Antibody 17-1A Research Articles

Carcinogenic heavy metals replace Ca 2+ for DNA binding and annealing activities of mono-ubiquitinated annexin A1 homodimer

Mono-ubiquitinated annexin A1 was purified from rat liver nuclei. The homodimer form of mono-ubiquitinated annexin A1 was able to unwind dsDNA in a Mg 2+- and ATP-dependent manner, and to anneal ssDNA in a Ca 2+-dependent manner. Phospholipids decreased the concentration of Ca 2+ required for maximal annealing activity. Heavy metals such as As 3+, Cr 6+, Pb 2+ and Cd 2+ substituted for Ca 2+ in the ssDNA binding and annealing activities of annexin A1. While these metals inhibited the unwinding of dsDNA by nuclear annexin A1 in the presence of Mg 2+ and ATP, they enhanced dsDNA-dependent ATPase activity of annexin A1. Heavy metals may have produced dsDNA, a substrate for the DNA unwinding reaction, via the DNA annealing reaction. DNA synthesomes were isolated from L5178Y tk(+/−) mouse lymphoma cells in exponential growth, and were found to contain helicase activities. The As 3+- or Cr 6+-induced increases in ssDNA binding activity of DNA synthesomes were reduced by a mono-specific anti-annexin A1 antibody, but not by anti-Ig antibody. Anti-annexin A1 antibody also blocked the inhibitory and stimulatory effects of As 3+ or Cr 6+ towards DNA unwinding and annealing activities of DNA synthesomes. Based on these observations, it can be concluded that the effects of heavy metals on DNA annealing and unwinding activities are mediated, at least in substantial part, through actions of the mono-ubiquitinated annexin A1 homodimer.

Integrin α5 expression induced by epidermal growth factor affect proliferation and migration of human retinal pigment epithelium cells

Objective To observe the effect of epidermal growth factor (EGF) on integrin α5 expression and its influence on human retinal pigment epithelium (RPE) cells. Methods Human RPE cells were treated in vitro with 0.1, 1.0, 10.0, 20.0 and 100.0 ng/ml of EGF, the mRNA and protein of integrin α5 was measured by reverse transcription polymerase chain reaction(RT-PCR)and flow cytometry. Human RPE cells were cultured under 4 conditions including DMEM/F12, DMEM/F12 + 10 ng/ml EGF, DMEM/F12 + 10 ng/ml EGF+ rabbit anti-human integrin α5 antibody (1: 100), DMEM/F12 + 10 ng/ml EGF+ rabbit anti-human vimentin antibody (1: 100), and their proliferation and migration were measured by methyl-thiazole tetrazolium(MTT)and Boyden chamber. Results The integrin α5 mRNA level of human RPE cells was not changed after 12 hours of EGF stimulation (F=0.618, P = 0. 687), however it was induced in a dose-dependent manner after 24 and 48 hours of EGF stimulation (F=465. 303, 212. 340; P= 0. 000, 0. 000). The protein level of integrinα5 was higher in 10 ng/ml EGF stimulation compared with the control group and 0. 1 ng/ml group (P<0. 01). MTT and Boyden chamber showed that the integrin α5 expression increased the proliferation and migration of human RPE cells. Conclusion EGF can induce integrin α5 expression, thus increase the proliferation and migration of human RPE cells. Key words: Epidermal growth factor; Pigment epithelium of eye; Integrin alphα5; Cell proliferation; Cell movement

Abstract LB-373: Elucidation of the mechanisms involved in IL-6 induced cell transformation in prostate cancer

