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Pancreatic cancer is among the deadliest malignancies; however, the genetic events that lead to pancreatic carcinogenesis in adults remain unclear. In vivo models in which these genetic alterations occur in adult animals may more accurately reflect the features of human cancer. In this study, we demonstrate that inactivation of Cdkn2b (p15ink4b) is necessary for induction of pancreatic cancer by oncogenic KRASG12D expression and inactivation of Tp53 and Cdkn2a in adult mouse pancreatic ductal cells (P60 or older). KRASG12D overexpression in these cells activated transforming growth factor-β signaling and expression of CDKN2B, which, along with CDKN2A, led to cellular senescence and protected cells from KRAS-mediated transformation via inhibition of retinoblastoma phosphorylation. These results show a critical role of CDKN2B inactivation in pancreatic carcinogenesis, and provide a useful adult animal model by genetic engineering via lentiviral delivery.

The persistence of cholesterol-engorged macrophages (foam cells) in the artery wall fuels the development of atherosclerosis. However, the mechanism that regulates the formation of macrophage foam cells and impedes their emigration out of inflamed plaques is still elusive. Here, we report that adhesion receptor CD146 controls the formation of macrophage foam cells and their retention within the plaque during atherosclerosis exacerbation. CD146 is expressed on the macrophages in human and mouse atheroma and can be upregulated by oxidized low-density lipoprotein (oxLDL). CD146 triggers macrophage activation by driving the internalization of scavenger receptor CD36 during lipid uptake. In response to oxLDL, macrophages show reduced migratory capacity toward chemokines CCL19 and CCL21; this capacity can be restored by blocking CD146. Genetic deletion of macrophagic CD146 or targeting of CD146 with an antibody result in much less complex plaques in high-fat diet-fed ApoE−/− mice by causing lipid-loaded macrophages to leave plaques. Collectively, our findings identify CD146 as a novel retention signal that traps macrophages within the artery wall, and a promising therapeutic target in atherosclerosis treatment.

Background Busulfan/cyclophosphamide (Bu/Cy) is commonly used as a standard conditioning regimen without total body irradiation for patients with hematological myeloid malignancies undergoing hematopoietic stem cell transplantation (HSCT). Objective To develop a new myeloablative conditioning regimen incorporating fludarabine (Flu) and cytarabine (Ara-c). Setting A tertiary blood disease hospital in Tianjin, China. Methods A Bu/Cy preparative regimen was used, modified by Flu 90 mg/m2 and Ara-c 6 g/m2 in 57 unselected patients (median age 37 years) with hematological myeloid malignancies. The patients were to receive allogeneic hematopoietic stem cell transplantation (allo-HSCT). Thirteen patients had high-risk leukemia, fifty patients had HLA matched sibling donors while seven patients had HLA mismatched sibling donors. Cy was given 50 mg/kg/day for 2 days while Bu was given 3.2 mg/kg/day intravenously for 3 days. Main outcome measure Post-transplant donor chimerism, relapse tendency and minimal residual disease. Results Extramedullar toxicity was relatively limited; the incidence of treatment-related mortality (TRM) within 100 days was 3.5 %. The incidence of grade II–IV, grade III–IV acute graft versus host disease (GVHD) and chronic GVHD of the evaluable patients were 21.1, 8.8 and 36.4 %, respectively. With a median follow up of 59 (13–96.5) months, TRM and relapse rate (RR) at eight years were 24.1 ± 5.8 and 14.7 ± 4.8 %, respectively. Disease free survival at eight years was 67.9 ± 6.2 % for the entire group, 60.0 ± 8.9 % for patients with AML, 77.3 ± 8.9 % for patients with CML, 70.0 ± 6.5 and 42.9 ± 18.7 % or matched sibling and mismatched sibling HSCT respectively. Conclusion The new regimen was associated with a low relapse rate, low incidence and severity of graft versus host disease and satisfactory survival for patients with myeloid malignancies.

FXYD6, FXYD domain containing ion transport regulator 6, has been reported to affect the activity of Na+/K+-ATPase and be associated with mental diseases. Here, we demonstrate that FXYD6 is up-regulated in hepatocellular carcinoma (HCC) and enhances the migration and proliferation of HCC cells. Up-regulation of FXYD6 not only positively correlates with the increase of Na+/K+-ATPase but also coordinates with the activation of its downstream Src-ERK signaling pathway. More importantly, blocking FXYD6 by its functional antibody significantly inhibits the growth potential of the xenografted HCC tumors in mice, indicating that FXYD6 represents a potential therapeutic target toward HCC. Altogether, our results establish a critical role of FXYD6 in HCC progression and suggest that the therapy targeting FXYD6 can benefit the clinical treatment toward HCC patients. Electronic supplementary material The online version of this article (doi:10.1007/s13238-014-0045-0) contains supplementary material, which is available to authorized users.

