Cholecystokinin type 2 receptor in colorectal cancer: diagnostic and therapeutic target.

INTRODUCTION: Cholecystokinin type 2 receptor (CCK2R), which mediates the action of gastrin and cholecystokinin (CCK), has been demonstrated to promote the proliferation of colorectal cancer (CRC). A number of studies showed that CCK2R overexpressed in gastric cancer and pancreatic cancer but few in CRC. The correlation between CCK2R expression and clinicopathological characteristics is also not clear. METHODS: This study investigated CCK2R expression in a wide range of cell lines and clinical CRC samples, and explored expression pattern and prognostic value of CCK2R in relation to clinicopathological parameters. The location and expression levels of CCK2R were measured by immunocytochemical (ICC), qRT-PCR and Western blot. The druggability and antineoplastic effects of CCK2R as a therapeutic target were investigated using an anti-CCK2R targeting recombinant toxin named rCCK8PE38 by CCK-8 assay. RESULTS: Compared with paracarcinoma tissues, tumor samples showed overexpression of CCK2R (p = 0.028) including both CRC tissue and plasma samples, with plasma detection showing a significant indication for CCK2R evaluation. Aberrant expression correlated significantly with histological type (p = 0.032) and p53 status (p < 0.01), and patients with CCK2R overexpression had significantly lower disease-free survival. Application of rCCK8PE38 demonstrated the specificity and druggability of CCK2R as a therapeutic target, providing a strategy for clinical case screening of drugs targeting CCK2R. CONCLUSION: This study highlighted the aberrant expression and clinical correlation of CCK2R and reveals its diagnostic, prognostic and treatment value in CRC. We hypothesize that CCK2R serve as a target for the diagnosis and treatment of this cancer.

Host Prdx6 contributing to the intracellular survival of Brucella suis S2 strain.

BACKGROUND: Brucellosis is a worldwide zoonotic infectious disease that is transmitted in various ways and causes great harm to humans and animals. The brucellosis pathogen is Brucella, which mainly resides in macrophage cells and survives and replicates in host cells. However, the mechanisms underlying Brucella survival in macrophage cells have not been thoroughly elucidated to date. Peroxiredoxin 6 (Prdx6) is a bifunctional protein that shows not only GSH peroxidase activity but also phospholipase A2 activity and plays important roles in combating oxidative damage and regulating apoptosis. RESULTS: Recombinant mouse (Mus musculus) Prdx6 (MmPrdx6) was expressed and purified, and monoclonal antibodies against MmPrdx6 were prepared. Using the Brucella suis S2 strain to infect RAW264.7 murine macrophages, the level of intracellular Prdx6 expression first decreased and later increased following infection. Overexpressing Prdx6 in macrophages resulted in an increase in B. suis S2 strain levels in RAW264.7 cells, while knocking down Prdx6 reduced the S2 levels in cells. CONCLUSIONS: Host Prdx6 can increase the intracellular survival of B. suis S2 strain and plays a role in Brucella infection.

An efficient scheme for purification of a novel recombinant immunotoxin, rCCK8PE38, for anti-tumour experiments.

rCCK8PE38 is a novel immunotoxin that targets choleystokinin B receptor, which is over-expressed in some tumor tissues. Although we constructed a prokaryotic expression vector to express rCCK8PE38 in our laboratory, thorough purification was necessary to quantitatively assess its anti-tumor effect. In this study, we established a purification protocol to obtain rCCK8PE38 with high purity from E. coli. Three different types of chromatography, hydrophobic chromatography, ion exchange chromatography and size exclusion chromatography, were used in combination. The purification technological parameters of each chromatography type were optimized. The whole process of purification was arranged to minimize the purification steps and achieve purity and bioactivity. Finally, through this optimized scheme, we obtained a recombinant protein with a purity of >95%; then, the protein was stored at -80°C after lyophilization. The purified protein was used in a tumor inhibition experiment and was effective in killing tumor cells that over-expressed choleystokinin B receptor. The results of this study may provide some valuable information about protein purification and lay the foundation for further clinical experiments with rCCK8PE38.

Clinical targeting recombinant immunotoxins for cancer therapy.

