A Novel Immunological Assay for Hepcidin Quantification in Human Serum

Contains fulltext : 81054.pdf (publisher's version ) (Open Access)BACKGROUND: Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera. METHODS AND FINDINGS: An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10-1500 microg/L and a detection limit of 5.4 microg/L. The intra- and interassay coefficients of variance ranged from 8-15% and 5-16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 microg/L) and 10 patients with iron deficiency anemia (15.7 microg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 microg/L) compared to 32 age-matched healthy controls (42.7 microg/L). CONCLUSIONS: We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility

Connection between Telomerase Activity in PBMC and Markers of Inflammation and Endothelial Dysfunction in Patients with Metabolic Syndrome

Metabolic syndrome (MS) is a constellation of metabolic derangements associated with vascular endothelial dysfunction and oxidative stress and is widely regarded as an inflammatory condition, accompanied by an increased risk for cardiovascular disease. The present study tried to investigate the implications of telomerase activity with inflammation and impaired endothelial function in patients with metabolic syndrome. Telomerase activity in circulating peripheral blood mononuclear cells (PBMC), TNF-α, IL-6 and ADMA were monitored in 39 patients with MS and 20 age and sex-matched healthy volunteers. Telomerase activity in PBMC, TNF-α, IL-6 and ADMA were all significantly elevated in patients with MS compared to healthy volunteers. PBMC telomerase was negatively correlated with HDL and positively correlated with ADMA, while no association between TNF-α and IL-6 was observed. IL-6 was increasing with increasing systolic pressure both in the patients with MS and in the healthy volunteers, while smoking and diabetes were positively correlated with IL-6 only in the patients' group. In conclusion, in patients with MS characterised by a strong dyslipidemic profile and low diabetes prevalence, significant telomerase activity was detected in circulating PBMC, along with elevated markers of inflammation and endothelial dysfunction. These findings suggest a prolonged activity of inflammatory cells in the studied state of this metabolic disorder that could represent a contributory pathway in the pathogenesis of atherosclerosis

The 4C5 Cell-Impermeable Anti-HSP90 Antibody with Anti-Cancer Activity, Is Composed of a Single Light Chain Dimer

MAb 4C5 is a cell impermeable, anti-HSP90 murine monoclonal antibody, originally produced using hybridoma technology. We have previously shown that mAb 4C5 specifically recognizes both the α- and to a lesser extent the β-isoform of HSP90. Additionally, in vitro and in vivo studies revealed that by selectively inhibiting the function of cell-surface HSP90, mAb 4C5 significantly impairs cancer cell invasion and metastasis. Here we describe the reconstitution of mAb 4C5 into a mouse-human chimera. More importantly we report that mAb 4C5 and consequently its chimeric counterpart are completely devoid of heavy chain and consist only of a functional kappa light chain dimer. The chimeric antibody is shown to retain the original antibody's specificity and functional properties. Thus it is capable of inhibiting the function of surface HSP90, leading to reduced cancer cell invasion in vitro. Finally, we present in vivo evidence showing that the chimeric 4C5 significantly inhibits the metastatic deposit formation of MDA-MB-453 cells into the lungs of SCID mice. These data suggest that a chimeric kappa light chain antibody could be potentially used as an anti-cancer agent, thereby introducing a novel type of antibody fragment, with reduced possible adverse immunogenic effects, into cancer therapeutics

The mannosylated extracellular domain of Her2/neu produced in <it>P. pastoris </it>induces protective antitumor immunity

Abstract Background Her2/neu is overexpressed in various human cancers of epithelial origin and is associated with increased metastatic potential and poor prognosis. Several attempts have been made using the extracellular domain of Her2/neu (ECD/Her2) as a prophylactic vaccine in mice with no success in tumor prevention. Methods The extracellular domain of Her2/neu (ECD/Her2) was expressed in yeast P. pastoris, in a soluble highly mannosylated form. The immune response of the immunization with this recombinant ECD/Her2 was analyzed using immunoprecipitation and western blot analysis, proliferation and cytotoxicity assays as well as specific tumor growth assays. Results Mannosylated ECD/Her2 elicited a humoral response with HER2/neu specific antibodies in vaccinated mice, which were able to reduce the proliferation rate of cancer cells in vitro. Moreover, it elicited a cellular response with Her2/neu-specific CTL capable of lysing tumor cells, in vitro. When immunized Balb/c and HHD mice were challenged with Her2/neu-overexpressing cells, tumor growth was inhibited. Conclusion Here we report on the efficacy of the extracellular domain of human Her2/neu produced in yeast P. pastoris, which confers mannosylation of the protein, to act as a potent anti-tumor vaccine against Her2/neu overexpressing tumors. Specific cellular and humoral responses were observed as well as efficacy.</p

