Nonspecific IgG Research Articles

OxLDL-targeted iron oxide nanoparticles for in vivo MRI detection of perivascular carotid collar induced atherosclerotic lesions in ApoE-deficient mice

Atherosclerotic disease is a leading cause of morbidity and mortality in developed countries, and oxidized LDL (OxLDL) plays a key role in the formation, rupture, and subsequent thrombus formation in atherosclerotic plaques. In the current study, anti-mouse OxLDL polyclonal antibody and nonspecific IgG antibody were conjugated to polyethylene glycol-coated ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, and a carotid perivascular collar model in apolipoprotein E-deficient mice was imaged at 7.0 Tesla MRI before contrast administration and at 8 h and 24 h after injection of 30 mg Fe/kg. The results showed MRI signal loss in the carotid atherosclerotic lesions after administration of targeted anti-OxLDL-USPIO at 8 h and 24 h, which is consistent with the presence of the nanoparticles in the lesions. Immunohistochemistry confirmed the colocalization of the OxLDL/macrophages and iron oxide nanoparticles. The nonspecific IgG-USPIO, unconjugated USPIO nanoparticles, and competitive inhibition groups had limited signal changes (p < 0.05). This report shows that anti-OxLDL-USPIO nanoparticles can be used to directly detect OxLDL and image atherosclerotic lesions within 24 h of nanoparticle administration and suggests a strategy for the therapeutic evaluation of atherosclerotic plaques in vivo.

Abstract 2848: Preclinical activity of OM-RCA-01, a humanized anti-FGFR1 antibody, in renal cell carcinoma (RCC)

Abstract Background: Fibroblast growth factor (FGF) receptor 1 (FGFR1) is a potential therapeutic target for treatment of metastatic RCC. Early we reported that anti-FGFR1 monoclonal antibody inhibited ligand-induced phosphorylation of FGFR1, activation of MAP kinase and Akt downstream signaling pathways in FGFR1 expressing endothelial (bovine adrenal cortex capillary endothelial cells) and human RCC (Caki-1) lines (Tsimafeyeu et al. AACR/JCA joint conference 2010). Today we investigated the preclinical activity of OM-RCA-01, a novel therapeutic humanized anti-FGFR1 antibody with high affinity (Kd of 1.59 nM), in RCC. Methods: To assess the effect of anti-FGFR1 antibody on FGF-mediated signaling, the human renal carcinoma Caki-1 FGFR1-expressing cells were incubated (0.5% FBS) and were dosed with OM-RCA-01 at 100, 10, and 1 mcg/ml. Control wells were left untreated. Three hours after dosing, basic FGF was added at a concentration of 50 ng/ml. Additional control wells were treated with OM-RCA-01 without FGF-stimulation. Cell growth inhibition was determined using Promega's Cell Titer-Glo® assay. Charles River female NCr nu/nu mice (6-12 weeks of age) were set up with 1 mm3 Caki-1 tumor fragments sc in flank. Tumor sizes were measured in a blind fashion twice a week with a vernier caliper. Mice with established tumors (an average size of 80 - 120 mg) were randomly divided into vehicle, non-specific IgG or OM-RCA-01 groups per 10 animals in group. Animals were treated with antibody using different doses (1 or 10 mg/kg). Endpoint was significant differences in tumor growth delay. Results: In vitro study showed that basic FGF significantly increased proliferation of the human FGFR1-expressing renal carcinoma cells (P=0.011). No effect of basic FGF on cell line proliferation was observed when the cells were incubated with OM-RCA-01 antibody at any concentrations up to 100 mcg/kg in comparison with FGF-untreated control (P=0.855). In vivo, the tumors in untreated mice or mice treated with non-specific IgG continued their aggressive growth to reach the size of 2000 cm3, at which point the mice were killed. In contrast, treatment with OM-RCA-01 not only significant arrested further growth of the tumors (P=0.006) but also demonstrated differences in tumor volume compared with vehicle already on Day 13. A similar anti-tumor activity of OM-RCA-01 was observed when the antibody was given in low (1 mg/kg) or high (10 mg/kg) doses (P=0.917). Conclusions: High-affinity humanized anti-FGFR1 antibody OM-RCA-01 suppressed ligand-induced proliferation of human RCC cells in vitro. OM-RCA-01 has significant early anti-tumor efficacy in Caki-1 xenograft model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2848. doi:1538-7445.AM2012-2848