Recombinant immunotoxins (RITs) are proteins that contain a toxin fused to an antibody or small molecules and are constructed by the genetic engineering technique. RITs can bind to and be internalized by cells and kill cancerous or non-cancerous cells by inhibiting protein synthesis. A wide variety of RITs have been tested against different cancers in cell culture, xenograft models, and human patients during the past several decades. RITs have shown activity in therapy of several kinds of cancers, but different levels of side effects, mainly related to vascular leak syndrome, were also observed in the treated patients. High immunogenicity of RITs limited their long-term or repeat applications in clinical cases. Recent advances in the design of immunotoxins, such as humanization of antibody fragment, PEGylation, and modification of human B- and T-cell epitopes, are overcoming the above mentioned problems, which predict the use of these immunotoxins as a potential therapeutic method to treat cancer patients.

Characterization of surface antigen protein 1 (SurA1) from Acinetobacter baumannii and its role in virulence and fitness.

Acinetobacter baumannii is a Gram-negative bacillus that causes nosocomial infections, such as bacteremia, pneumonia, and meningitis and urinary tract and wound infections. In the present study, the surface antigen protein 1 (SurA1) gene of A. baumannii strain CCGGD201101 was identified, cloned and expressed, and then its roles in fitness and virulence were investigated. Virulence was observed in the human lung cancer cell lines A549 and HEp-2 at one week after treatment with recombinant SurA1. One isogenic SurA1 knock-out strain, GR0015, which was derived from the A. baumannii strain CCGGD201101 isolated from diseased chicks in a previous study, highlighted the effect of SurA1 on fitness and growth. Its growth rate in LB broth and killing activity in human sera were significantly decreased compared with strain CCGGD201101. In the Galleria mellonella insect model, the isogenic SurA1 knock-out strain exhibited a lower survival rate and decreased dissemination. These results suggest that SurA1 plays an important role in the fitness and virulence of A. baumannii.

Expression, purification and characterization of recombinant toxins consisting of truncated gastrin 17 and pseudomonas exotoxin.

Gastric cancer is a major cause of mortality and morbidity around world. However the effectiveness of the current approaches to the diagnosis and treatment of gastric cancer is limited. Recombinant targeted toxins may represent a novel direction of cancer therapy. In this study, we aimed to explore whether recombinant toxins fused with the truncated forms of G17 could target to kill cancer cells by recognizing CCK2R. Four recombinant Pseudomonas toxins PE38 fused with the forward or reverse truncated forms of G17 (G14 and G13) were successfully constructed, expressed, and purified. Their characteristics were further analyzed by SDS-PAGE, western blot and indirect immunofluorescence assay. The cytotoxicity assay demonstrated that only reversely fused recombinant toxins rG14PE38 and rG13PE38 exhibited certain toxicity on several cancer cell lines, and a competition assay indicated that the binding of the reverse gastrin-endotoxin to CCK2R (+) cells may be mediated by interaction between gastrin/gastrin-like and CCK2R.

A magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin.

Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles-chemiluminescence enzyme immunoassay (MPs-CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol-H2O2 chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs-CLEIA method had a linear range from 0.31 to 100ng/ml with a detection limit of 0.24ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA.

A novel analytical probe binding to a potential carcinogenic factor of N-glycolylneuraminic acid by SELEX.

N-glycolylneuraminic acid (Neu5Gc) is an abundant sialic acid in many mammals and is present in the glycoconjugates of most deuterostome animals. Neu5Gc also occurs in fresh samples of human tumors and fetuses. However, very little is known about the expression level and biologic functions of Neu5Gc due to the limitations of available analytical probes for detection methods. In this study, we first report the development of aptamers specific to Neu5Gc screened by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). After 15 selection rounds, cloning, sequencing and enzyme-linked immuno sorbent assay (ELISA) analysis, 6 different selected aptamers showed specificity for Neu5Gc. Among these 6 aptamers, N8 showed the best half maximal inhibitory concentration (IC50) value (127 ng mL(-1)) and had a relatively high affinity constant (Ka=6.68 × 10(9)M(-1)). The aptamers selected in this study will provide a novel analytical probe for the development of a biosensor to detect Neu5Gc in tissues and sera from patients with tumors as well as to detect Neu5Gc in animal-derived foods. In addition, the successful aptamer candidate can solve the problem that antibody is difficult to prepare in immunological assays. Thus, the discovery of novel aptamers specific for Neu5Gc is important for developing new methods of detecting Neu5Gc for the diagnosis and prevention of cancer as well as food poisoning.

