cell lung cancer: implications for therapeutic intervention

Here we investigate the expression of OCT4 human lung adenocarcinoma and bronchioloalveolar carcinoma (BAC) tumor biopsies and tumor-derived primary cell cultures. OCT4 has been detected in several human tumors suggesting a potentially critical role in tumorigenesis. We assessed the presence of OCT4 in clinical tumor samples of both adenocarcinoma and BAC at the cellular and transcriptional levels, respectively. Furthermore, we evaluated tumor-derived cell cultures for potential differences in OCT4 expression. Immunohistochemical analysis depicted OCT4 in 2 of 8 adenocarcinoma tumor samples and 3 of 5 BAC tumor samples, with no apparent difference in the degree of expression among the sections examined. These results were validated by transcript analysis. Flow cytometric assessment of 11 adenocarcinoma-derived cell cultures and 3 BAC-derived cell cultures revealed significantly higher OCT4 expression in adenocarcinoma tumors compared to their normal counterparts. This, however, was not observed in the BAC cultures. Comparative studies of OCT4 in adenocarcinoma and BAC tumor cell cultures demonstrated a dramatically higher expression in the former. The expression of OCT4 may represent a specific and effective target for therapeutic intervention in adenocarcinoma and BAC. In addition, the aberrant expression and distribution of OCT4 may indicate important parameters concerning the differences between adenocarcinoma and BAC.

Development of a bead-based multiplex assay for the simultaneous detection of porcine inflammation markers using xMAP technology

Commercially available assays for the simultaneous detection of multiple inflammatory and cardiac markers in porcine blood samples are currently lacking. Therefore, this study was aimed at developing a bead-based, multiplexed flow cytometric assay to simultaneously detect porcine cytokines [interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor alpha], chemokines (IL-8 and monocyte chemotactic protein 1), growth factors [basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and platelet-derived growth factor-bb], and injury markers (cardiac troponin-I) as well as complement activation markers (C5a and sC5b-9). The method was based on the Luminex xMAP technology, resulting in the assembly of a 6- and 11-plex from the respective individual singleplex situation. The assay was evaluated for dynamic range, sensitivity, cross-reactivity, intra-assay and interassay variance, spike recovery, and correlation between multiplex and commercially available enzyme-linked immunosorbent assay as well as the respective singleplex. The limit of detection ranged from 2.5 to 30,000 pg/ml for all analytes (6- and 11-plex assays), except for soluble C5b-9 with a detection range of 2-10,000 ng/ml (11-plex). Typically, very low cross-reactivity (<3% and <1.4% by 11- and 6-plex, respectively) between analytes was found. Intra-assay variances ranged from 4.9 to 7.4% (6-plex) and 5.3 to 12.9% (11-plex). Interassay variances for cytokines were between 8.1 and 28.8% (6-plex) and 10.1 and 26.4% (11-plex). Correlation coefficients with singleplex assays for 6-plex as well as for 11-plex were high, ranging from 0.988 to 0.997 and 0.913 to 0.999, respectively. In this study, a bead-based porcine 11-plex and 6-plex assay with a good assay sensitivity, broad dynamic range, and low intra-assay variance and cross-reactivity was established. These assays therefore represent a new, useful tool for the analysis of samples generated from experiments with pigs.