CD146, an endothelial biomarker, has been shown to be aberrantly upregulated during pathological angiogenesis and functions as a coreceptor for vascular endothelial growth factor receptor 2 (VEGFR-2) to promote disease progression. However, the regulatory mechanisms of CD146 expression during angiogenesis remain unclear. Using a microRNA screening approach, we identified a novel negative regulator of angiogenesis, microRNA 329 (miR-329), that directly targeted CD146 and inhibited CD146-mediated angiogenesis in vitro and in vivo. Endogenous miR-329 expression was downregulated by VEGF and tumor necrosis factor alpha (TNF-α), resulting in the elevation of CD146 in endothelial cells. Upregulation of CD146 facilitated an endothelial response to VEGF-induced SRC kinase family (SKF)/p38 mitogen-activated protein kinase (MAPK)/NF-κB activation and consequently promoted endothelial cell migration and tube formation. Our animal experiments showed that treatment with miR-329 repressed excessive CD146 expression on blood vessels and significantly attenuated neovascularization in a mouse model of pathological angiogenesis. Our findings provide the first evidence that CD146 expression in angiogenesis is regulated by miR-329 and suggest that miR-329 could present a potential therapeutic tool for the treatment of angiogenic diseases.

The soluble form of CD146 has been reported to be present in various inflammatory diseases and displays pro-inflammatory properties. However, little is known about sCD146 in multiple sclerosis (MS). Here we show that sCD146 is significantly elevated in the cerebrospinal fluid of patients with active MS compared with that of inactive MS or patients with non-demyelinating diseases. Moreover, abnormally increased sCD146 in the CSF of active MS patients correlated with albumin quotient, MBP antibody and MOG antibody from both CSF and sera. Importantly, the level of CSF sCD146 is correlated with levels of inflammatory factors, such as TNFα, IFNγ, IL-2, and IL-17A in the CSF. We also found that CSF sCD146 might originate from membrane-bound CD146 on inflamed blood-brain barrier (BBB) endothelial cells. In addition, sCD146 promotes leukocyte transmigration in vitro, at least in part by stimulating the expression of ICAM-1 and VCAM-1 on endothelial cells. Our findings suggest that CSF levels of sCD146 may provide a potential marker for monitoring disease activity in MS patients.

Despite efforts, a decline in breastfeeding rates has been documented in China recently. This study explored the awareness of the World Health Organization (WHO) guidelines for breastfeeding and intention to breastfeed among first-time mothers and identified the gap between mothers' needs and perinatal care provision regarding breastfeeding promotion.In total, 653 women at 5-22 gestational weeks were recruited from four community health centers in Shanghai, China. They completed a self-administered questionnaire at recruitment. Two focus group discussions were held among third-trimester pregnant women who had received prenatal education. Twenty-four in-depth interviews were conducted among postpartum mothers.During early pregnancy, a substantial proportion of mothers were not aware of the nutritional value of breastmilk (40%) or the value of exclusive breastfeeding for 6 months (80%) or any breastfeeding for 24 months (98%). The awareness of the WHO guidelines for breastfeeding was associated with intention to breastfeed (adjusted odds ratio [OR] 2.67, 95% confidence interval [CI] 1.88, 3.78) or intention to breastfeed exclusively (adjusted OR 3.31, 95% CI 1.81, 6.06). In late pregnancy and postpartum, most mothers were still not fully aware of the breastfeeding recommendations and nutritional value of breastmilk. Limited communications with healthcare providers and lack of support for dealing with breastfeeding difficulties were reported.Low awareness of the WHO breastfeeding guidelines was found among first-time mothers in Shanghai. Awareness of breastfeeding guidelines was independently associated with mothers' intention to breastfeed and intention to breastfeed exclusively. The health benefits of breastfeeding and the recommended duration of breastfeeding should be emphasized in prenatal education programs.