Abstract Background: Prostate cancer is the most common non-cutaneous malignant neoplasm in men in Western countries. Several studies have shown a significant correlation between the formation of a tumor and chronic prostatic inflammation, generally triggered by infectious agents or exposure to environmental factors (smoking, alcohol, burned meat, etc.), and/or various combination thereof. The incidence of chronic inflammation of the prostate is associated with the presence of inflammatory cells involved in the secretion of cytokines, which promote the migration and proliferation of cells implicated in the immune response. Our main objective of this project is to elucidate the mechanism involved in the promotion of prostate cancer mediated by IL-6 using different approaches such as Western Blots, qPCR, luciferase based reporter assays and immunofluorescence. Results: The effects of cytokines on prostate epithelial cells are not well understood, however some evidence showed that its presence may play a role in the initiation of prostate cancer of patients with chronic prostate inflammation. Preliminary data generated in our laboratory have shown that when the prostate cell lines p69 and BPH-1 (non- tumorigenic prostate cell lines) are treated with the cytokines IL-6 and TNF-α, their proliferation rate is higher when compared with non-treated cells. In addition we have observed that P69 cells cultured in presence of IL-6 suffer significant changes in their morphology as well as changes in the distribution of E-cadherin in the cell membrane. The activation of the MAPK pathway also was detected after the cytokine treatment but when we used the MAPK inhibitors (LY294002 and PD98059) the expression levels of E-cadherin and Vimentin were closed to those observed on non-treated cells. Previous reports have shown that the Snail transcription factor is involved in the regulation of the expression of E-cadherin and Vimentin. Interestingly, we have detected this transcription higher levels of this transcription factor (mRNA as well as protein) after stimulation of p69 and BPH-1 cells with IL-6. The presence of this transcription factor was not noticed in non-treated cells. Finally, we have performed a luciferase based assay to establish the occurrence of cell transformation and we have found a higher activation of the luciferase reporter in these cells when they were treated with IL-6. Interestingly, when we block the activity of the IGF-IR using the antibody A12, we have a reduction of the EMT phenotype observed in cells that were treated only with IL-6. Low effects of the antibody were detected in non-treated cells. It is possible that such modifications might help to facilitate epithelial cell invasion into the stromal and vasculature compartments. In summary, we are showing novel mechanisms by which IL-6 may play a key role in the initiation of prostate cancer in patients with chronic prostate inflammation. To have a better understanding of this event may help in the early treatment of this type of cancer. Further “in vivo” experiments will be performed to establish the importance of IL-6 in the development of tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-373.

Antitumor effects of EMAP II against pancreatic cancer through inhibition of fibronectin-dependent proliferation

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to conventional chemotherapy. The presence of both cellular and stromal fibronectin (FN) and its interaction with integrins is necessary for PDAC progression. We tested the efficacy of endothelial monocyte-activating polypeptide II (EMAP II) to inhibit PDAC progression and its ability to interfere with FN-integrin angiogenesis signaling. In heterotopic PDAC tumors EMAP II caused a significant reduction (>65%) in tumor growth, accompanied by a >50% and 44% decrease in microvessel density and proliferative activity, respectively. EMAP II therapy caused a 62% and 56% reduction in host and tumor cell FN expression. Cultured PDAC cells expressed aVb3 and a5b1 integrins. In vitro EMAP II had limited antiproliferative effects on ASPC-1, but a pronounced antiproliferative effect on endothellial cells. 3D FN matrices increased ASPC-1 cell proliferation by > 50%, and this induction was significantly blocked by a3, a5, a6 and aV integrin funtional blocking antibodies, while a1, a2 and a4 antibodies had no effect. EMAP II also inhibited 3D FN-matrix induced ASPC-1 proliferation by >43% at 20 mM. These findings suggest that EMAP II demonstrates significant antitumor activity against PDAC cells, and that this effect may be in part mediated through targeted interference with stromal FN-integrin dependent PDAC cell proliferation.

Abstract 2329: Alterations in laminin expression modulate prostate cancer cell behavior through changes in integrin and insulin-like growth factor receptor actions