Abstract 2448: Development of a red fluorescent labeled agent for assessing HER2 expression in vitro and in vivo

Abstract Upregulation of tumor HER2 occurs in approximately 25% women with breast cancer and is often associated with poor prognosis. We developed a red fluorescent imaging agent to non-invasively image and quantify tumor-associated HER2 expression in vivo. A red fluorescent dye (VivoTag 645; α=210,000 μ/cm; abs/em max 643/660 nm) was used to label trastuzumab, which is currently used to treat breast and stomach cancer. The red-labeled trastuzumab (VM4003) preferentially labeled HER2+ SKOV-3 human ovarian adenocarcinoma cells over HER2− human colorectal adenocarcinoma Colo-205 cells (10 fold), and the specificity of binding was confirmed by control experiments using free dye, labeled non-specific IgG, and competitive blockade with unlabeled excess trastuzumab. Fluorescence microscopy confirmed the expected membrane localization of fluorescence. In vivo and ex vivo imaging by fluorescence molecular tomography (FMT), showed significantly higher signal within the tumors, peaking at 6-72 hours following intravenous injection of 2 mg/kg VM4003, decreasing thereafter with a tissue half-life of 3 days. In vivo quantification of tumor signal in nude mice showed significantly higher tumor signal in HER2+ than in HER2− tumors (14.36 + 4 versus 2.39+ 0.74 pmol, at 6h imaging time, p=0.007; 18.77 + 4.45 versus 3.50+ 0.98 pmol, at 24h, p=0.001). Specificity of targeting was confirmed by competition with excess intravenous unlabeled trastuzumab, which achieved 70% signal inhibition in the tumors (tumor signal 16.12 + 3.03 versus 4.72 + 1.96 pmol, p=0.022 at 24h). Tumor volumes, as determined by direct measurements of tumor size, were comparable between both groups of mice (p=0.193). Fluorescence microscopy of ex vivo frozen tissue sections confirmed tumor fluorescence and signal localization associated to cell membranes and cytoplasm. In summary, red fluorescent-labeled trastuzumab selectively targets αER2, allowing both imaging in vitro and the non-invasive real-time tomographic imaging and quantification in vivo of HER-2 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2448. doi:1538-7445.AM2012-2448

Identification of new alternative pathways and therapeutic targets in tnf-α-mediated inflammation and osteolysis induced by ultra-high molecular weight polyethylene

Identification of new alternative pathways and therapeutic targets in tnf-α-mediated inflammation and osteolysis induced by ultra-high molecular weight polyethylene

Toll-like receptor 4 contributes to blood pressure regulation and vascular contraction in spontaneously hypertensive rats

Activation of Toll-like receptors (TLR) induces gene expression of proteins involved in the immune system response. TLR4 has been implicated in the development and progression of cardiovascular diseases. Innate and adaptive immunity contribute to hypertension-associated end-organ damage, although the mechanism by which this occurs remains unclear. In the present study we hypothesize that inhibition of TLR4 decreases blood pressure and improves vascular contractility in resistance arteries from spontaneously hypertensive rats (SHR). TLR4 protein expression in mesenteric resistance arteries was higher in 15 weeks-old SHR than in same age Wistar controls or in 5 weeks-old SHR. In order to decrease activation of TLR4, 15 weeks-old SHR and Wistar rats were treated with anti-TLR4 antibody or non-specific IgG control antibody for 15 days (1µg per day, i.p.). Treatment with anti-TLR4 decreased mean arterial pressure as well as TLR4 protein expression in mesenteric resistance arteries and interleukin-6 (IL-6) serum levels from SHR when compared to SHR treated with IgG. No changes in these parameters were found in Wistar treated rats. Mesenteric resistance arteries from anti-TLR4-treated SHR exhibited decreased maximal contractile response to noradrenaline compared to IgG-treated-SHR. Inhibition of cyclooxygenase-1 (Cox) and Cox-2, enzymes related to inflammatory pathways, decreased noradrenaline responses only in mesenteric resistance arteries of SHR treated with IgG. Cox-2 expression and thromboxane A2 release were decreased in SHR treated with anti-TLR4 compared with IgG-treated-SHR. Our results suggest that TLR4 activation contributes to increased blood pressure, low grade inflammation and plays a role in the augmented vascular contractility displayed by SHR.

