Rapid detection and quantitation of hepatitis B virus DNA by real-time PCR using a new fluorescent (FRET) detection system.

BACKGROUND: The diagnosis of hepatitis B virus (HBV) has until recently been based on traditional serologic methods targeting viral antigens and antibodies to viral proteins. The development of molecular methods allowing for the quantitation of HBV DNA is proving clinically valuable for monitoring therapy and detecting early treatment failures. OBJECTIVES: Here we report a new real-time (LightCycler) quantitative PCR for the detection of HBV DNA based on sequence specific hybridisation probes (designed in-house), targeting the HBV surface antigen. STUDY DESIGN: The assay was evaluated using a 10-fold dilution series of standard HBV DNA [Eurohep standard reference 1, genotype A, HBsAg subtype adw with a unitage of 10(6) WHO. i.u./ml] and 89 clinical serum samples. The performance was measured against a quantified standard HBV DNA working reagent (NIBSC code 98/780) and the sensitivity compared with our conventional thermal-block PCR. RESULTS AND CONCLUSION: Real-time PCR detected HBV DNA in 45% (40/89) and thermal-block PCR in 16% (14/75) of clinical samples. Results for 26 samples were below the detection limit of the thermal-block PCR but could be quantified by real-time (LightCycler) PCR. The LightCycler assay was at least 5 logs more sensitive than thermal-block PCR and could detect HBV in a linear range between 5 and 10(7) i.u. per reaction. The broad generic nature of the PCR primers coupled with the enhanced sensitivity and specificity of the fluorescent hybridisation probes makes this assay potentially valuable for both routine diagnostic and epidemiological work.     

Hypoglycaemic Activity of Nauclea Latifolia Sm. (Rubiaceae) in Experimental Animals

Aqueous, ethanolic and hexane extracts of the leaves of Nauclea latifolia (Rubiaceae) were assessed for their fasting blood glucose lowering effect in normoglycaemic and streptozotocin - diabetic rats. Wistar strain albino rats were given different doses of the extracts after 18 hrs fast and their blood glucose measured at 0,1,2,4 and 6 hours after treatment. The aqueous and ethanolic extracts significantly lowered the fasting blood glucose levels of the STZ-diabetic rats in a dose-dependent manner. The highest dose administered (400mg/kg) lowered the fasting blood glucose of the diabetic rats by 31.7% (aqueous) and 36.1% (ethanolic) extracts. The aqueous extract did not significantly lower the glucose levels of normoglycaemic rats (maximum 6.6%), nor was any significant decrease seen in the rats administered with the hexane (maximum of 4.0% for normoglycaemic and 2.4% for diabetics) extract. The hypoglycaemic and antihyperglycaemic potentials of the aqueous and ethanolic extracts were comparable to that of glibenclamide (1mg/kg).These results further support the traditional use of the plant in the treatment of diabetes mellitus.     

Correlation among [h-3] thymidine, flow-cytometry and proliferating cell nuclear antigen indexes in colorectal tumors

In colorectal cancer and other tumors, proliferating cell nuclear antigen (PCNA) has been found to be a useful marker in immunohistochemical studies of cell proliferation, The presence of a correlation among PCNA labeling index (PCNA LI) and values of cell proliferation expressed by [H-3]thymidine labeling index (TLI) and flow cytometry (FC), the most common techniques used in the evaluation of proliferative activity in colorectal cancer were studied. In 50 operable colorectal cancer patients, TLI, PCNA LI and determination of S-phase fraction by FC were carried out in order to evaluate the correlation among these three indices. A good correlation was obtained between PCNA LI and both TLI (r=0.667; P=0.0001) and percent of cells in S-phase as determined by FC (r=0.819; P=0.0001). It is concluded that PCNA immunohistochemistry can be a reliable marker of the proliferative activity in colorectal rumours. Furthermore, because of its technical simplicity and lower cost it can be more advantageous than TLI or FC.     

Characterization of endothelin-1 receptor subtypes in isolated human cardiomyocytes.

On cardiac membranes and isolated cardiomyocytes from the human heart, cell-type distribution and functional activities of endothelin-1 (ET-1) receptor subtypes were investigated by using binding methods and messenger RNA (mRNA) in situ hybridization. The ET-receptor antagonist BMS-182874 selectively and competitively inhibits ET(A) receptors both on isolated myocytes and ventricular membranes with approximately 1,300 times greater affinity for ET(A) than ET(B) subtypes. The [125I]-ET-1 specific binding revealed 42.851+/-2,546 receptors/myocyte with a prevalent proportion of ET(A)-receptor subtypes on both myocytes (84+/-2%) and ventricular membranes (66+/-3%). In situ hybridization studies revealed that mRNA for ET(A) receptors was expressed on both myocytes and nonmyocyte cells, whereas mRNA for ET(B) receptors was almost exclusively expressed on fibroblasts and endothelial cells. Specific binding of [125I]-ET-1 to both myocytes and ventricular membranes in the presence of specific ET(A) (BMS-182874) and ET(B) (BQ-788)-receptor antagonists showed a displacement of [125I]-ET-1 by unlabeled ET-1, which were significantly faster from ET(B) than from ET(A). This suggests a clearance function of ventricular ET(B) receptors.     

Effect of microfluidization parameters on the physical properties of PEG-PLGA nanoparticles prepared using high pressure microfluidization

The objective of this work was to develop uniformly distributed poly(ethylene glycol) grafted poly(lactide-co-glycolide) (PEG-PLGA) nanoparticles of mean size range ～100–200 nm using ethyl acetate as the solvent. In the multiple emulsion solvent evaporation method a high pressure microfluidization process was adopted to produce the W/O/W multiple emulsion. Non-toxic ethyl acetate was used to solubilize PEG-PLGA. The mean size of nanoparticles obtained was less than 180 nm. The particle size and size distribution were dependent on the microfluidization conditions applied. Mean particle size steadily increased from 121 nm at three passes to 172 nm at 20 passes of the microfluidizer, indicating that over-processing may be detrimental to PEG-PLGA nanoparticles prepared using this technique. There was no significant alteration of the PEG-PLGA matrix, as evidenced from the differential scanning calorimetric studies.     

Barriers to Effective Drug Treatment for Brain Metastases: A Multifactorial Problem in the Delivery of Precision Medicine

The treatment of metastatic lesions in the brain represents a serious unmet medical need in the field of neuro-oncology. Even though many effective compounds have demonstrated success in treating peripheral (non-CNS) tumors with targeted agents, one aspect of this lack of success in the brain may be related to poor delivery of otherwise effective compounds. Many factors can influence the brain delivery of these agents, but one key barrier is a heterogeneously “leaky” BBB that expresses efflux transporters that limit the BBB permeability for many targeted agents. Future success in therapeutics for brain metastases must take into account the adequate delivery of “active, free drug” to the target, and may include combinations of targeted drugs that are appropriate to address each individual patient’s tumor type. This review discusses some issues that are pertinent to precision medicine for brain metastases, using specific examples of tumor types that have a high incidence of brain metastases.