Abstract Introduction: During development of primary prostate carcinomas alterations to the extracellular matrix occur, including a loss of laminin 332 (LM332) and one of its cell surface receptors, β4 integrin. There are few published studies on what the laminin composition of metastases may be or how alterations in laminin expression affect prostate cancer cell behavior. Expression of the insulin-like growth factor receptor (IGF-IR) also is altered in prostate cancer progression and some studies suggest cross-talk between integrins and IGF-IR. Purpose: To determine if laminin and integrin expression is altered in prostate cancer metastases and how these alterations affect prostate cancer cell behavior in light of the interactions between integrins and IGF-IR. Methods: We performed immunofluorescent (IF) staining on tissue samples of primary PCa and metastases. We also used various prostate cancer cell lines, including M12 cells transfected with the LMα4, β2, or α4β2 chains, to perform adhesion, migration, and proliferation assays in the presence of inhibitory integrin and IGF-IR antibodies. Results: Our IF staining demonstrated that in addition to a loss in LM332 expression, the LMα4 and β2 chains were absent from the prostate basal membrane in primary prostate tumors. Expression of the LMα5 and β1γ1 chains was retained in primary tumors as well as in lymph node metastases. We saw a loss of β4 integrin in primary tumors as well as in metastatic cancer, but α6 and β1 integrins were retained; in lymph node mets, α2 and α3 integrins also were expressed. For M12 laminin cells, enhanced adhesion to both collagen and fibronectin was further increased by treatment with the inhibitory IGF-IR antibody A12. An inhibitory β1 integrin antibody, but not individual alpha integrin antibodies, blocked this enhancement. Inhibitory β1 as well as alpha integrin antibodies blocked adhesion of the empty-vector control cells. Similar results were obtained for cell migration studies using a scratch-assay. Staining of cells following exposure to A12 showed there was an increase in LM332 and LMβ1γ1 expression compared to untreated cells, which may account for the increased adhesion and migration following A12 treatment. We then examined laminin expression of various prostate cancer cell lines: LNCaP, LNCaP C4-2, PC3, and DU145. We found no expression of LM332 in any of these lines. Cytoplasmic expression of the LMβ1γ1 and LMβ2 (LNCaP lines) chains was detected, but deposition of these laminins was not present. These lines demonstrated increased adhesion to laminin-rich ECM compared with adhesion to fibronectin or collagen. And these lines showed enhanced proliferation when grown on laminin-rich ECM from the M12 laminin cells. Summary: Alterations in laminins affect the adhesion, migration, and proliferation of prostate cancer cells, in part, through integrin and IGF-IR actions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2329.

Abstract LB-233: IGF activation in a molecular subclass of hepatocellular carcinoma and preclinical efficacy of IGF-1R blockage

Abstract Background: IGF signaling contributes to proliferation and cell survival through PI3K/AKT and MAPK pathways. It has a relevant role in a variety of human malignancies. Aims: (1) To analyze the role of IGF signaling in early hepatocellular carcinoma (HCC; BCLC class 0 or A) and (2) To asses the antitumoral activity of IGF pathway targeted therapies. Methods: An integrative molecular dissection of the axis was performed in a cohort of 104 HCCs, including gene (quantitative PCR and Affymetrix U133 2.0® microarray) and miRNA expression (ligation-mediated amplification method), structural aberrations (Affymetrix STY Mapping Array®) and protein activation (immunohistochemistry). The therapeutic potential of a selective IGF-1R inhibitor, the monoclonal antibody A12, was assessed in vitro through viability and proliferation assays. A xenograft model of HCC was used to evaluate A12 activity in vivo. Results: Activation of the IGF axis was observed in 21% of early HCCs. Several molecular aberrations were identified, such as overexpression of IGF2 -resulting from reactivation of fetal promoters-, IGFBP3 downregulation and allelic losses of IGF2R (25% of cases). A gene signature defining IGF-1R activation was developed. Overall, activation of IGF signaling in HCC was significantly associated with IGF2 overexpression (p=0.03), mTOR signaling (p=0.035) and was enriched in a molecularly distinct HCC subclass defined through unsupervised hierarchical clustering of the same cohort of samples (Proliferation subclass) (p=0.001). We also found an inverse correlation between IGF activation and miR-100/miR-216 levels (FDR&amp;lt;0.05). In vitro studies showed that A12-induced abrogation of IGF-1R activation and downstream signaling significantly decreased cell viability and proliferation. In vivo, A12 treated mice exhibited delayed tumor growth and prolonged survival in comparison to control mice, a reduction in proliferation rates and induction of apoptosis was observed in treated mice tumoral samples. Conclusions: Integrative genomic analysis showed enrichment of activation of IGF signaling in the Proliferation subclass of HCC. Effective blockage of IGF signaling with A12 provides the rationale for testing this therapy in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-233.