A nanoparticle-based immunoassay was developed for the rapid and sensitive detection of avian influenza virus (AIV). In this method, AIV-specific pentabody (pVHH3B) was conjugated to magnetic nanoparticles (MNPs) and used to capture AIV. Gold nanoparticles (GNPs), labelled with the anti-AIV mouse monoclonal antibody 3C8, were used as a detector. In the presence of target samples, the pentabody pVHH3B enriched AIV on the MNPs. Thereafter, mAb 3C8-labelled GNPs (GNPs-mAb3C8) bound to MNPs via AIV and were separated using a magnetic field. GNPs in the complex catalyzed the oxidation of hydroquinone to quinone, and the OD value of quinone was measured. The developed assay displayed substantial signal change after incubation in an AIV sample in a concentration-dependent manner. The detection limit was 10 ng/ml, which is 10 times more sensitive than conventional double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). In conclusion, by combining MNPs and a novel pentabody pVHH3B, this study provided a sensitive influenza viral detection assay that has the potential to become a rapid, sensitive and inexpensive diagnostic tool for infectious diseases.

Background: Cell adhesion molecules have been implicated to be up-regulated in systemic vasculitis and could be potentially used as clinical diagnostic biomarkers. As a newly identified adhesion molecule, the expression level of CD146 in vasculitis has never been determined. Methods: Clinical samples were collected and levels of soluble CD146 were measured in sera using sandwich ELISA and neutrophil CD146 using FACS analysis in 59 patients with active systemic vasculitis and 34 healthy controls. Results: Expression of CD146 was significantly elevated in circulating neutrophils of patients with active small-vessel vasculitis (WG and MPA), but not in patients with large-vessel vasculitis (TA), compared with samples from healthy controls. Interestingly, elevated levels of neutrophil CD146 were dramatically decreased after treatment with immunosuppressive drugs. Moreover, a significantly increased level of soluble CD146 was found in sera from patients with MPA and vasculitis-induced renal failure. Conclusion: The clinical study indicated that CD146 expression was elevated in active vasculitis and correlated with its pathological progression and post-treatment remission.

We evaluated the inhibitory effect of 3,7-dinitrodibenzobromonium salts (cBr) on the proliferation of human chronic myelogenous leukemia K562 cell by trypan blue exclusion test and MTT colorimetric assay. The degree of DNA damage in K562 cells treated with cBr, was detected by isotopic tracer method (3H-TdR). The morphological changes of these K562 cells were examined by fluorescence and electron microscopy. Biochemical characteristics of K562 cells were detected by flow cytometry and 3H-thymidine incorporation assay. Findings indicated that cBr could significantly inhibit cell proliferation and result in DNA damage of K562 cells. cBr is a new type of immunostimulant and can induce cell apoptosis.

Our previous study showed that the adhesion molecule CD146 as a biomarker is over-expressed on activated endothelium during angiogenesis, which was induced by tumor conditional medium and inhibited by anti-CD146 monoclonal antibody (mAb AA98). However, the CD146 molecular organization on the cells is unknown. Here, using immunoprecipitation, we found that the dimerization of CD146 occurs in both normal and tumor cells. However, the dimer/monomer ratio was higher in tumor cells than in normal cells. Moreover, we found that CD146 dimerization was up-regulated by tumor conditional medium through the NF-kappa B pathway and down-regulated by mAb AA98. To further confirm that CD146 dimerization occurs in living cells, we used fluorescence resonance energy transfer (FRET) with melanoma Mel888 cells co-expressing CFP/YFP-tagged CD146 fusion proteins. By acceptor photobleaching, we observed a strong FRET signal produced by these two fluorescence-tagged proteins. The FRET efficiency reached 20.1%. Our data provide the first evidence that CD146 dimerization occurs in living cells and is regulated within the tumor microenvironment, implying that dimerization of CD146 may be associated with malignancy.

The monoclonal antibody T2-2 was originally raised against the colorectal carcinoma cell line LS174T and was found to bind to several other human carcinomas, including hepatoma and ovarian cancer. The goal of this study was to investigate the antitumor activity of mAb T2-2 in human tumor models and further characterize the antigen. mAb T2-2 inhibited the growth of human hepatocellular cell line SMMC 7721 in vivo and in vitro. Western blot analysis revealed that this mAb recognizes an unique Mr 45,000 band from tissue extracts of human hepatocellular carcinoma (HCC), which localizes to the cell periphery. In vitro cell assays indicate that T2-2 decreases cell adhesion to laminin, implying the functional role of T2-2 antigen in cell-matrix interaction and cell migration.