577 CD63 Expression, IL3 Receptor, IGG Autoantibody and Autologous Serum Skin Test Accuracy in Patients with Chronic Urticaria in Brazil

BackgroundRecently, a laboratory technique called basophil activation test (BAT) using flow cytometry (FC) got demonstrated through the expression of CD63 molecules that basophils of atopics donors can be activated when stimulated by serum of patients with CU (supposedly autoimmune). This paper aims to analyze the autologous serum skin test (ASST) in relation to the BAT as well as evaluating the IL3 receptor (CD123) and nonspecific autoantibodies IgG bound to basophils of patients with chronic urticaria.MethodsWe studied 33 adults (24 women) with CU with a mean age of 42.5 + 14 years, of which 22 with ASST positive and 11 with ASST negative. It was done through the analysis by FC of CD63 molecules expression on basophils from an atopic donor after stimulation by serum of these patients. We used as control the serum from 4 volunteers (without urticaria). Also we researched the CD123 molecule expression and IgG nonspecific autoantibodies in basophils from patients with CU.ResultsWe found 21 (63.6%) patients with positive BAT, of these 14 (66.6%) were ASST positive and 7 (33.3%) were ASST negative. Taking as parameter the BAT, we found an accuracy of 54.5% for the ASST, a sensitivity of 66%, specificity 33%, positive predictive value of 63% and negative predictive value of 36%. Comparing the expression intensity (mean with SD) of IgG autoantibodies in patients' basophils with positive and negative ASST there was not statistical difference (for a P < 0.05); the same was true when comparing the autoantibodies (IgG) between groups with BAT positive and with BAT negative. We also didn't find statistical difference (for a P < 0.05) of receptor expression of IL3 (CD123) between the groups.ConclusionsTaking as parameter the BAT for diagnosis of autoimmune CU this study found that ASST is accurate about 55%. There was no statistical difference when comparing the expression of IgG nonspecific autoantibodies and CD123 molecule, between groups.

Therapeutic efficacy of CXCR3 blockade in an experimental model of severe sepsis

IntroductionIn our previous studies we demonstrated that CXC chemokine receptor 3 (CXCR3) participates in the regulation of lymphocyte trafficking during cecal ligation and puncture (CLP)-induced sepsis. In this study, we evaluated the effects of treatment with anti-CXCR3 immunoglobulin (IgG) and antibiotics on outcome during septic shock caused by CLP.MethodsC57BL/6J mice were treated with neutralizing IgG against CXCR3 plus Primaxin either 24 hours prior to, 2 hours after or 6 hours after CLP. Control mice received nonspecific IgG plus Primaxin in the same regimen. Survival, core body temperature, bacterial clearance and systemic cytokine production were evaluated.ResultsOur results show that treatment with anti-CXCR3 IgG plus Primaxin significantly improved survival when administered 24 hours prior to CLP (50% vs. 10%), 2 hours after CLP (55% vs. 10%) or 6 hours after CLP (55% vs. 25%) compared with mice receiving nonspecific IgG plus Primaxin. Treatment with anti-CXCR3 plus Primaxin 24 hours prior to CLP attenuated hypothermia and IL-6 and macrophage inflammatory protein 2 (MIP-2) production but did not alter bacterial clearance. Treatment with anti-CXCR3 IgG and Primaxin 2 hours after CLP did not improve bacterial clearance and systemic cytokine production compared with mice treated with IgG and Primaxin, whereas 6 hours after CLP the bacterial clearance and IL-6 and MIP-2 concentrations, both in plasma and peritoneal lavage fluid, were significantly improved in mice receiving anti-CXCR3 IgG and Primaxin compared with mice that only received nonspecific IgG and Primaxin.ConclusionThe results from this study indicate that neutralization of CXCR3 prior to, 2 hours after or 6 hours after the initiation of CLP-induced septic shock improves survival and attenuates CLP-induced inflammation and physiologic dysfunction.

