Peter J. Bugelski

Extracellular Matrix Metalloproteinase Inducer Stimulates Tumor Angiogenesis by Elevating Vascular Endothelial Cell Growth Factor and Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) are endopeptidases that play pivotal roles in promoting tumor disease progression, including tumor angiogenesis. In many solid tumors, MMP expression could be attributed to tumor stromal cells and is partially regulated by tumor-stroma interactions via tumor cell-associated extracellular matrix metalloproteinase inducer (EMMPRIN). The role of EMMPRIN during tumor angiogenesis and growth was explored by modulating EMMPRIN expression and activity using recombinant DNA engineering and neutralizing antibodies. In human breast cancer cells, changes in EMMPRIN expression influenced vascular endothelial growth factor (VEGF) production at both RNA and protein levels. In coculture of tumor cells and fibroblasts mimicking tumor-stroma interactions, VEGF expression was induced in an EMMPRIN- and MMP-dependent fashion, and was further enhanced by overexpressing EMMPRIN. Conversely, VEGF expression was inhibited by suppressing EMMPRIN expression in tumor cells, by neutralizing EMMPRIN activity, or by inhibiting MMPs. In vivo, EMMPRIN overexpression stimulated tumor angiogenesis and growth; both were significantly inhibited by antisense suppression of EMMPRIN. Expression of both human and mouse VEGF and MMP, derived from tumor and host cells, respectively, was regulated by EMMPRIN. These results suggest a novel tumor angiogenesis mechanism in which tumor-associated EMMPRIN functionally mediates tumor-stroma interactions and directly contributes to tumor angiogenesis and growth by stimulating VEGF and MMP expression.

Troglitazone-induced intracellular oxidative stress in rat hepatoma cells : a flow cytometric assessment

Troglitazone (TRO), a thiazolidinedione (TZD) peroxisome proliferator‐activated receptor γ agonist, was recently withdrawn from the market because of rare but serious hepatotoxicity. Previous studies investigating the cytotoxicity of TRO in cultured rat hepatocytes have conjectured about the role of oxidative stress in TRO‐induced hepatotoxicity. Therefore, we investigated whether TRO induces oxidative stress and, if so, the portion of the TRO molecule responsible for the induction of oxidative stress.

Pharmacodynamics of Recombinant Human Erythropoietin in Murine Bone Marrow

PurposeOriginally approved for three times/week dosing, recombinant human erythropoietin (rhEPO) is now often used at weekly intervals. We have studied rhEPO in mice to better understand why the extended dosing interval retains efficacy.MethodsC57Bl/6 mice received a single sc. dose of rhEPO (3,000xa0IU/kg). Bone marrow and blood were collected at 8xa0h and 1, 2, 5 and 7xa0days. Staining for TER-119 and CD71, pulse labeling with bromodeoxyuridine, annexin-V binding and vital staining with 7-aminoactinomycin d were used cell cycle and apoptosis in erythroblasts by four color flow cytometry.ResultsA wave of proliferation and/or maturation progressed through all erythroblasts, resulting in the emigration of immature reticulocytes into the periphery. An increase in the fraction of erythroblasts in S and G2M was found, but suppression of apoptosis was not.ConclusionsMost of the effects of rhEPO occurred 48xa0h after dosing, when the concentration of rhEPO was less than 1% of Cmax, suggesting that the processes set in motion by rhEPO can continue after rhEPO concentrations fall. Our observation of apoptosis in erythroblasts even when rhEPO concentrations were high suggests that regulatory mechanisms which down-regulate erythropoiesis are also engaged.

Identification of natural antibodies to interleukin-18 in the sera of normal humans and three nonhuman primate species.