An ideal nanocarrier for efficient drug delivery must be able to target specific cells and carry high doses of therapeutic drugs and should also exhibit optimized physicochemical properties and biocompatibility. However, it is a tremendous challenge to engineer all of the above characteristics into a single carrier particle. Here, we show that natural H-ferritin (HFn) nanocages can carry high doses of doxorubicin (Dox) for tumor-specific targeting and killing without any targeting ligand functionalization or property modulation. Dox-loaded HFn (HFn-Dox) specifically bound and subsequently internalized into tumor cells via interaction with overexpressed transferrin receptor 1 and released Dox in the lysosomes. In vivo in the mouse, HFn-Dox exhibited more than 10-fold higher intratumoral drug concentration than free Dox and significantly inhibited tumor growth after a single-dose injection. Importantly, HFn-Dox displayed an excellent safety profile that significantly reduced healthy organ drug exposure and improved the maximum tolerated dose by fourfold compared with free Dox. Moreover, because the HFn nanocarrier has well-defined morphology and does not need any ligand modification or property modulation it can be easily produced with high purity and yield, which are requirements for drugs used in clinical trials. Thus, these unique properties make the HFn nanocage an ideal vehicle for efficient anticancer drug delivery.

Recently, enhanced CD146 expression was reported on endothelial cells in intestinal biopsies from patients with inflammatory bowel disease. However, the underlying mechanism remains unknown. Here, we found that overexpressed endothelial CD146 promoted the inflammatory responses in inflammatory bowel disease, which further potentiated the occurrence of colitis-associated colorectal carcinogenesis. Eliminating endothelial CD146 by conditional knockout significantly ameliorated the severity of inflammation in two different murine models of colitis, and decreased tumor incidence and tumor progression in a murine model of colitis-associated colorectal carcinogenesis. Mechanistic study showed that cytokine tumor necrosis factor-α (TNF-α) up-regulated the expression of endothelial CD146 through NF-κB transactivation. In turn, the enhanced endothelial CD146 expression promoted both angiogenesis and proinflammatory leukocyte extravasations, contributing to inflammation. Using an anti-CD146 antibody, AA98, alone or together with an anti–TNF-α antibody significantly attenuated colitis and prevented colitis-associated colorectal carcinogenesis in mice. Our study provides the first evidence that CD146 plays a dual role on endothelium, facilitating leukocyte extravasations and angiogenesis, thus promoting inflammation. This finding not only reveals the function and regulating mechanism of CD146 in inflammatory bowel disease, but also provides a promising therapeutic strategy for treating inflammatory bowel disease and preventing colitis-associated colorectal carcinogenesis.

The ability to selectively block the entry of leukocytes into the central nervous system (CNS) without compromising the immune system is an attractive therapeutic approach for treating multiple sclerosis (MS). Using endothelial CD146-deficienct mice as a MS model, we found that endothelial CD146 plays an active role in the CNS-directed extravasation of encephalitogenic T cells, including CD146(+) TH1 and TH17 lymphocytes. Moreover, treating both active and passive MS models with the anti-CD146 antibody AA98 significantly decreased the infiltrated lymphocytes in the CNS and decreased neuroinflammation. Interestingly, the ability of AA98 to inhibit the migration of CD146(+) lymphocytes was dependent on targeting endothelial CD146, but not lymphocytic CD146. These results suggest a key molecular target located on the blood-brain barrier endothelium that mediates the extravasation of inflammatory cells into the CNS. In addition, our data suggest that the AA98 is a promising candidate for treating MS and other CNS autoimmune diseases.

A novel ‘pipeline’ system for the preparation of therapeutic monoclonal antibodies (mAb) in a non-GMP compliant environment has been developed. We used sterile silica-gel pipes to connect individual process units, in order to form a fully-enclosed and seamlessly connected system. This ‘pipeline’ system was used to implement downstream preparation processes for a humanized anti-CD146 mAb, huAA98, which is a therapeutic mAb generated to inhibit cancer-related angiogenesis. The quality assessment of the huAA98 end-product indicated that endotoxin levels were 0.016 EU/ml, protein A levels were 1.08 ng/ml and host cell protein (HCP) was undetectable. Thus, all measures were below the clinical criteria set by the Chinese Pharmacopoeia (Edition 2010). Having passed our proof-of-concept test, this ‘pipeline’ system can be used as a universal platform for the preparation of mAbs suitable for pre-clinical studies, in a non-GMP compliant laboratory environment.