Role of Thiol Isomerase ERp5 in Thrombus Formation

Abstract 370Protein disulfide isomerase is required for thrombus formation in various in vivo models of thrombosis. Another member of the thiol isomerase family, endoplasmic reticulum protein 5 (ERp5), is released from activated platelets and co-immunoprecipitates with beta 3 integrin (Jordan et al, 2005). We further investigated the association of ERp5 with the platelet fibrinogen receptor alpha IIb beta 3 and the significance of ERp5 release in thrombus formation in vivo. Recombinant purified ERp5 was labeled with Alexa 488 and used in direct binding assays to CHO cells expressing wild type (WT) alpha IIb beta 3, CHO cells expressing mutant alpha IIb beta 3 (containing an Asp119Tyr substitution in the beta 3 subunit) and to control CHO cells. The mutant alpha IIb beta 3 does not bind fibrinogen. ERp5 bound to CHO cells expressing wild type (WT) alpha IIb beta 3 in a dose-dependent manner but did not bind to CHO cells expressing mutant alpha IIb beta 3 or to control CHO cells. The relative increase in the geomean of Alexa 488-labeled ERp5 binding to 0.5 ×106 WT alpha IIb beta 3 CHO cells over that bound to control CHO cells was 20, 45 and 85% for ERp5 concentrations of 80, 160 and 400 nM respectively. Binding of ERp5 (160 nM) to WT alpha IIb beta 3 expressing CHO cells was further increased by 75% when the integrin was activated with 2 mM Mn2+ compared to non-activated WT alpha IIb beta 3 CHO cells. A role for ERp5 in thrombus formation was studied in the laser injury model of thrombosis in mouse cremaster arterioles using a rabbit polyclonal anti-ERp5 antibody, immunoaffinity purified against recombinant ERp5. This antibody detected ERp5 in the releasate of thrombin-activated mouse platelets in vitro by Western blot and on the surface of thrombin-activated mouse platelets by flow cytometry. Dylight 649-labeled anti-CD42b was infused into the mouse circulation to detect platelet accumulation and Alexa 488-labeled anti-ERp5 antibody at 0.05 ug/g, a dose that does not inhibit thrombus formation, was infused to detect ERp5. The fluorescent anti-ERp5 signal detected at the thrombus site was compared to the signal produced by a non-specific IgG labeled with Alexa 488 infused into a control mouse. Anti-ERp5 fluorescence was detected in the thrombus with kinetics that followed platelet accumulation whereas there was minimal signal from the control IgG. We examined whether higher doses of anti-ERp5 affect thrombus formation. Platelet and fibrin accumulation were detected using fluorescently labeled anti-CD42b antibody and monoclonal anti-fibrin-specific antibody respectively before or after the injection of unlabeled anti-ERp5 antibody or pre-immune IgG at 2.5 ug/g. Platelet and fibrin accumulation, expressed as area under the curve of the median integrated fluorescence over time, was obtained from 14 thrombi in 6 mice formed before infusion of antibody, 18 thrombi in 2 mice formed after infusion of control IgG and 29 thrombi in 3 mice formed after infusion of anti-ERp5. Anti-ERp5 infusion caused a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (p<0.01). There was no difference in platelet and fibrin accumulation before infusion of antibody and after infusion of control antibody. These results provide evidence for a role of a second thiol isomerase, ERp5, in thrombus formation, a function which may be mediated through its association with alpha IIb beta 3. Disclosures:No relevant conflicts of interest to declare.

Binding of Anti-Membrane-Proximal gp41 Monoclonal Antibodies to CD4-Liganded and -Unliganded Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus Virions

The broadly neutralizing monoclonal antibodies (MAbs) 4E10, 2F5, and Z13e1 target membrane-proximal external region (MPER) epitopes of HIV-1 gp41 in a manner that remains controversial. The requirements for initial lipid bilayer binding and/or CD4 ligation have been proposed. To further investigate these issues, we probed for binding of these MAbs to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) virions with protein A-conjugated gold (PAG) nanoparticles using negative-stain electron microscopy. We found moderate levels of PAG associated with unliganded HIV-1 and SIV virions incubated with the three MAbs. Significantly higher levels of PAG were associated with CD4-liganded HIV-1 (epitope-positive) but not SIV (epitope-negative) virions. A chimeric SIV virion displaying the HIV-1 4E10 epitope also showed significantly higher PAG association after CD4 ligation and incubation with 4E10. MAbs accumulated rapidly on CD4-liganded virions and slowly on unliganded virions, although both reached similar levels in time. Anti-MPER epitope-specific binding was stable to washout. Virions incubated with an irrelevant MAb or CD4-only (no MAb) showed negligible PAG association, as did a vesicle-rich fraction devoid of virions. Preincubation with Fab 4E10 inhibited both specific and nonspecific 4E10 IgG binding. Our data provide evidence for moderate association of anti-MPER MAbs to viral surfaces but not lipid vesicles, even in the absence of cognate epitopes. Significantly greater MAb interaction occurs in epitope-positive virions following long incubation or CD4 ligation. These findings are consistent with a two-stage binding model where these anti-MPER MAbs bind first to the viral lipid bilayer and then to the MPER epitopes following spontaneous or induced exposure.