Abstract 5121: Laminin 511 on human gastric carcinoma cells is a ligand for hepatic asialoglycoprotein receptor involved in liver metastasis

Abstract – Background and Aim – Gastric cancer often metastasizes to the liver and galactose-type lectins were previously proposed to play an important role in hepatic metastases because intravenous administration of galactose at the time of surgical removal of gastric cancer was reported to extend the survival of the patients. Asialoglycoprotein receptor (Asgpr) is a galactose-type calcium-type lectin and a primary candidate molecule involved in this process. Variant cell lines of human gastric carcinoma, AZ521 cells, were obtained by sorting for high and low levels of cell surface bindings of recombinant Asgpr and designated as AZhigh and AZlow cells, respectively. The present report is to assess the metastatic potential of these cell lines and determine the ligand for Asgpr on the surfaces of these gastric carcinoma cells. – Materials and Methods – AZhigh and AZlow cells were injected into nude mice via portal vein and metastatic growth in the liver were evaluated 14 days later. AZhigh and AZlow cells were biotinylated at the cell surfaces and the lysates were prepared. Components bound to immobilized Asgpr were collected and analyzed by SDS-PAGE then differentially expressed components were subjected to MALDI-TOF MS analysis after trypsin digestion. Specific monoclonal antibodies specific for particular components such as laminin α5 chain were used to confirm the identified proteins from the carcinoma cells. – Results and Discussion – Metastatic growth in the liver was more prominent with AZhigh cells than AZlow cells. AZhigh cells adhered to immobilized Asgpr more rapidly than AZlow cells. From components eluted from immobilized Asgpr, laminin α5, β1, and γ1 chains were identified. By flow cytometric analysis, anti-human laminin α5 antibody bound to AZhigh cells but the binding to AZlow were negligible. Similar difference was observed by immunoprecipitation. Laminin α5 chain was also in the culture supernatants of AZhigh cells, but not those of AZlow cells. These results suggest that laminin 511 differentially expressed between AZhigh and AZlow cells and acted as the ligands for Asgpr. Further study focusing on laminin 511 will be needed for elucidating the mechanism of hepatic metastases of gastric cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5121.

High-Resolution X-Ray Structure and Functional Analysis of the Murine Norovirus 1 Capsid Protein Protruding Domain

Murine noroviruses (MNV) are closely related to the human noroviruses (HuNoV), which cause the majority of nonbacterial gastroenteritis. Unlike HuNoV, MNV grow in culture and in a small-animal model that represents a tractable model to study norovirus biology. To begin a detailed investigation of molecular events that occur during norovirus binding to cells, the crystallographic structure of the murine norovirus 1 (MNV-1) capsid protein protruding (P) domain has been determined. Crystallization of the bacterially expressed protein yielded two different crystal forms (Protein Data Bank identifiers [PDB ID], 3LQ6 and 3LQE). Comparison of the structures indicated a large degree of structural mobility in loops on the surface of the P2 subdomain. Specifically, the A'-B' and E'-F' loops were found in open and closed conformations. These regions of high mobility include the known escape mutation site for the neutralizing antibody A6.2 and an attenuation mutation site, which arose after serial passaging in culture and led to a loss in lethality in STAT1(-/-) mice, respectively. Modeling of a Fab fragment and crystal structures of the P dimer into the cryoelectron microscopy three-dimensional (3D) image reconstruction of the A6.2/MNV-1 complex indicated that the closed conformation is most likely bound to the Fab fragment and that the antibody contact is localized to the A'-B' and E'-F' loops. Therefore, we hypothesize that these loop regions and the flexibility of the P domains play important roles during MNV-1 binding to the cell surface.

IGF activation in a molecular subclass of hepatocellular carcinoma and pre-clinical efficacy of IGF-1R blockage

IGF signaling has a relevant role in a variety of human malignancies. We analyzed the underlying molecular mechanisms of IGF signaling activation in early hepatocellular carcinoma (HCC; BCLC class 0 or A) and assessed novel targeted therapies blocking this pathway. An integrative molecular dissection of the axis was conducted in a cohort of 104 HCCs analyzing gene and miRNA expression, structural aberrations, and protein activation. The therapeutic potential of a selective IGF-1R inhibitor, the monoclonal antibody A12, was assessed in vitro and in a xenograft model of HCC. Activation of the IGF axis was observed in 21% of early HCCs. Several molecular aberrations were identified, such as overexpression of IGF2 -resulting from reactivation of fetal promoters P3 and P4-, IGFBP3 downregulation and allelic losses of IGF2R (25% of cases). A gene signature defining IGF-1R activation was developed. Overall, activation of IGF signaling in HCC was significantly associated with mTOR signaling (p=0.035) and was clearly enriched in the Proliferation subclass of the molecular classification of HCC (p=0.001). We also found an inverse correlation between IGF activation and miR-100/miR-216 levels (FDR<0.05). In vitro studies showed that A12-induced abrogation of IGF-1R activation and downstream signaling significantly decreased cell viability and proliferation. In vivo, A12 delayed tumor growth and prolonged survival, reducing proliferation rates and inducing apoptosis. Integrative genomic analysis showed enrichment of activation of IGF signaling in the Proliferation subclass of HCC. Effective blockage of IGF signaling with A12 provides the rationale for testing this therapy in clinical trials.

