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van der Heyde H.C.,La Jolla Infectious Disease Institute
Methods in molecular biology (Clifton, N.J.) | Year: 2011

The Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot analysis have been workhorse techniques for the analysis of protein levels and state, such as phosphorylation. The ELISA is also useful for measuring the affinity of a molecule for its ligand. The disadvantage of these techniques is that only a single protein can be analyzed for ELISA and a few (up to three) proteins for Western Blotting. Exact quantification is difficult with Western Blotting and changes are often reported as fold differences. We present here protocols for using fluorescent microspheres coated with the selected capture molecule to perform in essence several hundred mini ELISAs with each microsphere representing an ELISA; this reduces the variability of the assay. In addition, it is possible to analyze up to 100 analytes simultaneously using microspheres because each fluorescent microsphere exhibits distinct fluorescence in the red and far red channels: the fluorescence intensity in the channels in the red and far red channels (up to ten different intensities for each channel leading to a 10 × 10 matrix of intensities) constitutes the address for each analyte.

van der Heyde H.C.,La Jolla Infectious Disease Institute
Methods in molecular biology (Clifton, N.J.) | Year: 2011

Cell-derived microparticles (MPs) are increasingly recognized as important cell-to-cell signaling mechanisms and may exhibit important functions in homeostasis but also in pathogenesis. Indeed, MPs are associated with a number of diseases inhibiting their production that protects against pathogenesis. MPs are distinct from exosomes and apoptotic bodies, often exhibiting the membrane proteins of the activated or apoptotic cell from which they are derived. Electron microscopic analyses have shown that MPs are produced by all cell types tested to date, and ELISA-based assays have established that increased numbers of MPs are produced following cell activation. These approaches do not, however, determine the exact number of MPs and distribution of functional proteins on their surface. Flow cytometry represents an obvious approach to analyze MPs, and we present here a method to assess the number and phenotype of MPs by using a conventional flow cytometer. We also present the caveats with this method and describe a new imaging flow cytometry approach that overcomes these limitations.

Hsieh Y.-H.,University of Alabama at Birmingham | van der Heyde H.,La Jolla Infectious Disease Institute | Oh E.-S.,Ewha Womans University | Guan J.-L.,University of Michigan | Chang P.-L.,University of Alabama at Birmingham
Molecular Carcinogenesis | Year: 2015

Osteopontin (OPN), an adhesive, matricellular glycoprotein, is a rate-limiting factor in tumor promotion of skin carcinogenesis. With a tumor promotion model, the JB6 Cl41.5a cell line, we have shown that suppressing 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced OPN expression markedly inhibits TPA-induced colony formation in soft agar, an assay indicative of tumorigenic transformation. Further, the addition of exogenous OPN promotes colony formation of these cells. These findings support a function of OPN in mediating TPA-induced neoplastic transformation of JB6 cells. In regard to the mechanism of action by OPN, we hypothesized that, for JB6 cells grown in soft-agar, secreted OPN induced by TPA stimulates cell proliferation and/or prevents anoikis to facilitate TPA-induced colony formation. Analyses of cell cycle and cyclin D1 expression, and direct cell counting of JB6 cells treated with OPN indicate that OPN does not stimulate cell proliferation relative to non-treated controls. Instead, at 24h, OPN decreases anoikis by 41%, as assessed by annexin V assays. Further, in suspended cells OPN suppresses caspase-8 activation, which is mediated specifically through its RGD-cell binding motif that transduces signals through integrin receptors. Transfection studies with wild-type and mutant focal adhesion kinases (FAK) and Western blot analyses suggest that OPN suppression of caspase-8 activation is mediated through phosphorylation of FAK at Tyr861. In summary, these studies indicate that induced OPN is a microenvironment modulator that facilitates tumorigenic transformation of JB6 cells by inhibiting anoikis through its RGD-dependent suppression of caspase-8 activity, which is mediated in part through the activation of FAK at Tyr861. © 2013 Wiley Periodicals, Inc.

Grau G.E.R.,University of Sydney | Grau G.E.R.,La Jolla Infectious Disease Institute
Future Microbiology | Year: 2012

Cerebral malaria is one of a number of clinical syndromes associated with infection by human malaria parasites of the genus Plasmodium. The etiology of cerebral malaria derives from sequestration of parasitized red cells in brain microvasculature and is thought to be enhanced by the proinflammatory status of the host and virulence characteristics of the infecting parasite variant. In this article we examine the range of factors thought to influence the development of Plasmodium falciparum cerebral malaria in humans and review the evidence to support their role. © 2012 Future Medicine Ltd.

Hsieh Y.-H.,University of Alabama at Birmingham | Margaret Juliana M.,University of Alabama at Birmingham | Ho K.-J.,University of Alabama at Birmingham | Kuo H.-C.,University of Alabama at Birmingham | And 3 more authors.
International Journal of Cancer | Year: 2012

The matricellular protein osteopontin (OPN), expressed in various cancer types and elevated in the blood of cancer patients, is thought to have different functions when derived from host versus cancer cells. To assess the effect of host-derived OPN on growth of cancers of epithelial origin, we established a line of cutaneous squamous cell carcinoma (SCC) cells, named ONSC, which lacks the OPN gene and develops SCC in syngeneic wild-type (WT) and OPN-null mice. At 8 and/or 10 week after subcutaneous injection of ONSC cells in mice, however, there was a lower tumor incidence in WT mice, suggesting that host-derived OPN is associated with suppression of early growth of extrinsic cancer cells. Histological, immunohistochemical, biochemical and hematological analyses were performed on the tumor microenvironment and blood from tumor-bearing mice during the first week after implantation. Host-derived OPN suppression of extrinsic ONSC cell progression is likely mediated through elicitation of an early innate inflammatory response, through its function as a chemoattractant and/or by enhancing survival of inflammatory cells. Further, consistent with a previous report, the serum levels of host-derived OPN, which are elevated during the early phase of tumor growth in mice implanted with ONSC, appear to reflect an anti-tumor progression effect. Copyright © 2011 UICC.

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