Natural antibodies to cytokines can be found in the sera of normal healthy individuals in the absence of specific immunostimulation. However, the function, impact, and purpose of natural antibody development have yet to be fully elucidated. Interleukin (IL)-18 is a cytokine that exerts proinflammatory activities and induces natural killer (NK) cell activity. Recombinant human IL-18 (rHuIL-18) is currently in development as a cancer immunotherapy. In this study, the presence of natural antibodies to IL-18 in the sera of normal humans and three nonhuman primate species was evaluated by electrochemiluminescence immunoassay (ECLIA). Of the human sera tested, 6 of 47 samples were positive for natural antibodies to IL-18. Of the nonhuman primate sera tested, 22 of 80 cynomolgus monkey samples, 4 of 31 rhesus monkey samples, and 2 of 20 chimpanzee samples were positive for natural antibodies to IL-18. Natural anti-IL-18 antibodies were neutralizing in 5 of 22 cynomolgus and 2 of 4 rhesus sera. None of the chimpanzee or human sera were able to neutralize IL-18 induction of interferon (IFN)-gamma in vitro. In vivo activity of rHuIL-18 was compared in IL-18 natural antibody-positive and -negative cynomolgus monkeys. The presence of natural antibodies to IL-18 did not alter rHuIL-18 systemic exposure levels, induction of neopterin, or induction of treatment-induced antibodies following intravenous administration of rHuIL-18. In conclusion, our data indicate that, as has been found with other cytokines, natural anti-IL-18 antibodies are relatively common. Moreover, natural anti-IL-18 antibodies do not appear to influence rHuIL-18 activity in vivo and are not predictive of a heightened immune response, suggesting that natural anti-IL-18 antibodies do not impact IL-18 therapy. Finally, our data suggest that the ability to detect natural anti-cytokine antibodies may be a useful measure of the adequacy of an assay for deployment in clinical trials.

Chemometric strategies for normalisation of gene expression data obtained from cDNA microarrays

Abstract DNA microarray technology is emerging as an important method in Functional Genomics. Microarrays enable rapid, large scale gene expression analysis but the cumulative, systematic errors which occur during experimentation result in large variances in the results. Thus data pretreatment, or normalisation, has been recognised as a major challenge in the analysis of microarray data. In this study, chemometric strategies were applied to microarray data in order to develop an appropriate normalisation method. In total, 192 pretreatment methods were systematically designed based on using the empty spots as background. In order to find the best method, partial least squares (PLS-1) was used to classify microarray data into compound treated and control groups. The optimal technique — a robust normal method (RNM), was identified by it’s correct classification rate (CCR) based on cross-validation. In the RNM, the microarray data were normalised using signals of the k nearest neighbour (kNN) empty spots to compensate for localised background variation. The results of our study showed that RNM greatly reduced the variances within slides and between slides, performing better than any other studied normalisation methods, increasing the CCR by 17% and greatly improving the effective reproducibility of microarray data. Thus, RNM provides an effective method for normalisation of gene expression data obtained from DNA microarrays.

Sequential univariate gating approach to study the effects of erythropoietin in murine bone marrow.

Analysis of multicolor flow cytometric data is traditionally based on the judgment of an expert, generally time consuming, sometimes incomplete and often subjective in nature. In this article, we investigate another statistical method using a Sequential Univariate Gating (SUG) algorithm to identify regions of interest between two groups of multivariate flow cytometric data. The metric used to differentiate between the groups of univariate distributions in SUG is the Kolmogorov‐Smirnov distance (D) statistic. The performance of the algorithm is evaluated by applying it to a known three‐color data set looking at activation of CD4+ and CD8+ lymphocytes with anti‐CD3 antibody treatment and comparing the results to the expert analysis. The algorithm is then applied to a four‐color data set used to study the effects of recombinant human erythropoietin (rHuEPO) on several murine bone marrow populations. SUG was used to identify regions of interest in the data and results compared to expert analysis and the current state‐of‐the‐art statistical method, Frequency Difference Gating (FDG). Cluster analysis was then performed to identify subpopulations responding differently to rHuEPO. Expert analysis, SUG and FDG identified regions in the data that showed activation of CD4+ and CD8+ lymphocytes with anti‐CD3 treatment. In the rHuEPO treated data sets, the expert and SUG identified a dose responsive expansion of only the erythroid precursor population. In contrast, FDG resulted in identification of regions of interest both in the erythroid precursors as well as in other bone marrow populations. Clustering within the regions of interest defined by SUG resulted in identification of four subpopulations of erythroid precursors that are morphologically distinct and show a differential response to rHuEPO treatment. Greatest expansion is seen in the basophilic and poly/orthochromic erythroblast populations with treatment. Identification of populations of interest can be performed using SUG in less subjective, time efficient, biologically interpretable manner that corroborates with the expert analysis. The results suggest that basophilic erythroblasts cells or their immediate precursors are an important target for the effects of rHuEPO in murine bone marrow. The MATLAB implementation of the method described in the article, both experimental data and other supplemental materials are freely available at http://web.mac.com/acidrap18.