Differential Sensitivity of A2A and Especially D2 Receptor Trafficking to Cocaine Compared with Lipid Rafts in Cotransfected CHO Cell Lines. Novel Actions of Cocaine Independent of the DA Transporter

The effects of low and high concentrations of cocaine have been studied in vitro on the trafficking of plasma membrane A(2A) and D(2) immunoreactivities in previously characterized A(2A)-D(2) CHO cell lines. Receptor double immunofluorescence staining was performed with D(2) and A(2A) antibodies, planar lipid rafts immunolabeling with biotinylated cholera toxin subunit B and membrane invaginations with an anti-caveolin-1 antibody. A computer-assisted image analysis demonstrated a substantial and highly significant rise of membrane-associated D(2) immunoreactivity (IR) after 8 h of exposure to a low concentration of cocaine (150 nM). At this low concentration of cocaine, there was also an increase of membrane associated A(2A) immunoreactivity but smaller and less significant. However, this increase became considerably larger and highly significant at 150 microM at which concentration the rise of D(2) immunoreactivity had begun to disappear. It may be suggested that an allosteric action of cocaine at 150 nM on the D(2) receptors may primarily increase the insertion of D(2) monomers, homomers and also of a subpopulation of A(2A)-D(2) heteromers from the cytoplasm into the plasma membrane due to the conformational change induced by cocaine in the D(2) receptor. The planar lipid rafts and the caveolae are only affected by the higher concentrations of cocaine. It is proposed that changes in D(2) and A(2A)-D(2) trafficking induced by allosteric actions of cocaine at D(2) receptors may contribute to the alterations of D(2) signaling found in cocaine abusers.

The Ubiquitin Ligase Nedd4-1 Is Required for Heart Development and Is a Suppressor of Thrombospondin-1

Nedd4 (Nedd4-1) is a Hect domain E3 ubiquitin ligase that also contains a C2 domain and three WW domains. Despite numerous in vitro studies, its biological function in vivo is not well understood. Here we show that disruption of Nedd4-1 in mice (leaving Nedd4-2 intact) caused embryonic lethality at mid gestation, with pronounced heart defects (double-outlet right ventricle and atrioventricular cushion defects) and vasculature abnormalities. Quantitative mass spectrometry and immunoblot analyses of lysates from the wild type and knock-out mouse embryonic fibroblasts to identify Nedd4-1 in vivo targets revealed dramatically increased amounts of thrombospondin-1 (Tsp-1) in the knock-out mouse embryonic fibroblasts and embryos. Tsp-1 is an inhibitor of angiogenesis, and its elevated level was mediated primarily by enhanced transcription. Interestingly, the administration of aspirin (an inhibitor of Tsp-1) to the pregnant heterozygote mothers led to a reduction in Tsp-1 levels and a substantial rescue of the embryonic lethality. These results suggest that Nedd4-1 is a suppressor of Tsp1 and that increased levels of Tsp-1 in the Nedd4-1 knock-out mice may have contributed to the developmental defect observed in the embryos.

Proteomics Analysis of A33 Immunoaffinity-purified Exosomes Released from the Human Colon Tumor Cell Line LIM1215 Reveals a Tissue-specific Protein Signature