Auto-activation of c-JUN Gene by Amino Acid Deprivation of Hepatocellular Carcinoma Cells Reveals a Novel c-JUN-mediated Signaling Pathway

Mammalian cells respond to protein or amino acid (AA) limitation by activating a number of signaling pathways, collectively referred to as the AA response (AAR), that modulate a range of cellular functions, including transcriptional induction of target genes. This study demonstrates that in hepatocellular carcinoma cells, expression of c-JUN, JUN-B, c-FOS, and FOS-B was induced by the AAR, whereas JUN-D, FRA-1, and FRA-2 were not. Of the four activated FOS/JUN members, c-JUN made the largest contribution to the induction of several known AAR target genes. For several human liver, prostate, and ovarian cell lines, the AAR-induced increase in c-JUN expression was greater in transformed cells compared with nontransformed counterparts, an effect independent of cell growth rate. Thus far, the best characterized AA-responsive genes are all transcriptionally activated by ATF4, but the AAR-dependent induction of c-JUN transcription was ATF4-independent. The increased expression of c-JUN was dependent on ATF2 and on activation of the MEK-ERK and JNK arms of the MAPK signaling pathways. Formation of c-JUN-ATF2-activated heterodimers was increased after AA limitation, and c-JUN or ATF2 knockdown suppressed the induction of c-JUN and other AAR target genes. AA deprivation triggers a feed-forward process that involves phosphorylation of existing c-JUN protein by JNK and subsequent auto-activation of the c-JUN gene by recruitment of c-JUN and ATF2 to two AP-1 sites within the proximal promoter. The results document the novel observation that AP-1 sequences within the c-JUN gene can function as transcriptional amino acid-response elements.

USP1 Deubiquitinates ID Proteins to Preserve a Mesenchymal Stem Cell Program in Osteosarcoma

Inhibitors of DNA binding (IDs) antagonize basic-helix-loop-helix (bHLH) transcription factors to inhibit differentiation and maintain stem cell fate. ID ubiquitination and proteasomal degradation occur in differentiated tissues, but IDs in many neoplasms appear to escape degradation. We show that the deubiquitinating enzyme USP1 promotes ID protein stability and stem cell-like characteristics in osteosarcoma. USP1 bound, deubiquitinated, and thereby stabilized ID1, ID2, and ID3. A subset of primary human osteosarcomas coordinately overexpressed USP1 and ID proteins. USP1 knockdown in osteosarcoma cells precipitated ID protein destabilization, cell-cycle arrest, and osteogenic differentiation. Conversely, ectopic USP1 expression in mesenchymal stem cells stabilized ID proteins, inhibited osteoblastic differentiation, and enhanced proliferation. Consistent with USP1 functioning in normal mesenchymal stem cells, USP1-deficient mice were osteopenic. Our observations implicate USP1 in preservation of the stem cell state that characterizes osteosarcoma and identify USP1 as a target for differentiation therapy.