THE ROLE OF PLATELETS IN OVARIAN CARCINOMA

Platelets represent one of the largest storage pools of angiogenic and oncogenic growth factors in the human body. The observation that thrombocytosis (platelet count >450,000/uL) occurs in patients with solid malignancies was made over 100 years ago. However, the clinical and biological implications as well as the underlying mechanism of paraneoplastic thrombocytosis associated with ovarian carcinoma remains unknown and were the focus of the current study. Following IRB approval, patient data were collected on 619 patients from 4 U.S. centers and used to test associations between platelet count at initial diagnosis, clinicopathologic factors, and outcome. In vitro effects of plasma-purified platelets on ovarian cancer cell proliferation, docetaxel-induced apoptosis, and migration were evaluated using BrdU-PI flow cytometric and two-chamber chemotaxis assays. In vivo effects of platelet depletion on tumor growth, proliferation, apoptosis, and angiogenesis were examined using an anti-platelet antibody (anti-mouse glycoprotein 1ba, Emfret) to reduce platelets by 50%. Complete blood counts and number of mature megakaryocytes in the spleen and bone marrow were compared between control mice and ovarian cancer-bearing mice. Plasma levels of key megakaryo- and thrombopoietic factors including thrombopoietin (TPO), IL-1a, IL-3, IL-4, IL-6, IL-11, G-CSF, GM-CSF, stem cell factor, and FLT-3 ligand were assayed in a subset of 150 patients at the time of initial diagnosis with advanced stage, high grade epithelial ovarian cancer using immunobead-based cytokine profiling coupled with the Luminex® xMAP platform. Plasma cytokines significantly associated with thrombocytosis in ovarian cancer patients were subsequently evaluated in mouse models of ovarian cancer using ELISA immunoassays. The results of human and mouse plasma cytokine profiling were used to inform subsequent in vivo studies evaluating the effect of siRNA-induced silencing of select megakaryo- and thrombopoietic cytokines on paraneoplastic thrombocytosis. Thirty-one percent of patients had thrombocytosis at initial diagnosis. Compared to patients with normal platelet counts, women with thrombocytosis were significantly more likely to have advanced stage disease (p<0.001) and poor median progression-free (0.94 vs 1.35 years, p<0.001) and overall survival (2.62 vs 4.65 years, p<0.001). On multivariate analysis, thrombocytosis remained an independent predictor of decreased overall survival. Our analysis revealed that thrombocytosis significantly increases the risk of VTE in ovarian cancer patients and that thrombocytosis is an independent predictor of increased mortality in women who do develop a blood clot. Platelets increased ovarian cancer cell proliferation and migration by 4.1- and 2.8-fold (p<0.01), respectively. Platelets reduced docetaxel-induced apoptosis in ovarian cancer cells by 2-fold (p<0.001). In vivo, platelet depletion reduced tumor growth by 50%. Staining of in vivo specimens revealed decreased tumor cell proliferation (p<0.001) and increased tumor and endothelial cell apoptosis (p<0.01). Platelet depletion also significantly decreased microvessel density and pericyte coverage (p<0.001). Platelet counts increase by 31-130% in mice with invasive ovarian cancer compared to controls (p<0.01) and strongly correlate with mean megakaryocyte counts in the spleen and bone marrow (r=0.95, p<0.05). Plasma levels of TPO, IL-6, and G-CSF were significantly increased in ovarian cancer patients with thrombocytosis. Plasma levels of the same cytokines were found to be significantly elevated in orthotopic mouse models of ovarian cancer, which consistently develop paraneoplastic thromocytosis. Silencing TPO, IL-6, and G-CSF significantly abrogated paraneoplastic thrombocytosis in vivo. This study provides new understanding of the clinical and biological significance of paraneoplastic thrombocytosis in ovarian cancer and uncovers key humoral factors driving this process. Blocking the development of paraneoplastic thrombocytosis and interfering with platelet-cancer cell interactions could represent novel therapeutic strategies.

STUDIES OF THE MOLECULAR MECHANISMS OF CHROMOSOME ABERRATION FORMATION (DNA REPAIR, CROSSLINKS, MITOMYCIN C)

The objective of this research has been to study the molecular basis for chromosome aberration formation. Predicated on a variety of data, Mitomycin C (MMC)-induced DNA damage has been postulated to cause the formation of chromatid breaks (and gaps) by preventing the replication of regions of the genome prior to mitosis. The basic protocol for these experiments involved treating synchronized Hela cells in G(,1)-phase with a 1 (mu)g/ml dose of MMC for one hour. After removing the drug, cells were then allowed to progress to mitosis and were harvested for analysis by selective detachment. Utilizing the alkaline elution assay for DNA damage, evidence was obtained to support the conclusion that Hela cells can progress through S-phase into mitosis with intact DNA-DNA interstrand crosslinks. A higher level of crosslinking was observed in those cells remaining in interphase compared to those able to reach mitosis at the time of analysis. Dual radioisotope labeling experiments revealed that, at this dose, these crosslinks were associated to the same extent with both parental and newly replicated DNA. This finding was shown not to be the result of a two-step crosslink formation mechanism in which crosslink levels increase with time after drug treatment. It was also shown not to be an artefact of the double-labeling protocol. Using neutral CsCl density gradient ultracentrifugation of mitotic cells containing BrdU-labeled newly replicated DNA, control cells exhibited one major peak at a heavy/light density. However, MMC-treated cells had this same major peak at the heavy/light density, in addition to another minor peak at a density characteristic for light/light DNA. This was interpreted as indicating either: (1) that some parental DNA had not been replicated in the MMC treated sample or; (2) that a recombination repair mechanism was operational. To distinguish between these two possibilities, flow cytometric DNA fluorescence (i.e., DNA content) measurements of MMC-treated and control cells were made. These studies revealed that the mitotic cells that had been treated with MMC while in G(,1)-phase displayed a 10-20% lower DNA content than untreated control cells when measured under conditions that neutralize chromosome condensation effects (i.e., hypotonic treatment). These measurements were made under conditions in which the binding of the drug, MMC, was shown not to interfere with the stoichiometry of the ethidium bromide-mithramycin stain. At the chromosome level, differential staining techniques were used in an attempt to visualize unreplicated regions of the genome, but staining indicative of large unreplicated regions was not observed. These results are best explained by a recombinogenic mechanism. A model consistent with these results has been proposed.^

