A recombinant influenza virus with a CTLA4-specific scFv inhibits tumor growth in a mouse model.

Oncolytic viruses (OV) have shown excellent safety and efficacy in preclinical and clinical studies. Influenza A virus (IAV) is considered a promising oncolytic virus. In this report, we generated a recombinant influenza virus expressing an immune checkpoint blockade agent targeting CTLA4. Using reverse genetics, a recombinant influenza virus, termed rFlu-CTLA4, encoding the heavy chain of a CTLA4 antibody on the PB1 segment and the light chain of the CTLA4 antibody on the PA segment was produced. RFlu-CTLA4 could replicate to high titers, and antibodies were produced in the allantoic fluid of infected eggs. Furthermore, the selective cytotoxicity of the virus was higher in various hepatocellular carcinoma cancer cell lines than in the normal cell line L02 in vitro, as indicated by MTS assays. More importantly, in a subcutaneous H22 mouse hepatocarcinoma model, intratumoral injections of rFlu-CTLA4 inhibited the growth of treated tumors and increased the overall survival of mice compared with injections of the PR8 virus. Taken together, these results warrant further exploration of this novel recombinant influenza virus for its potential use as a single or combination agent for cancer immunotherapy.

Simultaneous Determination of 6 Residual Organic Solvents in Omeprazole Magnesium by Headspace GC / 中国药房

OBJECTIVE:To establish a method for the simultaneous determination of 6 kinds of residual organic solvents in Omeprazole magnesium,such as methanol,isopropanol,acetonitrile,dichloromethane,ethyl acetate and toluenein. METHODS:Headspace GC was adopted. The determination was performed on the column of DB-624 column by temperature programming. The inlet temperature was 200 ℃,and hydrogen flame ionization detector was adopted with the temperature of 250 ℃;nitrogen gas was used as carrier gas with flow rate of 2.0 mL/min;the splitting-radio was 5 : 1,and sample size was 1 mL;the headspace tem-perature was 80 ℃ and the equilibrium time was 20 min. RESULTS:The linear range were 12.56-628.00 μg/mL for methanol(r=0.9997),20.22-1011.20 μg/mL for isopropanol(r=0.9999),1.96-97.76 μg/mL for acetonitrile(r=0.9997),3.10-154.88 μg/mL for dichloromethane(r=0.9998),20.69-1034.56 μg/mL for ethyl acetate(r=0.9998),and 3.53-176.72 μg/mL for toluene(r=0.9998);the limits of quantitation were 1.00,0.91,0.47,0.93,0.41 and 0.35 μg/mL respectively;the limits of detection were 0.31,0.30,0.14,0.31,0.12 and 0.11 μg/mL respectively;RSDs of precision,stability and reproducibility tests were lower than 3%;the recoveries were 94.53%-101.29%(RSD=2.15%,n=9),97.78%-103.42%(RSD=1.77%,n=9),96.99%-105.76%(RSD=2.59%,n=9),96.83%-102.05%(RSD=1.86%,n=9),97.98%-101.13%(RSD=0.88%,n=9)and 97.80%-102.40%(RSD=1.41%,n=9). CONCLUSIONS:The method is sensitive and accurate,can be used for the simultaneous determination of 6 kinds of residual organic solvents in Omeprazole magnesium,such as methanol,isopropanol,acetonitrile,dichloromethane,eth-yl acetate and toluenein.

hSav1 interacts with HAX1 and attenuates its anti-apoptotic effects in MCF-7 breast cancer cells.

It has been reported that Salvador (SAV) is a core component of the Salvador-Warts-Hippo (SWH) pathway that restricts cell number, by functioning as a dual regulator of cell proliferation and apoptosis in Drosophila. However, the function of its human ortholog hSav1 (also called hWW45) in mammalian cells is poorly understood. In this study, we identified hematopoietic cell-specific protein 1 (HS1)-associated protein X-1 (HAX1), a 35-kDa protein localized to cell mitochondria, as a novel binding partner of hSav1 using a yeast two-hybrid screening technique. Our finding was confirmed by immunoprecipitation and glutathione-S-transferase (GST) pull-down assays of both proteins. Using immunofluorescence staining, we showed that HAX1 and hSav1 interact with each other. Analysis of the anti-apoptotic function of HAX1 revealed that the presence of hSav1 attenuated the HAX1 protective effects from hydrogen peroxide (H2O2)-induced cell death in MCF-7 cells, while knockdown of hSav1 by small interfering RNAs (siRNAs) significantly enhanced the anti-apoptotic function of HAX1. Also, using the Oncomine database, we found several studies in which HAX1 levels were significantly up-regulated and hSav1 expression was down-regulated in breast cancer samples compared to normal breast tissue. In summary, we conclude that hSav1 interacts with HAX1 and attenuates its protective role against apoptosis in MCF-7 breast cancer cells.

Use of green fluorescent protein to monitor Lactobacillus in the gastro-intestinal tract of chicken.

Lactobacilli, like other gut commensal bacteria, are well known for their use in industrial food fermentations and for their probiotic properties. However, little is known about the interaction of these microorganisms with the gastro-intestinal epithelia when administered in vivo. To specifically monitor the passage of lactobacilli after oral administration, the gfp gene was cloned downstream from the constitutive l-lactate dehydrogenase promoter (pldhL) in the experiment. The recombinant expression vector pLEM415::gfp was electroporated into different lactobacilli isolated from chicken. Green fluorescent protein (GFP) was expressed successfully in Lactobacillus delbrueckii ssp. lactis D17 (D17-GFP) and Lactobacillus fructosus C2 (C2-GFP). Moreover, oral administration of D17-GFP in chickens allowed us to trace it in the gastro-intestinal tract. Six hours after ingestion, D17-GFP was detectable in all luminal contents (stomach, jejunum, ileum and caecum). At 42 h post-administration the microorganism was present throughout the intestine with maximum concentrations about 10(5.5) in all intestinal sections. No fluorescent lactobacilli were detected in the spleen or liver of chickens at any time. Using fluorescence microscopy, it became apparent that the D17-GFP were mainly embedded in the mucus, localized close to the epithelial surface of the intestine and scattered in the intestine lamina propria.