Preclinical Strategies to Optimize Optical Imaging in Head and Neck Cancer

Objective: There is a need for cancer imaging in the clinic and operating room. We have shown that fluorescently labeled antibodies can detect microscopic islands of head and neck cancer cells using modified intraoperative microscopes in preclinical animal studies. Here we evaluated the optimal targeting molecule for clinical translation. Method: We assessed in vitro binding of fluorescently labeled antibodies targeting EGFR (cetuximab/Erbitux and panitumumab/Vectibix) and an antibody mimetic (anti-EGFR affibody) in multiple cell lines (SCC5, OSC19, FADU). To compare in vivo imaging characteristics, HNSCC xenografts (SCC5, SCC1, FADU) were imaged after systemic injection of fluorescently labeled antibodies over 5 days. Results: The binding affinity was lower for the control antibody IgG (KD = 9.55 nM) compared with either panitumumab (KD = 6.82 nM) or cetuximab (KD = 2.86 nM). In vitro, panitumumab had a two fold increase in relative fluorescence compared with cetuximab and a four fold increase in fluorescence compared with control antibody (nonspecific IgG). Xenograft imaging in vivo demonstrated a variation in fluorescence intensity that correlated with circulating half-lives: peaks in fluorescence intensity were seen at 1.5 h (affibody) and 48 to 72 h (cetuximab, panitunumab). The pantitumumab and affibody images demonstrated greater tumor fluorescence and lower background signal compared to cetuximab. Conclusion: Panitumumab/Vectibix demonstrated optimal imaging characteristics which appeared to be related to its binding affinity and increased circulation time. These characteristics may allow for increased localization to the tumor site. Panitumumab may be the optimal antibody for clinical translation since it will decrease false positive rates associated with non-specific uptake.

Differential Impact of Caveolae and Caveolin-1 Scaffolds on The Membrane Raft Proteome

Caveolae, a class of cholesterol-rich lipid rafts, are smooth invaginations of the plasma membrane whose formation in nonmuscle cells requires caveolin-1 (Cav1). The recent demonstration that Cav1-associated cavin proteins, in particular PTRF/cavin-1, are also required for caveolae formation supports a functional role for Cav1 independently of caveolae. In tumor cells deficient for Golgi β-1,6N-acetylglucosaminyltransferase V (Mgat5), reduced Cav1 expression is associated not with caveolae but with oligomerized Cav1 domains, or scaffolds, that functionally regulate receptor signaling and raft-dependent endocytosis. Using subdiffraction-limit microscopy, we show that Cav1 scaffolds are homogenous subdiffraction-limit sized structures whose size distribution differs from that of Cav1 in caveolae expressing cells. These cell lines displaying differing Cav1/caveolae phenotypes are effective tools for probing the structure and composition of caveolae. Using stable isotope labeling by amino acids in cell culture, we are able to quantitatively distinguish the composition of caveolae from the background of detergent-resistant membrane proteins and show that the presence of caveolae enriches the protein composition of detergent-resistant membrane, including the recruitment of multiple heterotrimeric G-protein subunits. These data were further supported by analysis of immuno-isolated Cav1 domains and of methyl-β-cyclodextrin-disrupted detergent-resistant membrane. Our data show that loss of caveolae results in a dramatic change to the membrane raft proteome and that this change is independent of Cav1 expression. The proteomics data, in combination with subdiffraction-limit microscopy, indicates that noncaveolar Cav1 domains, or scaffolds are structurally and functionally distinct from caveolae and differentially impact on the molecular composition of lipid rafts.

Long-period gratings in photonic crystal fiber as an optofluidic label-free biosensor

Using long-period gratings (LPG) inscribed in photonic crystal fiber (PCF) and coupling this structure with an optically aligned flow cell, we have developed an optofluidic refractive index transduction platform for label-free biosensing. The LPG–PCF scheme possesses extremely high sensitivity to the change in refractive index induced by localized binding event in different solution media. A model immunoassay experiment was carried out inside the air channels of PCF by a series of surface modification steps in sequence that include adsorption of poly(allylamine hydrochloride) monolayer, immobilization of anti-rat bone sialoprotein monoclonal primary antibody, and binding interactions with non-specific goat anti-rabbit IgG (H + L) and specific secondary goat anti-mouse IgG (H + L) antibodies. These adsorption and binding events were monitored in situ using the LPG–PCF by measuring the shift of the core-to-cladding mode coupling resonance wavelength. Steady and significant resonance changes, about 0.75 nm per nanometer-thick adsorbed/bound bio-molecules, have been observed following the sequence of the surface events with monolayer sensitivity, suggesting the promising potential of LPG–PCF for biological sensing and evaluation.