Amiloride attenuates lipopolysaccharide-accelerated atherosclerosis via inhibition of NHE1-dependent endothelial cell apoptosis.

AIM: To investigate the effects of the potassium-sparing diuretic amiloride on endothelial cell apoptosis during lipopolysaccharide (LPS)-accelerated atherosclerosis. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to LPS (100 ng/mL) in the presence of drugs tested. The activity of Na(+)/H(+) exchanger 1 (NHE1) and calpain, intracellular free Ca(2+)level ([Ca(2+)](i)), as well as the expression of apoptosis-related proteins in the cells were measured. For in vivo study, ApoE-deficient (ApoE(-/-)) mice were fed high-fat diets with 0.5% (w/w) amiloride for 4 weeks and LPS (10 µg/mouse) infusion into caudal veins. Afterwards, atherosclerotic lesions, NHE1 activity and Bcl-2 expression in the aortic tissues were evaluated. RESULTS: LPS treatment increased NHE1 activity and [Ca(2+)](i) in HUVECs in a time-dependent manner, which was associated with increased activity of the Ca(2+)-dependent protease calpain. Amiloride (1-10 µmol/L) significantly suppressed LPS-induced increases in NHE1 activity, [Ca(2+)](i). and calpain activity. In the presence of the Ca(2+) chelator BAPTA (0.5 mmol/L), LPS-induced increase of calpain activity was also abolished. In LPS-treated HUVECs, the expression of Bcl-2 protein was significantly decreased without altering its mRNA level. In the presence of amiloride (10 µmol/L) or the calpain inhibitor ZLLal (50 µmol/L), the down-regulation of Bcl-2 protein by LPS was blocked. LPS treatment did not alter the expression of Bax and Bak proteins in HUVECs. In the presence of amiloride, BAPTA or ZLLal, LPS-induced HUVEC apoptosis was significantly attenuated. In ApoE(-/-) mice, administration of amiloride significantly suppressed LPS-accelerated atherosclerosis and LPS-induced increase of NHE1 activity, and reversed LPS-induced down-regulation of Bcl-2 expression. CONCLUSION: LPS stimulates NHE1 activity, increases [Ca(2+)](i), and activates calpain, which leads to endothelial cell apoptosis related to decreased Bcl-2 expression. Amiloride inhibits NHE1 activity, thus attenuates LPS-accelerated atherosclerosis in mice.

In vitro and in vivo antitumor effects of the recombinant immunotoxin IL6(T23)-PE38KDEL in multiple myeloma.

IL6(T23)-PE38KDEL is a chimeric molecule composed of interleukin 6 (IL6), missing the N-terminal 23 amino acids, and fused to a truncated mutant form of Pseudomonas exotoxin (PE38KDEL). The aim of this study was to evaluate this recombinant immunotoxin in terms of its specific cytotoxicity to IL6R-overexpressing multiple myeloma (MM) cells in vitro, as well as its antitumor effects and side effects in vivo. IL6(T23)-PE38KDEL was expressed in Escherichia coli, refolded and purified from inclusion bodies. The purified IL6(T23)-PE38KDEL was found to be selectively cytotoxic to IL6 receptor-positive tumor cells in vitro. IC(50) values of IL6(T23)-PE38KDEL were evaluated by MTS assay. Toxicity and maximum-tolerated dose of IL6(T23)-PE38KDEL were determined in mice. The antitumor activity of IL6(T23)-PE38KDEL was evaluated in mice with MM through intravenous injection and interventional therapy. Intravenous administration of IL6(T23)-PE38KDEL caused a significantly increased survival time in treated mice, and exhibited dose- and time-dependent antitumor effects against MM mice. Moreover, complete tumor regression was observed in 30 and 80% of mice treated intravenously and intraperitoneally, respectively, with 0.4 mg/kg/day for 10 days. These results demonstrated that the recombinant immunotoxin IL6(T23)-PE38KDEL kills IL6R-overexpressing cancer cells, and causes significant tumor regression.

A screening lateral flow immunochromatographic assay for on-site detection of okadaic acid in shellfish products.