Exosomes are 40-100-nm-diameter nanovesicles of endocytic origin that are released from diverse cell types. To better understand the biological role of exosomes and to avoid confounding data arising from proteinaceous contaminants, it is important to work with highly purified material. Here, we describe an immunoaffinity capture method using the colon epithelial cell-specific A33 antibody to purify colorectal cancer cell (LIM1215)-derived exosomes. LC-MS/MS revealed 394 unique exosomal proteins of which 112 proteins (28%) contained signal peptides and a significant enrichment of proteins containing coiled coil, RAS, and MIRO domains. A comparative protein profiling analysis of LIM1215-, murine mast cell-, and human urine-derived exosomes revealed a subset of proteins common to all exosomes such as endosomal sorting complex required for transport (ESCRT) proteins, tetraspanins, signaling, trafficking, and cytoskeletal proteins. A conspicuous finding of this comparative analysis was the presence of host cell-specific (LIM1215 exosome) proteins such as A33, cadherin-17, carcinoembryonic antigen, epithelial cell surface antigen (EpCAM), proliferating cell nuclear antigen, epidermal growth factor receptor, mucin 13, misshapen-like kinase 1, keratin 18, mitogen-activated protein kinase 4, claudins (1, 3, and 7), centrosomal protein 55 kDa, and ephrin-B1 and -B2. Furthermore, we report the presence of the enzyme phospholipid scramblase implicated in transbilayer lipid distribution membrane remodeling. The LIM1215-specific exosomal proteins identified in this study may provide insights into colon cancer biology and potential diagnostic biomarkers.

Annexin A2 Is a Molecular Target for TM601, a Peptide with Tumor-targeting and Anti-angiogenic Effects

TM601 is a synthetic form of chlorotoxin, a 36-amino acid peptide derived from the venom of the Israeli scorpion, Leirius quinquestriatus, initially found to specifically bind and inhibit the migration of glioma cells in culture. Subsequent studies demonstrated specific in vitro binding to additional tumor cell lines. Recently, we demonstrated that proliferating human vascular endothelial cells are the only normal cell line tested that exhibits specific binding to TM601. Here, we identify annexin A2 as a novel binding partner for TM601 in multiple human tumor cell lines and human umbilical vein endothelial cell (HUVEC). We demonstrate that the surface binding of TM601 to the pancreatic tumor cell line Panc-1 is dependent on the expression of annexin A2. Identification of annexin A2 as a binding partner for TM601 is also consistent with the anti-angiogenic effects of TM601. Annexin A2 functions in angiogenesis by binding to tissue plasminogen activator and regulating plasminogen activation on vascular endothelial cells. We demonstrate that in HUVECs, TM601 inhibits both vascular endothelial growth factor- and basic fibroblast growth factor-induced tissue plasminogen activator activation, which is required for activation of plasminogen to plasmin. Consistent with inhibition of cell surface protease activity, TM601 also inhibits platelet-derived growth factor-C induced trans-well migration of both HUVEC and U373-MG glioma cells.

Antibodies against Crandell Rees Feline Kidney (CRFK) Cell Line Antigens, α-Enolase, and Annexin A2 in Vaccinated and CRFK Hyperinoculated Cats

Cats inoculated with feline herpesvirus 1, calicivirus, and panleukopenia (FVRCP) vaccines grown on the Crandell Rees feline kidney (CRFK) cell line have been shown to develop anti-CRFK antibodies. The identities of common CRFK antigens are unknown. Cats inoculated with CRFK lysates and FVRCP vaccines will develop autoantibodies measurable by Western blot immunoassay. Antigens associated with these antibodies can be isolated for further study. One CRFK hyperinoculated rabbit, 44 age-matched unvaccinated kittens purchased from a commercial vendor. Commonly recognized CRFK antigens were identified by comparison of Western blot immunoassays using sera from a hyperinoculated rabbit and kittens inoculated with CRFK lysate or 1 of 4 commercially available FVRCP vaccines. Antigens were purified from CRFK lysates and sequenced. Antigen recognition was confirmed by Western blot immunoassay and indirect ELISA for 2 proteins using sera from CRFK and FVRCP inoculated kittens. CRFK antigens 47, 40, and 38 kD in size were identified. Protein isolation and sequencing identified 3 CRFK proteins as alpha-enolase, annexin A2, and macrophage capping protein (MCP). Sera from FVRCP and CRFK inoculated cats were confirmed to recognize annexin A2 and alpha-enolase by Western blot immunoassay and indirect ELISA. This study validated the use of Western blot immunoassay for detection of antibodies against CRFK proteins and identified 3 CRFK antigens. In humans, alpha-enolase antibodies are nephritogenic; alpha-enolase and annexin A2 antibodies have been associated with autoimmune diseases. Further research will be necessary to determine the clinical relevance of these findings.