Pore water geochemistry of Håkon Mosby mud volcano sediments measured at station PS74/169-1_PUC-15 (Z1-S1, new flow surface)

Pore water and turnover rates were determined for surface sediment cores obtained in 2009 and 2010. The pore water was extracted with Rhizons (Rhizon CSS: length 5 cm, pore diameter 0.15 µm; Rhizosphere Research Products, Wageningen, Netherlands) in 1 cm-resolution and immediately fixed in 5% zinc acetate (ZnAc) solution for sulfate, and sulfide analyses. The samples were diluted, filtered and the concentrations measured with non-suppressed anion exchange chromatography (Waters IC-Pak anion exchange column, waters 430 conductivity detector). The total sulfide concentrations (H2S + HS- + S**2-) were determined using the diamine complexation method (doi:10.4319/lo.1969.14.3.0454). Samples for dissolved inorganic carbon (DIC) and alkalinity measurements were preserved by adding 2 µl saturated mercury chloride (HgCl2) solution and stored headspace-free in gas-tight glass vials. DIC and alkalinity were measured using the flow injection method (detector VWR scientific model 1054) (doi:10.4319/lo.1992.37.5.1113). Dissolved sulfide was eliminated prior to the DIC measurement by adding 0.5 M molybdate solution (doi:10.4319/lo.1995.40.5.1011). Nutrient subsamples (10 - 15 ml) were stored at - 20 °C prior to concentration measurements with a Skalar Continuous-Flow Analyzer (doi:10.1002/9783527613984).

Pore water geochemistry of Håkon Mosby mud volcano sediments measured at station MSM16/2_847-1 (Z2-S2, aged flow surface)

Pore water and turnover rates were determined for surface sediment cores obtained in 2009 and 2010. The pore water was extracted with Rhizons (Rhizon CSS: length 5 cm, pore diameter 0.15 µm; Rhizosphere Research Products, Wageningen, Netherlands) in 1 cm-resolution and immediately fixed in 5% zinc acetate (ZnAc) solution for sulfate, and sulfide analyses. The samples were diluted, filtered and the concentrations measured with non-suppressed anion exchange chromatography (Waters IC-Pak anion exchange column, waters 430 conductivity detector). The total sulfide concentrations (H2S + HS- + S**2-) were determined using the diamine complexation method (doi:10.4319/lo.1969.14.3.0454). Samples for dissolved inorganic carbon (DIC) and alkalinity measurements were preserved by adding 2 µl saturated mercury chloride (HgCl2) solution and stored headspace-free in gas-tight glass vials. DIC and alkalinity were measured using the flow injection method (detector VWR scientific model 1054) (doi:10.4319/lo.1992.37.5.1113). Dissolved sulfide was eliminated prior to the DIC measurement by adding 0.5 M molybdate solution (doi:10.4319/lo.1995.40.5.1011). Nutrient subsamples (10 - 15 ml) were stored at - 20 °C prior to concentration measurements with a Skalar Continuous-Flow Analyzer (doi:10.1002/9783527613984).