ch-4C5 inhibits the metastatic deposition of MDA-MB-453 cancer cells into the lungs of SCID mice.

<p>Control or ch-4C5 treated MDA-MB-453 cells were labeled with the fluorescent dye DiO and injected into SCID mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023906#s4" target="_blank">Materials and Methods</a>. Evaluation of metastatic deposits was performed several hours later. A. Representative cryosections of the lungs of control and ch-4C5 treated mice. The arrows show MDA-MB-453 cells stained with DiO present in the lung tissue. A significant decrease in the deposition of cancer cells was observed in the lungs of ch-4C5 treated mice. Scale bar: 100 µm <b>B</b>. Quantitative effect of ch-4C5 on the metastatic deposition of MDA-MB-453 cells into the lungs, showed a 38% inhibition when compared to control animals. The bars represent the average of two independent experiments ±SEM. Statistical significance of differences was tested by Student's t test (p<0.01).</p

Nucleotide and aminoacid sequences of mAb 4C5.

<p>Nucleotide and amino acid sequences of the L-chain variable region of mAb 4C5.</p

mAb 4C5 is a kappa L- chain dimer.

<p><b>A</b>. Electrophoretic analysis of mAb 4C5, followed by immunoblotting with an anti-mouse kappa chain, an anti-mouse Fab and an anti-Fcγ antibody. Intact IgG1 immunoglobulin produced by the 2D10 hybridoma cells serves as positive control. Under reducing electrophoresis followed by western blot with the anti-Fab antibody, a single 25 kDa-immunoreactive band is observed, instead of the 25- and 50-kDa bands corresponding to the L- and H-chain, respectively, of an intact IgG1. This 25 kDa band is identical to the band corresponding to the kappa L-chain as shown by western blot with the anti-mouse kappa chain antibody. Under-non reducing electrophoresis followed by western blot with both the anti-kappa and the anti-Fab antibodies, mAb 4C5 is shown to migrate at approximately 50 kDa, and not at 150 kDa as expected for an intact IgG1 molecule. No mAb 4C5 immunoreactivity is detected after electrophoresis under both reducing and non-reducing conditions followed by western blot with an anti-Fcγ antibody. In the case of the IgG1 immunoglobulin under the same conditions a 50 kDa and a 150 kDa band is observed, respectively. <b>B</b>. No radioactivity is detected after northern blot analysis of 4C5 hybridoma-derived RNA with a H-chain radiolabelled probe. RNA derived from the IgG1-producing 2D10 hybridoma cells and the NSO myeloma cells served as positive and negative control, respectively.</p

Electrophoretic mobility and specificity of antibodies.

<p><b>A</b>. <i>Left panel:</i> SDS-PAGE of purified antibodies under reducing conditions followed by Coomasie Brilliant Blue-R staining revealed in all cases an approximately 25 kDa band corresponding to the L-chain. <i>Right panel:</i> Under non-reducing conditions the antibodies are shown to migrate as a L-chain dimer. <b>B</b>. Western blot of MDA-MB-453 cell lysates using mAb 4C5, rec-4C5, ch-4C5 and a commercial anti-HSP90α antibody, serving as positive control. In all cases a single 90 kDa immunoreactive band corresponding to HSP90 is observed. <b>C</b>. Immunoprecipitation in MDA-MB-453 cell lysates with anti-HSP90α, followed by immunoblotting with either the murine or the recombinant antibodies. In all cases a single immunoreactive band is observed. <b>D</b>. Reverse immunoprecipitation experiments in MDA-MB-453 cell lysates using mAb 4C5, rec-4C5 and ch-4C5, followed by western blot with anti-HSP90α. In all cases a single immunoreactive band is observed indicating that both recombinant antibodies recognize HSP90.</p

ch-4C5 inhibis cancer cell invasion in <i>vitro</i>.