Cell adhesion molecules (CAMs) are crucial effectors for the development and maintenance of the nervous system. Mutations in human CAM genes are linked to brain disorders and psychological diseases, and CAM knockout mice always exhibit similar behavioral abnormalities. CD146 is a CAM of the immunoglobulin superfamily that interacts with Neurite Outgrowth Factor and involved in neurite extension in vitro. However, little is known about its in vivo function in the nervous system. In this study, we used a murine CD146 nervous system knockout (CD146(ns-ko)) model. We found that the brains of some CD146(ns-ko) mice were malformed with small olfactory bulbs. CD146(ns-ko) mice exhibited lower body weights and smaller food intake when compared with wild type littermates. Importantly, behavior tests revealed that CD146(ns-ko) mice exhibited significant decreased locomotor activity and impaired capacity for spatial learning and memory. Our results demonstrate that CD146 is important for mammalian nervous system development and proper behavior patterns.

Summary: The aim of this study is to raise tumor-suppressing monoclonal antibodies (mAbs) against colorectal carcinomas. Here, we generated a novel mAb 3P9, targeted a cancer-associated carbohydrate antigen sialyl-Tn (sTn), which showed significant inhibitory effect on proliferation and migration of sTn + cells and tumor growth by inducing apoptosis. We also demonstrated that mAb 3P9 showed higher sensitivity and specificity in immunohistochemistry assay on colonic adenocarcinoma than the broadly used commercial anti-sTn antibody B72.3. These results provide the first evidence that mAb 3P9 has potential applications, not only for diagnosis but also for antibody-based tumor therapy.

Rapid and sensitive detection methods are in urgent demand for the screening of extensively used organophosphorus pesticides and highly toxic nerve agents for their neurotoxicity. In this study, we developed a novel Fe(3)O(4) magnetic nanoparticle (MNP) peroxidase mimetic-based colorimetric method for the rapid detection of organophosphorus pesticides and nerve agents. The detection assay is composed of MNPs, acetylcholinesterase (AChE), and choline oxidase (CHO). The enzymes AChE and CHO catalyze the formation of H(2)O(2) in the presence of acetylcholine, which then activates MNPs to catalyze the oxidation of colorimetric substrates to produce a color reaction. After incubation with the organophosphorus neurotoxins, the enzymatic activity of AChE was inhibited and produced less H(2)O(2), resulting in a decreased catalytic oxidation of colorimetric substrates over MNP peroxidase mimetics, accompanied by a drop in color intensity. Three organophosphorus compounds were tested on the assay: acephate and methyl-paraoxon as representative organophosphorus pesticides and the nerve agent Sarin. The novel assay displayed substantial color change after incubation in organophosphorus neurotoxins in a concentration-dependent manner. As low as 1 nM Sarin, 10 nM methyl-paraoxon, and 5 μM acephate are easily detected by the novel assay. In conclusion, by employing the peroxidase-mimicking activity of MNPs, the developed colorimetric assay has the potential of becoming a screening tool for the rapid and sensitive assessment of the neurotoxicity of an overwhelming number of organophosphate compounds.

The epithelial-mesenchymal transition (EMT) plays an important role in breast cancer metastasis, especially in the most aggressive and lethal subtype, “triple-negative breast cancer” (TNBC). Here, we report that CD146 is a unique activator of EMTs and significantly correlates with TNBC. In epithelial breast cancer cells, overexpression of CD146 down-regulated epithelial markers and up-regulated mesenchymal markers, significantly promoted cell migration and invasion, and induced cancer stem cell-like properties. We further found that RhoA pathways positively regulated CD146-induced EMTs via the key EMT transcriptional factor Slug. An orthotopic breast tumor model demonstrated that CD146-overexpressing breast tumors showed a poorly differentiated phenotype and displayed increased tumor invasion and metastasis. We confirmed these findings by conducting an immunohistochemical analysis of 505 human primary breast tumor tissues and found that CD146 expression was significantly associated with high tumor stage, poor prognosis, and TNBC. CD146 was expressed at abnormally high levels (68.9%), and was strongly associated with E-cadherin down-regulation in TNBC samples. Taken together, these findings provide unique evidence that CD146 promotes breast cancer progression by induction of EMTs via the activation of RhoA and up-regulation of Slug. Thus, CD146 could be a therapeutic target for breast cancer, especially for TNBC.