The Hsp90 Kinase Co-chaperone Cdc37 Regulates Tau Stability and Phosphorylation Dynamics

The microtubule-associated protein tau, which becomes hyperphosphorylated and pathologically aggregates in a number of these diseases, is extremely sensitive to manipulations of chaperone signaling. For example, Hsp90 inhibitors can reduce the levels of tau in transgenic mouse models of tauopathy. Because of this, we hypothesized that a number of Hsp90 accessory proteins, termed co-chaperones, could also affect tau stability. Perhaps by identifying these co-chaperones, new therapeutics could be designed to specifically target these proteins and facilitate tau clearance. Here, we report that the co-chaperone Cdc37 can regulate aspects of tau pathogenesis. We found that suppression of Cdc37 destabilized tau, leading to its clearance, whereas Cdc37 overexpression preserved tau. Cdc37 was found to co-localize with tau in neuronal cells and to physically interact with tau from human brain. Moreover, Cdc37 levels significantly increased with age. Cdc37 knockdown altered the phosphorylation profile of tau, an effect that was due in part to reduced tau kinase stability, specifically Cdk5 and Akt. Conversely, GSK3β and Mark2 were unaffected by Cdc37 modulation. Cdc37 overexpression prevented whereas Cdc37 suppression potentiated tau clearance following Hsp90 inhibition. Thus, Cdc37 can regulate tau in two ways: by directly stabilizing it via Hsp90 and by regulating the stability of distinct tau kinases. We propose that changes in the neuronal levels or activity of Cdc37 could dramatically alter the kinome, leading to profound changes in the tau phosphorylation signature, altering its proteotoxicity and stability.

Abstract 2704: A role of the ADCC in the antibody therapy for gastrointestinal carcinoma

Abstract (Background and Aim) Both cetuximab and trastuzumab inhibit growth signal by binding to growth factor receptors, and also have an alternative function regarded as ADCC. In this study, we examined cetuximab-mediated ADCC against EGFR-positive colon cancer cells and trastuzumab-mediated ADCC against HER2-positive gastric cancer cells. Moreover, we investigated the new methods to enhance those ADCC activities. (Materials and Methods) We used human gastric cancer cell lines (MKN1, MKN45) and colon cancer cells (HT-29), cetuximab and trastuzumab as an antibody which potentially had ADCC, and non-specific IgG1 antibody as a control. ADCC was measured by Europium release assay: First, we used peripheral blood mononuclear cells (PBMCs) from healthy volunteers as effector, and we prepared those cancer cell lines combined with those kinds of antibodies as target. Next, we used PBMCs activated by IL-2 instead of normal PBMCs. At same time, CD16, perforin, granzyneB on PBMCs were evaluated by Flow Cytometer and PCR. NK activity of PBMN and activated PBMC against for K562 cell and ADCC activity of PBMN and activated PBMC were evaluated. (Results) Cetuximab and trastuzumab didn't kill cancer cells by themselves, but the cytotoxicity of PBMCs was increased when the target cells were treated with these antibodies in advance. This increase was further strengthened by activated PBMCs with IL-2. But IL-2 didn't enhance CD16 expression on PBMCs. Finally PBMC, which have high potential of NK activity shows high ADCC activity, (Conclusion) These two antibody therapy shows ADCC in vitro, As a next step, we have to start in vivo study Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2704. doi:10.1158/1538-7445.AM2011-2704

Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells

The solute carrier family 11 member 1 (SLC11A1) gene is strictly regulated and exclusively expressed in myeloid lineage cells. However, little is known about the transcriptional regulation of the SLC11A1 gene during myeloid development. In this study, we used HL-60 cells as a model to investigate the regulatory elements/factors involved in the transactivation of the SLC11A1 gene during phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation of HL-60 cells. Promoter deletion analysis showed that a 7-base AP-1-like element (TGACTCT) was critical for the responsiveness of the SLC11A1 promoter to PMA. Stimulation by PMA induced the binding of ATF-3 and the recruitment of two components of the SWI/SNF complex, BRG1 and β-actin, to this element in an ATF-3-dependent manner. RNAi-mediated depletion of ATF-3 or BRG1 markedly decreased SLC11A1 gene expression and its promoter activity induced by PMA. Luciferase reporter experiments demonstrated that ATF-3 cooperated with BRG1 and β-actin to activate the SLC11A1 promoter. Furthermore, we showed that PMA can induce the proximal (GT/AC)(n) repeat sequence to convert to the Z-DNA structure in the SLC11A1 gene promoter, and depletion of BRG1 resulted in a significant decrease of Z-DNA formation. Our results demonstrated that recruitment of the SWI/SNF complex initiated Z-DNA formation and subsequently helped to transactivate the SLC11A1 gene.