<p><b>A</b>. Wound healing assay. Photographs represent phase-contrast images obtained at zero time (left panel) and at 24 h (right panel) after scratch formation, showing MDA-MB-453 cell migration either in control cultures or cultures including anti-HSP90α, mAb 4C5, rec-4C5 or ch-4C5. Scale bar: 200 µm. <b>B</b>. Quantitative effect of antibodies on the closure of the wound. Addition of 200 µg/ml of anti-HSP90α and mAb 4C5 in the culture medium resulted in a 48% and 55% reduction of wound closure, respectively when compared to control cultures that were considered as resulting in 100% wound closure. Addition of 200 µg/ml rec-4C5 and ch-4C5 in the culture medium resulted in a 46% and 46% inhibition of wound closure, respectively. Statistical significance of differences was assessed by Student's t test. The presence of anti-HSP90α, mAb 4C5, rec-4C5 or ch-4C5 had a statistically significant effect on the wound closure (p<0.01 in each case). <b>C</b>. Phase-contrast images obtained at zero time (left panel) and at 24 h after scratch formation (right panel), showing B16 F10 melanoma cell invasion in a wound healing assay in the presence of 200 µg/ml of ch-4C5. Scale bar: 200 µm. <b>D</b>. Quantitative effect of increasing concentrations of ch-4C5 on the invasion level of B16 F10 melanoma cells. Presence of 50 µg/ml ch-4C5 resulted in 15% inhibition of invasion, while addition of 100 µg/ml and 200 µg/ml ch 4C5 resulted in 21% and 43% inhibition of migration, respectively when compared to control cultures that were considered as resulting in 100% wound closure. <b>E</b>. Visualization of dead cells using trypan blue dye. In ch-4C5 treated cultures, the cell death incidence is similar to that observed in the control cultures. In contrast, in the cultures treated with the anti-HSP90α antibody, a much greater number of cells are stained with Trypan blue, indicating an increased incidence of cell death Scale bar, 30 µm. <b>F</b>. Control and ch-4C5 treated cells were fixed permeabilized and stained with fluorescently labelled phalloidin. Scale bar 40 µm <b>G</b>. Higher magnification showing phalloidin staining (F-actin). Ch-4C5 effectively blocks spreading of lamellipodia. Scale bar: 16 µm.</p

ch-4C5 binds to the surface pool of HSP90 and is not internalized by MDA-MB-453 cells.

<p><b>A</b>. Immunofluoresence labelling of MDA-MB-453 cells using both the rec-4C5 and ch-4C5. The punctuate immunolabeling indicates the surface pool of HSP90. Negative controls were performed using an antibody against the intracellular protein β tubulin (data non shown). Scale bar: 20 µm <b>B</b>. Immunofluoresence detection of intracellular HSP90 in fixed MDA-MB-453 cells, permeabilized with 0.1% Triton X-100. Similarly to the murine antibody, rec-4C5 and ch-4C5 recognize the intracellular pool of HSP90. Scale bar: 20 µm <b>C</b>. For detection of antibody internalization, live cells were incubated with the antibodies for various time intervals, then fixed, permeabilized and fluorescently labelled. No internalization of mAb4C5, rec-4C5 and ch-4C5 is observed even after 24 h of incubation. In contrast the anti-HSP90α antibody could be detected intracellularly after 24 h of incubation. Scale bar: 20 µm <b>D</b>. MDA-MB-453 cell lysates, treated with mAb 4C5, rec-4C5, ch-4C5 and anti-HSP90α were analyzed by western blot using antibodies against ErbB2, Akt, cRaf and HSP90α. Actin served as a loading control. Presence of mAb 4C5, rec-4C5 and ch-4C5 did not affect the levels of the kinases compared to controls. In contrast the cell permeable anti-HSP90α antibody significantly reduced the levels of ErbB2, Akt and cRaf.</p