Pore water geochemistry of Håkon Mosby mud volcano sediments measured at station MSM16/2_838-1 (Z1-S3, new flow surface)

Pore water and turnover rates were determined for surface sediment cores obtained in 2009 and 2010. The pore water was extracted with Rhizons (Rhizon CSS: length 5 cm, pore diameter 0.15 µm; Rhizosphere Research Products, Wageningen, Netherlands) in 1 cm-resolution and immediately fixed in 5% zinc acetate (ZnAc) solution for sulfate, and sulfide analyses. The samples were diluted, filtered and the concentrations measured with non-suppressed anion exchange chromatography (Waters IC-Pak anion exchange column, waters 430 conductivity detector). The total sulfide concentrations (H2S + HS- + S**2-) were determined using the diamine complexation method (doi:10.4319/lo.1969.14.3.0454). Samples for dissolved inorganic carbon (DIC) and alkalinity measurements were preserved by adding 2 µl saturated mercury chloride (HgCl2) solution and stored headspace-free in gas-tight glass vials. DIC and alkalinity were measured using the flow injection method (detector VWR scientific model 1054) (doi:10.4319/lo.1992.37.5.1113). Dissolved sulfide was eliminated prior to the DIC measurement by adding 0.5 M molybdate solution (doi:10.4319/lo.1995.40.5.1011). Nutrient subsamples (10 - 15 ml) were stored at - 20 °C prior to concentration measurements with a Skalar Continuous-Flow Analyzer (doi:10.1002/9783527613984).

Pore water geochemistry of Håkon Mosby mud volcano sediments measured at station MSM16/2_855-1 (Z2-S3, aged flow surface)

Pore water and turnover rates were determined for surface sediment cores obtained in 2009 and 2010. The pore water was extracted with Rhizons (Rhizon CSS: length 5 cm, pore diameter 0.15 µm; Rhizosphere Research Products, Wageningen, Netherlands) in 1 cm-resolution and immediately fixed in 5% zinc acetate (ZnAc) solution for sulfate, and sulfide analyses. The samples were diluted, filtered and the concentrations measured with non-suppressed anion exchange chromatography (Waters IC-Pak anion exchange column, waters 430 conductivity detector). The total sulfide concentrations (H2S + HS- + S**2-) were determined using the diamine complexation method (doi:10.4319/lo.1969.14.3.0454). Samples for dissolved inorganic carbon (DIC) and alkalinity measurements were preserved by adding 2 µl saturated mercury chloride (HgCl2) solution and stored headspace-free in gas-tight glass vials. DIC and alkalinity were measured using the flow injection method (detector VWR scientific model 1054) (doi:10.4319/lo.1992.37.5.1113). Dissolved sulfide was eliminated prior to the DIC measurement by adding 0.5 M molybdate solution (doi:10.4319/lo.1995.40.5.1011). Nutrient subsamples (10 - 15 ml) were stored at - 20 °C prior to concentration measurements with a Skalar Continuous-Flow Analyzer (doi:10.1002/9783527613984).

Pore water geochemistry of Håkon Mosby mud volcano sediments measured at station PS74/168-1 (Z1-S2, new flow surface)

Pore water and turnover rates were determined for surface sediment cores obtained in 2009 and 2010. The pore water was extracted with Rhizons (Rhizon CSS: length 5 cm, pore diameter 0.15 µm; Rhizosphere Research Products, Wageningen, Netherlands) in 1 cm-resolution and immediately fixed in 5% zinc acetate (ZnAc) solution for sulfate, and sulfide analyses. The samples were diluted, filtered and the concentrations measured with non-suppressed anion exchange chromatography (Waters IC-Pak anion exchange column, waters 430 conductivity detector). The total sulfide concentrations (H2S + HS- + S**2-) were determined using the diamine complexation method (doi:10.4319/lo.1969.14.3.0454). Samples for dissolved inorganic carbon (DIC) and alkalinity measurements were preserved by adding 2 µl saturated mercury chloride (HgCl2) solution and stored headspace-free in gas-tight glass vials. DIC and alkalinity were measured using the flow injection method (detector VWR scientific model 1054) (doi:10.4319/lo.1992.37.5.1113). Dissolved sulfide was eliminated prior to the DIC measurement by adding 0.5 M molybdate solution (doi:10.4319/lo.1995.40.5.1011). Nutrient subsamples (10 - 15 ml) were stored at - 20 °C prior to concentration measurements with a Skalar Continuous-Flow Analyzer (doi:10.1002/9783527613984).