Enhanced Memory Responses to Seasonal H1N1 Influenza Vaccination of the Skin with the Use of Vaccine‐Coated Microneedles

Morbidity and mortality due to influenza could be reduced by improved vaccination. To develop a novel skin delivery method that is simple and allows for easy self-administration, we prepared microneedle patches with stabilized influenza vaccine and investigated their protective immune responses. Mice vaccinated with a single microneedle dose of trehalose-stabilized influenza vaccine developed strong antibody responses that were long-lived. Compared with traditional intramuscular vaccination, stabilized microneedle vaccination was superior in inducing protective immunity, as was evidenced by efficient clearance of virus from the lung and enhanced humoral and antibody-secreting cell immune responses after 100% survival from lethal challenge. Vaccine stabilization was found to be important, because mice vaccinated with an unstabilized microneedle vaccine elicited a weaker immunoglobulin G 2a antibody response, compared with the stabilized microneedle vaccine, and were only partially protected against viral challenge. Improved trafficking of dendritic cells to regional lymph nodes as a result of microneedle delivery to the skin might play a role in contributing to improved protective immunity. These findings suggest that vaccination of the skin using a microneedle patch can improve protective efficacy and induce long-term sustained immunogenicity and may also provide a simple method of administration to improve influenza vaccination coverage.

Annexin A2 on lung epithelial cell surface is recognized by severe acute respiratory syndrome-associated coronavirus spike domain 2 antibodies

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection causes lung failure characterized by atypical pneumonia. We previously showed that antibodies against SARS-CoV spike domain 2 (S2) in the patient sera can cross-react with human lung epithelial cells; however, the autoantigen is not yet identified. In this study, we performed proteomic studies and identified several candidate autoantigens recognized by SARS patient sera in human lung type II epithelial cell A549. Among the candidate proteins, annexin A2, which was identified by mass spectrometry analysis and had the highest score by Mascot data search, was further characterized and investigated for its role as an autoantigen. By confocal microscopic observation, SARS patient sera and anti-S2 antibodies were co-localized on A549 cells and both of them were co-localized with anti-annexin A2 antibodies. Anti-annexin A2 antibodies bound to purified S2 proteins, and anti-S2 bound to immunoprecipitated annexin A2 from A549 cell lysate in a dose-dependent manner. Furthermore, an increased surface expression and raft-structure distribution of annexin A2 was present in A549 cells after stimulation with SARS-induced cytokines interleukin-6 and interferon-γ. Cytokine stimulation increased the binding capability of anti-S2 antibodies to human lung epithelial cells. Together, the upregulated expression of annexin A2 by SARS-associated cytokines and the cross-reactivity of anti-SARS-CoV S2 antibodies to annexin A2 may have implications in SARS disease pathogenesis.

Transforming Growth Factor-β–Stimulated Clone-22 Is an Androgen-Regulated Gene That Enhances Apoptosis in Prostate Cancer following Insulin-Like Growth Factor-I Receptor Inhibition

PURPOSE: Inhibition of insulin-like growth factor (IGF) signaling using the human IGF-I receptor monoclonal antibody A12 is most effective at inducing apoptosis in prostate cancer xenografts in the presence of androgen. We undertook this study to determine mechanisms for increased apoptosis by A12 in the presence of androgens. Experimental Methods: The castrate-resistant human xenograft LuCaP 35 V was implanted into intact or castrate severe combined immunodeficient mice and treated with A12 weekly. After 6 weeks of tumor growth, animals were sacrificed and tumors were removed and analyzed for cell cycle distribution/apoptosis and cDNA arrays were done. RESULTS: In castrate mice, the tumors were delayed in G(2) with no apoptosis; in contrast, tumors from intact mice underwent apoptosis with either G(1) or G(2) delay. Transforming growth factor-beta-stimulated clone-22 (TSC-22) was significantly elevated in tumors from the intact mice compared with castrate mice, especially in those tumors with the highest levels of apoptosis. To further determine the function of TSC-22, we transfected various human prostate cancer cell lines with a plasmid expressing TSC-22. Cell lines overexpressing TSC-22 showed an increase in apoptosis and a delay in G(1). When these cell lines were placed subcutaneously in athymic nude mice, a decreased number of animals formed tumors and the rate of tumor growth was decreased compared with control tumors. CONCLUSIONS: These data indicate that IGF-I receptor inhibition in the presence of androgen has an enhanced effect on decreasing tumor growth, in part, through increased expression of the tumor suppressor gene TSC-22. (Clin Cancer Res 2009;15(24):7634-41).