Human T-lymphotropic Virus Type 1 p30 Interacts with REGγ and Modulates ATM (Ataxia Telangiectasia Mutated) to Promote Cell Survival

Human T-lymphotropic virus type 1 (HTLV-1) is a causative agent of adult T cell leukemia/lymphoma and a variety of inflammatory disorders. HTLV-1 encodes a nuclear localizing protein, p30, that selectively alters viral and cellular gene expression, activates G(2)-M cell cycle checkpoints, and is essential for viral spread. Here, we used immunoprecipitation and affinity pulldown of ectopically expressed p30 coupled with mass spectrometry to identify cellular binding partners of p30. Our data indicate that p30 specifically binds to cellular ATM (ataxia telangiectasia mutated) and REGγ (a nuclear 20 S proteasome activator). Under conditions of genotoxic stress, p30 expression was associated with reduced levels of ATM and increased cell survival. Knockdown or overexpression of REGγ paralleled p30 expression, suggesting an unexpected enhancement of p30 expression in the presence of REGγ. Finally, size exclusion chromatography revealed the presence of p30 in a high molecular mass complex along with ATM and REGγ. On the basis of our findings, we propose that HTLV-1 p30 interacts with ATM and REGγ to increase viral spread by facilitating cell survival.

Strongyloides stercoralis excretory/secretory protein strongylastacin specifically recognized by IgE antibodies in infected human sera

The infective, microscopic Strongyloides stercoralis larvae in contaminated soil can penetrate human skin with the help of excretory/secretory proteases. These proteases play a critical role in infection and transmigration of the parasite to the intestines. Strongylastacin is similar to astacin (from the digestive gland of the crayfish Astacus astacus), a multi-domain protein with a signal peptide, a pro-enzyme, a catalytic domain containing the zinc binding consensus astacin family signature sequence HEXXHXXGFXHEXXRXDR, and a second conserved zinc binding motif SIMHY at N- terminal region. An EGF-1 like domain and a CUB domain are located at the COOH- terminal. In this study, the excretory/secretory Strongylastacin gene from S. stercoralis infective larval stage was cloned and expressed as a 45 kDa in Escherichia coli. Immunoblot analysis showed the presence of natural IgG antibodies against strongylastacin in six infected and six non-endemic normal sera. These findings were confirmed in an ELISA of 32 S. stercoralis infected and 32 presumed normal human sera; all contained natural anti-strongylastacin IgG antibodies. By contrast, IgE antibodies specific to strongylastacin were present in sera from individuals infected with S. stercoralis but not in uninfected control sera. Moreover, recombinant strongylastacin did not cross-react with IgE antibodies either from patients infected with filaria or patients with tropical pulmonary eosinophilic (TPE) who had increased IgE antibodies. The present authors conclude that strongylastacin, an excretory/secretory antigen, elicits specific IgE antibodies in S. stercoralis infected humans. Non-specific IgG antibodies to strongylastacin are present in both infected and normal humans. Further investigation is needed to understand the role of the host protective response against strongylastacin.

NKT ligand-loaded, antigen-expressing B cells function as long-lasting antigen presenting cells in vivo

We had previously shown that activated NKT cells licensed B cells to be immunogenic antigen-presenting cells and helped to elicit a wide spectrum of cancer targeted immune responses. In the current study, we sought to verify the safety of αGalCer-loaded, and adenovirus-transduced B cell-based vaccines, together with mechanism of action. Intravenously injected αGalCer-loaded, antigen-expressing B cells rapidly localized in the spleen and directly primed CD8+ T cells in an antigen-specific manner. The transferred antigen was sustained for at least 30days. While some injected B cells produced nonspecific IgG, the antigen-specific IgG response was completely dependent on endogenous B cells. The liver was one of the main tissues where injected B cells were retained; however, we could not find the signs of liver toxicity. Our results demonstrate that αGalCer-loaded, antigen-expressing B cells behave as “antigen-presenting” cells that stimulate endogenous antigen-specific T cells and B cells in vivo without significant toxicity.