A lateral flow immunochromatographic (LFIC) test strip based on a colloidal gold-monoclonal antibody (McAb) conjugate was developed for on-site rapid detection of okadaic acid (OA) in shellfish. It applies a competitive format using an immobilized toxin conjugate and free toxin present in samples. The McAb against OA was conjugated with 20-nm colloidal gold as detector reagent. The toxin in the sample competed with the immobilized toxin to bind to the gold conjugated with McAb. The colloidal gold/McAb/toxin mobile complex was not captured by OA-bovine serum albumin (BSA) on the test line, but it was captured by goat anti-mouse immunoglobulin G (IgG) on the control line. The color density of the test line correlated with the concentration of toxin in the range of 10-50 ng ml(-1). The qualitative detection limit of 150 µg kg(-1) sample was close to the European Union (EU) regulatory limit (160 µg kg(-1)). Therefore, these strips were able to directly and qualitatively estimate the consuming safety of shellfish. They require no equipment because of available visual results, and they screened numerous samples within 10 min. The results were further confirmed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). As a food safety screening tool, the test strips are convenient and useful to rapidly on-site test the presence of OA in shellfish products.

Production of monoclonal antibody and application in indirect competitive ELISA for detecting okadaic acid and dinophytoxin-1 in seafood.

BACKGROUND, AIM, AND SCOPE: Okadaic acid (OA) and analogues of dinophysistoxin (DTX) are key diarrheic shellfish poisoning (DSP) toxins, which possibly arouse DSP symptoms by consuming the contaminated shellfish. Because of the stable toxicity in high temperature and the long-term carcinogenicity, the outbreaks of DSP related to consumption of bivalve mollusks contaminated by DSP toxins pose a hazard to public health. Therefore, it is worth developing a fast and reliable analytical method for the detection of OA and analogues in shellfish. In this paper, an indirect competitive enzyme-linked immunosorbent assay (ELISA) (icELISA) for detecting OA and DTX-1 in seafood was developed based on monoclonal antibody (McAb). METHODS: The OA was conjugated to human immunoglobulin G (IgG) and bovine serum albumin (BSA) by the active ester method as the immune antigen and the detective antigen. The spleen cells from BALB/c mice immunized with OA-IgG were fused with SP2/0 myeloma cells. A hybridoma cell line, which secreted McAb against OA, was selected by "limiting dilution" cloning. An icELISA was developed based on immobilized conjugate (OA-BSA) competing the McAb with the free OA in seafood sample. RESULTS: A hybridoma cell line, which secreted IgG1 subclass monoclonal antibody (McAb) against OA, was selected. The IC(50) of the McAb for OA and dinophytoxin-1 (DTX-1) were 4.40 and 3.89 ng/mL, respectively. Based on the McAb, an indirect competitive ELISA for detection of OA and DTX-1 in seafood was developed. The regression equation was y = 54.713x - 25.879 with a coefficient correlation of R (2) = 0.9729. The linear range and the limit of detection were 0.4-12.5 and 0.45 ng/mL, respectively. The average recovery of OA and DTX-1 spiked shellfish was 82.29% with the coefficient of variation of 7.67%. CONCLUSION: The developed icELISA is a fast, sensitive, and convenient assay for detecting of total amount of OA and DTX-1 in seafood.

Development of a novel antibody probe useful for domoic acid detection.

The generation of monoclonal antibody (mAb) against marine toxins can serve as a valuable probe to detect this kind of compounds by immunological methods. However, traditional approaches to mAb generation usually need a comparative large quantity of standard substance (more than 400 microg mouse(-1)), and a comparative long immunization period (more than 6 weeks). Here we report a simple, inexpensive and fast protocol for the generation of monoclonal antibody probe specific for domoic acid (DA). In the method, lymph node cells were harvested from the Balb/C mice of hind footpad injection and fused with murine myeloma cells SP2/0 for hybridoma generation. This method for the preparation of mAb for DA has two main advantages: (a) there is no need for large-scale expensive antigen (only 70 microg antigen for one mouse); (b) immunization protocol can be accomplished within 16 days. Some characteristics of the mAb were studied and a direct competitive ELISA for the detection of DA using the mAb as a probe was developed. The detection limit was 0.41 ng well(-1) in phosphate buffered saline (PBS) and 0.53 ng well(-1) in blue mussel Mytilus edulis. The recoveries of DA from mussel and PBS buffer were from 94.8% to 105.1% and from 96.2% to 103.7%, respectively. Thus, the newly developed direct competitive ELISA using the mAb appears to be a reliable and useful method for monitoring of DA in shellfish (228 words).