Invasive micropapillary carcinoma (IMPC) is a rare variant of ductal carcinoma of the breast, and is characterized by a high metastatic potential and an aggressive clinical course. Studies of CD146 expression and function in breast cancer remain scarce. The aim of this study was to evaluate the role of CD146 and microvessel density (MVD) in breast IMPC. CD146 mRNA expression and immunohistochemistry for CD146 and MVD measured by CD31 were assessed in 82 cases of IMPC and 137 cases of invasive ductal carcinoma, not otherwise specified (IDC-NOS). The mRNA level of CD146 in cancer specimens was higher in IMPC than in IDC-NOS. CD146 expression in tumor cells was up-regulated in IMPC as compared with that in IDC-NOS, and was positively correlated with histological grade, ER, PR status, and P53 expression in IMPC and IDC-NOS. CD146 expression in vascular endothelial cells was significantly higher than that in IDC, and was positively correlated with tumor progression in IMPC and IDC-NOS. MVD in IMPC was significantly higher than that in IDC. CD146 expression in tumor cells was positively correlated with that in vascular endothelial cells of IMPC and IDC-NOS. The association of CD146 expression with MVD and its correlation with progression in breast carcinoma indicated that CD146 is a potentially useful prognostic marker for breast cancer. CD146 could be a new drug target in the treatment of breast cancer.

Tumor cell migration is a well-orchestrated multistep process that drives cancer development and metastasis. Previous data indicated that CD146 expression correlates with malignant progression and metastatic potential of human melanoma cells. However, the exact molecular mechanism of how CD146 promotes melanoma cell migration still remains poorly understood. Here, we report that CD146 physically interacts with actin-linking ezrin-radixin-moesin (ERM) proteins and recruits ERM proteins to cell protrusions, promoting the formation and elongation of microvilli. Moreover, CD146-promoted melanoma cell migration is linked to RhoA activation and ERM phosphorylation. CD146 recruits Rho guanine nucleotide dissociation inhibitory factors 1 (RhoGDI1) through ERM proteins and thus sequesters RhoGDI1 from RhoA, which leads to upregulated RhoA activity and increased melanoma cell motility. CD146-activated RhoA also promotes further ERM phosphorylation and activation through Rho-phosphatidylinositol-4-phosphate-5-kinase-phosphatidylinositol 4,5-biphosphate pathway, which reinforces CD146/ERM association. Thus, our results provide a mechanistic basis to understand the role of CD146 in regulating human melanoma cell motility.

We developed a novel silica coating magnetic nanoparticle-based silver enhancement immunoassay (SEIA) for ricin toxin (RT) rapid electrical detection using interdigitated array microelectrodes (IDAMs) as electrodes. This novel system was developed by taking advantage of the separation and enrichment properties of magnetic nanoparticles (MNPs) and the catalytic properties of gold nanoparticles (GNPs). In this system, MNPs labeled with anti-ricin A chain antibody 6A6 were used to capture ricin and GNPs labeled with anti-ricin B chain antibody 7G7 were used as detectors. To enhance the electrical signal, the catalytic properties of GNPs were used to promote silver reduction. In the presence of ricin, a sandwich structure was formed which could be separated by a magnetic field. The sandwich complex was then transferred to IDAMs. The silver particles bridged the IDAM gaps and gave rise to an enhancing electrical signal that was detected by conductivity measurements. The results showed that the sensitivity of the SEIA for ricin electrical detection was five times greater than that of conventional colorimetric sandwich ELISA. Once the antibody used for detection was coated on the plates or MNPs, our system was three times more rapid than colorimetric sandwich ELISA. This rapid and sensitive detection system provides promising new potential for ricin detection.