Phenolic Compounds Prevent Alzheimer’s Pathology through Different Effects on the Amyloid-β Aggregation Pathway

Inhibition of amyloid-beta (Abeta) aggregation is an attractive therapeutic strategy for Alzheimer's disease (AD). Certain phenolic compounds have been reported to have anti-Abeta aggregation effects in vitro. This study systematically investigated the effects of phenolic compounds on AD model transgenic mice (Tg2576). Mice were fed five phenolic compounds (curcumin, ferulic acid, myricetin, nordihydroguaiaretic acid (NDGA), and rosmarinic acid (RA)) for 10 months from the age of 5 months. Immunohistochemically, in both the NDGA- and RA-treated groups, Abeta deposition was significantly decreased in the brain (P < 0.05). In the RA-treated group, the level of Tris-buffered saline (TBS)-soluble Abeta monomers was increased (P < 0.01), whereas that of oligomers, as probed with the A11 antibody (A11-positive oligomers), was decreased (P < 0.001). However, in the NDGA-treated group, the abundance of A11-positive oligomers was increased (P < 0.05) without any change in the levels of TBS-soluble or TBS-insoluble Abeta. In the curcumin- and myricetin-treated groups, changes in the Abeta profile were similar to those in the RA-treated group, but Abeta plaque deposition was not significantly decreased. In the ferulic acid-treated group, there was no significant difference in the Abeta profile. These results showed that oral administration of phenolic compounds prevented the development of AD pathology by affecting different Abeta aggregation pathways in vivo. Clinical trials with these compounds are necessary to confirm the anti-AD effects and safety in humans.

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

The expression of acetylcholinesterase (AChE) in skeletal muscle is regulated by muscle activity; however, the underlying molecular mechanisms are incompletely understood. We show here that the expression of the synaptic collagen-tailed AChE form (ColQ-AChE) in quail muscle cultures can be regulated by muscle activity post-translationally. Inhibition of thiol oxidoreductase activity decreases expression of all active AChE forms. Likewise, primary quail myotubes transfected with protein disulfide isomerase (PDI) short hairpin RNAs showed a significant decrease of both the intracellular pool of all collagen-tailed AChE forms and cell surface AChE clusters. Conversely, overexpression of PDI, endoplasmic reticulum protein 72, or calnexin in muscle cells enhanced expression of all collagen-tailed AChE forms. Overexpression of PDI had the most dramatic effect with a 100% increase in the intracellular ColQ-AChE pool and cell surface enzyme activity. Moreover, the levels of PDI are regulated by muscle activity and correlate with the levels of ColQ-AChE and AChE tetramers. Finally, we demonstrate that PDI interacts directly with AChE intracellularly. These results show that collagen-tailed AChE form levels induced by muscle activity can be regulated by molecular chaperones and suggest that newly synthesized exportable proteins may compete for chaperone assistance during the folding process.

HER2-testing in 538 consecutive breast cancer cases using FISH and immunohistochemistry

HER2 is amplified and overexpressed in up to 20% of all breast carcinomas, and accurate assessment of the HER2-status is essential for the appropriate use of anti-HER2 therapies, including trastuzumab. Here, we have evaluated the accuracy of two commercially available and commonly used HER2 antibodies (antibody A0485 and HercepTest), followed by FISH-test, in 538 consecutive breast cancers, to identify a reliable procedure for the determination of HER2-status. Immunohistochemical (IHC) analysis revealed that 282 cases (52.2%) were positive (2+/3+) for antibody A0485, while only 179 cases (33.3%) were positive (2+/3+) for HercepTest (81.2% concordance between A0485 and HercepTest). FISH-analysis of the 103 IHC discordant cases revealed 89.3% concordance with HercepTest. The results of the present study, which is the largest in Scandinavia, revealed a significantly higher concordance rate between HercepTest and HER2 copy number status than between antibody A0485 and HER2 copy number status. Eleven cases (11% of 103 discordant cases) with HER2-amplification were positive for antibody A0485 and false negative for HercepTest. Our findings indicate that HercepTest is a reliable and accurate IHC-assay and that in the absence of clinical outcome data for IHC-positive/FISH-negative and IHC-negative/FISH-positive patients, FISH-analysis should be performed on all IHC 2+ and 3+ cases.