Pore water geochemistry of Håkon Mosby mud volcano sediments measured at station PS74/172-1_PUC-136 (Z3-S2, old flow surface)

Pore water and turnover rates were determined for surface sediment cores obtained in 2009 and 2010. The pore water was extracted with Rhizons (Rhizon CSS: length 5 cm, pore diameter 0.15 µm; Rhizosphere Research Products, Wageningen, Netherlands) in 1 cm-resolution and immediately fixed in 5% zinc acetate (ZnAc) solution for sulfate, and sulfide analyses. The samples were diluted, filtered and the concentrations measured with non-suppressed anion exchange chromatography (Waters IC-Pak anion exchange column, waters 430 conductivity detector). The total sulfide concentrations (H2S + HS- + S**2-) were determined using the diamine complexation method (doi:10.4319/lo.1969.14.3.0454). Samples for dissolved inorganic carbon (DIC) and alkalinity measurements were preserved by adding 2 µl saturated mercury chloride (HgCl2) solution and stored headspace-free in gas-tight glass vials. DIC and alkalinity were measured using the flow injection method (detector VWR scientific model 1054) (doi:10.4319/lo.1992.37.5.1113). Dissolved sulfide was eliminated prior to the DIC measurement by adding 0.5 M molybdate solution (doi:10.4319/lo.1995.40.5.1011). Nutrient subsamples (10 - 15 ml) were stored at - 20 °C prior to concentration measurements with a Skalar Continuous-Flow Analyzer (doi:10.1002/9783527613984).

Detection and characterization of carcinoma cells in the blood

A highly sensitive assay combining immunomagnetic enrichment with multiparameter flow cytometric and immunocytochemical analysis has been developed to detect, enumerate, and characterize carcinoma cells in the blood. The assay can detect one epithelial cell or less in 1 ml of blood. Peripheral blood (10–20 ml) from 30 patients with carcinoma of the breast, from 3 patients with prostate cancer, and from 13 controls was examined by flow cytometry for the presence of circulating epithelial cells defined as nucleic acid+, CD45−, and cytokeratin+. Highly significant differences in the number of circulating epithelial cells were found between normal controls and patients with cancer including 17 with organ-confined disease. To determine whether the circulating epithelial cells in the cancer patients were neoplastic cells, cytospin preparations were made after immunomagnetic enrichment and were analyzed. Epithelial cells from patients with breast cancer generally stained with mAbs against cytokeratin and 3 of 5 for mucin-1. In contrast, no cells that stained for these antigens were observed in the blood from normal controls. The morphology of the stained cells was consistent with that of neoplastic cells. Of 8 patients with breast cancer followed for 1–10 months, there was a good correlation between changes in the level of tumor cells in the blood with both treatment with chemotherapy and clinical status. The present assay may be helpful in early detection, in monitoring disease, and in prognostication.

Increased platelet-derived microparticles in the coronary circulation of percutaneous transluminal coronary angioplasty patients

Platelet-derived microparticles that are produced during platelet activation are capable of adhesion and aggregation. Endothelial trauma that occurs during percutaneous transluminal coronary angioplasty (PTCA) may support platelet-derived microparticle adhesion and contribute to development of restenosis. We have previously reported an increase in platelet-derived microparticles in peripheral arterial blood with angioplasty. This finding raised concerns regarding the role of platelet-derived microparticles in restenosis, and therefore the aim of this study was to monitor levels in the coronary circulation. The study population consisted of 19 angioplasty patients. Paired coronary artery and sinus samples were obtained following heparinization, following contrast administration, and subsequent to all vessel manipulation. Platelet-derived microparticles were identified with an anti-CD61 (glycoprotein IIIa) fluorescence-conjugated antibody using flow cytometry. There was a significant decrease in arterial platelet-derived microparticles from heparinization to contrast administration (P=0.001), followed by a significant increase to the end of angioplasty (P=0.004). However, there was no significant change throughout the venous samples. These results indicate that the higher level of platelet-derived microparticles after angioplasty in arterial blood remained in the coronary circulation. Interestingly, levels of thrombin-antithrombin complexes did not rise during PTCA. This may have implications for the development of coronary restenosis post-PTCA, although this remains to be determined.