Tumor angiogenesis, induced by tumor-secreted pro-angiogenic factors, is an essential process for cancer development and metastasis. CD146 is identified as an endothelial cell adhesion molecule and implicated in blood vessel formation, however, its exact role in angiogenesis, particularly tumor angiogenesis, and its potential function of mediating downstream signaling are still unclear. In present study, we evidenced that silencing endogenous endothelial CD146 by RNAi significantly impaired hepatocarcinoma cell secretions-promoted tubular morphogenesis and -enhanced motility of endothelial cells. Biochemical studies revealed that CD146 was required for the activation of p38/IKK/NF kappaB signaling cascade and up-regulation of NF kappaB downstream pro-angiogenic genes, notably IL-8, ICAM-1 and MMP9, in response to tumor secretions. Interestingly, specific anti-CD146 mAb AA98, which bound a conformational epitope depending on C452-C499 disulfide bond, could abrogate NF kappaB activation and tumor angiogenesis, whereas another anti-CD146 mAb AA1 recognizing a linear epitope containing aa50-54 did not have such effects. Further structure-function analysis identified that C452-C499 disulfide bond within the fifth extracellular Ig domain was indispensible for CD146-mediated signaling and tube formation. Moreover, dimerization of CD146, which was enhanced by tumor secretions and suppressed by AA98 but not AA1, also relied on C452 and C499. Together, this study for the first time uncovered the pro-angiogenic role of CD146 and also pinpointed the key structural basis responsible for its signaling function and dimerization. These findings also suggested that CD146 might serve as not just a cell adhesion molecule but also a membrane signal receptor in tumor-induced angiogenesis.

CD146 (MUC18, Mel-CAM/MCAM) is a transmembrane protein, originally identified as a biomarker of melanoma, and plays an important role in cancer invasion and metastasis. Further studies revealed that CD146 as a novel endothelial marker was also involved in angiogenesis. Previous studies reported several anti-CD146 antibodies, such as MUC18, A32, S-endo1, and P1H12, showing different binding patterns to the endothelium of various types of blood vessels. To examine the possibility that antibodies targeting different epitopes on CD146 could have different behaviors, we generated a panel of anti-human CD146 monoclonal antibodies, named AA1-5 and AA7, by immunizing mice with human CD146 protein purified from HUVEC. Their specificity and binding affinity were intensively characterized using Western blotting, flow cytometry, and immunohistochemical assay. On the basis of epitope mapping, we divided the six monoclonal antibodies (MAb) into two groups, groups V1 and C2-2, corresponding to the different extracellular domains harboring these epitopes, the first IgV and the second IgC2 domains, respectively. Furthermore, owing to different epitopes, the two groups of antibodies behaved differentially in cellular and histological levels. Therefore, these anti-CD146 MAbs targeting different domains should be useful tools in studying the expression and function of human CD146.

An antibody phage-display library was constructed from the B cells of convalescent severe acute respiratory syndrome (SARS) patients and screened using inactivated SARS coronavirus (CoV) virions as antigens. More than 80 positive clones were isolated from the library and one of them, scFv H12, was extensively characterized. scFv H12 bound to SARS-CoV with high affinity (equilibrium dissociation constant, Kd=73.5 nM), and neutralized SARS virions in vitro. The facts that scFv H12 bound to the SARS-S1 protein under non-reducing conditions and that it did not bind to monomeric S1 protein under reducing conditions strongly suggest that scFv H12 recognizes a conformational epitope shared by oligomeric S1 proteins. This study should aid in the manufacture of neutralizing antibody, provide a better understanding the immunological characteristics of SARS protein and facilitate the design of a SARS vaccine.

The goal of our study was to raise monoclonal antibodies (mAbs) against endothelial cell-surface proteins specific for tumor vasculature. Here, we describe the generation and intensive characterization of mAb AA98, including its functional properties and its antigen identification. In our study, an enhanced mAb AA98 immunoreactivity was observed on stimulated human umbilical vein endothelial cells (HUVECs). In addition, mAb AA98 showed remarkably restricted immunoreactivity against intratumoral neovasculature compared with blood vessels of normal tissues. We identified the AA98 antigen as human CD146, an adhesion molecule belonging to the immunoglobulin superfamily. Data from in vitro experiments imply structural and signaling functions for endothelial CD146; however, the role of CD146 in vivo is largely unknown. Here, we show that mAb AA98 displays antiangiogenic properties in vitro and in vivo. Proliferation and migration of HUVECs were inhibited by mAb AA98 as was angiogenesis in chicken chorioallantoic membrane (CAM) assays and tumor growth in 3 xenografted human tumor models in mice. Our data provide new insights into the function of CD146 on endothelial cells, validate CD146 as a novel target for antiangiogenic agents, and demonstrate that mAb AA98 has potential as a diagnostic and therapeutic agent in vascular and cancer biology.

