PubMed · 12418334
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Margaret R Colyar. 2002. Testing for lupus.. https://pubmed.ncbi.nlm.nih.gov/12418334/
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OBJECTIVE: To develop an RNAi approach that specifically targets the core gene sequence of hepatitis B virus by synthesizing short interfering RNA (siRNA) in vivo, and to assess the inhibitory effect of this siRNA on HBV replication. METHODS: The eukaryotic expression plasmid pHBV1.3, which contains 1.3-fold-overlength genome of HBV, were cotransfected into HepG2 cells with either the RNAi plasmid pSIHBV/C or unrelated control plasmid pSI. At 24, 48, 72 hours post transfection (p.t.), the levels of HBsAg and HBeAg in the cell culture medium were determined with Abbott MEIA Kits. The expression of intracellular viral proteins was also determined by immunofluorescence staining. RESULTS: Successfully constructed expressing siRNA vector pSIHBV which targeted the core gene of Hepatitis B virus.The introduction of RNAi plasmid was shown to efficiently and specifically inhibit the synthesis of surface and e antigen of HBV by Abbot MEIA kits, with inhibitory rates at 92%, 85% peaking at 24 hours p.t. Immunofluorescence staining showed that intracellular synthesis of viral antigen was sharply reduced to nearly background levels when the ratio of pSIHBV/C and pHBV1.3 was at 1:20, whereas the control vector did not exhibit any inhibitory effect on the replication and expression of HBV. CONCLUSION: Our results demonstrate that the short interfering RNA targeting HBV core gene exerts robust inhibition on HBV viral replication, suggesting that RNAi-based anti-HBV replication strategy may represent a potentially efficacious approach to the clinical management of HBV infection.
BACKGROUND: Glutathione S-transferase (GST) pi is a detoxifying enzyme abundant in normal prostate basal cells but only rarely expressed in prostate cancer cells. The current studies are the first to focus on GST pi in the stromal compartment of prostate tumors. METHODS: We employed immunohistochemical, immunofluorescence, and Western blot analysis to measure GST pi expression and subcellular localization in 21 primary and metastatic tumors from patients with hormone independent prostate cancer, as well as seven lymph node metastases and six prostatectomy specimens. RESULTS: GST pi was detectable in stromal cells in 17 of the 21 hormone independent prostate tumors. GST pi tissue distribution in hormone independent tumors coincided with vimentin staining, suggesting that GST pi is expressed by reactive fibroblasts and/or myofibroblasts. CONCLUSIONS: The current results suggest that prostate cancer cells induce an injury response in the stroma during progression to hormone independence, which results in GST pi expression. Stromal GST pi may contribute to chemoresistence of advanced prostate cancer.
Conventional isolation methods, including the Health Products and Food Branch (HPFB), Health Canada method used for the isolation and identification of Listeria species and Listeria monocytogenes from foods and environmental samples, can take a week or more to complete and are usually labor-intensive. This has led to the development of various rapid methods which attempt to generate results comparable to standard methods but in a reduced time-frame with less hands-on operation. Our previous work with rapid detection systems indicated that the recommended enrichment protocols failed to grow the Listeria to detectable levels in a reliable and consistent manner. In the present study, a novel enrichment protocol is described and consists of samples being pre-enriched in Palcam broth (incubated at 35 degrees C for 26 h), enriched in UVM 2 (30 degrees C for 26 h) and then plated and analysed by a rapid detection kit, with results being generated after 52 h of incubation. In total, 200 naturally contaminated samples were analysed by both the HPFB standard method and the Palcam method. The results showed that the Palcam method is comparable to the HPFB method. Further analysis involved a rapid detection system, which applies ELISA techniques and automation in an enzyme-linked fluorescent assay (ELFA) system. This system, referred to as the Vitek Immuno Diagnostic Assay System or VIDAS, can identify Listeria to the genus or species (L. monocytogenes) level. In this comparison, an additional 324 naturally contaminated samples were analysed by both the Palcam and ELFA methods. The sensitivity and specificity of the ELFA method were 98.1% and 97.0%, respectively, while the efficiency was 97.5%. False-negative and false-positive rates were 1.9% and 3.0%, respectively. These results show that the ELFA method (when using the Palcam method for pre- and secondary enrichment) was efficient and gave reliable results after 52 h of incubation, and met Health Canada's criteria for approval as a rapid method.