Importance Appropriate infant feeding practices have the potential for long-term health effects. However, research findings on improving early infant feeding practices are limited. The wide use of mobile phone short message service (SMS) provides new opportunities for health promotion and services. Objective To assess the effect of an SMS intervention on infant feeding practices. Design and Setting Quasiexperimental design with follow-up measures scheduled at 4, 6, and 12 months at 4 community health centers in Shanghai, China. Two community health centers represented the intervention group, and 2 other community health centers represented the control group. Participants In total, 582 expectant mothers were recruited during the first trimester. Expectant mothers were eligible to participate if they owned a mobile phone, were first-time mothers, conceived a singleton fetus, were older than 20 years and less than 13 weeks’ gestation, had completed at least a compulsory junior high school education, and had no illness that limited breastfeeding after childbirth. Intervention Mothers in the intervention group received weekly SMS messages about infant feeding from the third trimester to 12 months’ post partum. Main Outcomes and Measures The primary outcome was the duration of exclusive breastfeeding (EBF). Survival analysis was used to compare the duration of EBF between the intervention group and the control group. Results Compared with the control group, the intervention group had a significantly longer median duration of EBF at 6 months (11.41 [95% CI, 10.25-12.57] vs 8.87 [95% CI, 7.84-9.89] weeks). The hazard ratio for stopping EBF in the intervention group was 0.80 (95% CI, 0.66-0.97). The intervention resulted in a significantly higher rate of EBF at 6 months (adjusted odds ratio, 2.67 [95% CI, 1.45-4.91]) and a significantly lower rate of the introduction of solid foods before 4 months (adjusted odds ratio, 0.27 [95% CI, 0.08-0.94]). Conclusions and Relevance An SMS intervention may be effective in promoting EBF, delaying the introduction of solid foods, increasing awareness of the World Health Organization breastfeeding guidelines, and improving knowledge of appropriate infant feeding practices for new mothers.

Background Infant feeding practices can have long-term health effects for children. Short message service (SMS) through mobile telephones has been increasingly used for health promotion and health services. We assessed the effectiveness of an SMS intervention for improvement of infant feeding practices. Methods We recruited participants from four community health centres (CHCs) in Shanghai, China. Two CHCs were designated the intervention group and two other CHCs were designated the control group. We recruited participants at the beginning of the third trimester. Mothers were eligible to participate if they: owned a mobile phone, were first-time mothers, were aged older than 20 years, were at less than 13 weeks gestation, had completed at least compulsory junior high school education (up to age 15 years), were carrying a singleton fetus, and had no illness that restricted breastfeeding after childbirth. All participants received routine maternal and child health care. Mothers in the intervention group also received weekly SMS messages for infant feeding from the third trimester to 12 months post-partum. Participants were followed up at 4, 6, and 12 months post-partum. The primary outcome was duration of exclusive breastfeeding (EBF). We compared the duration of EBF between groups by survival analysis. Findings We enrolled 582 participants (281 in the intervention group and 301 in the control group). At 6 months after birth, 265 mothers in the intervention and 284 in the control groups were followed up. Participants in the intervention group had a significantly longer median duration of EBF at 6 months compared with those in the control group (11·4 weeks, 95% CI 10·3–12·6 vs 8·9 weeks, 95% CI 7·8–9·9; p Interpretation An SMS intervention might help to promote EBF for new mothers. Large randomised controlled trials are needed to further explore this approach. Funding Nestle Foundation, Shanghai Municipal Health Bureau.

Carcinoembryonic antigen (CEA, CEACAM5, and CD66e) has been found to be associated with various types of cancers, particularly colorectal carcinoma, and developed to be a molecular target for cancer diagnosis and therapy. In present study, we generated a novel anti-CEACAM5 monoclonal antibody, namely mAb CC4, by immunizing mice with living colorectal cancer LS174T cells. Immunohistochemical studies found that mAb CC4 specifically and strongly binds to tumor tissues, especially colorectal adenocarcinoma. In xenografted mice, mAb CC4 is specifically accumulated in tumor site and remarkably represses colorectal tumor growth. In vitro functional analysis showed that mAb CC4 significantly suppresses cell proliferation, migration and aggregation of colorectal cancer cells and also raises strong ADCC reaction. More interestingly, mAb CC4 is able to enhance NK cytotoxicity against MHC-I-deficient colorectal cancer cells by blocking intercellular interaction between epithelial CEACAM5 and NK inhibitory receptor CEACAM1. These data suggest that mAb CC4 has the potential to be developed as a novel tumor-targeting carrier and cancer therapeutic.