Effects of hyposalivation on the oral microflora of rats fed sucrose or wheat flour diets.

The effects of hyposalivation on the induction of dental caries and on the composition of the oral microflora were examined in specific pathogen-free Sprague-Dawley rats fed either a sucrose or a wheat flour diet with or without inoculation of Streptococcus mutans. Significant dental caries was induced in hyposalivated rats fed diet 2000 containing 56% sucrose, irrespective of infection by S. mutans. Diets containing 56% wheat flour did not induce dental caries in either hyposalivated or sham-operated rats, irrespective of infection by S. mutans. Bacteriological examinations at the end of the experiment demonstrated that the total numbers of lactobacilli and staphylococci increased in hyposalivated rats irrespective of the diet given, while the inoculated stain of S. mutans decreased significantly in hyposalivated rats. These findings suggest that some acidogenic microorganisms such as lactobacilli and staphylococci that can utilize sucrose or glucose but not wheat flour may also promote dental caries in hyposalivated rats.     

Long-term in vivo gene expression in mouse kidney using ϕC31 integrase and electroporation

Background: Achieving long-term gene expression in kidney will be beneficial for gene therapy of renal and congenital diseases, genetic studies constructing animal disease models, and the functional analysis of disease-related genes.

Purpose: The purpose of this study was to develop an in vivo long-term gene expression system in murine kidney using ?C31 integrase.

Methods: Gene expression in cultured RENCA, TCMK-1, and HEK293 cells was assessed. The long-term in vivo gene expression system in the kidney was achieved by co-transfecting 5?µg of pORF-luc/attB as a donor plasmid and 20?µg of pCMV-luc as a helper plasmid into the right kidney of mice by electroporation. Luciferase expression levels were measured to determine longevity of the expression.

Results: Significantly high luciferase expression levels were observed in cultured RENCA, TCMK-1, and HEK293 cells over 1 month compared with controls (non-integrase system). The luciferase cDNA sequence was integrated at a pseudo attP site termed mpsL1. In vivo luciferase expression levels in the integrase group were sustained and significantly higher than those in the control group over 2 months. Furthermore, ?C31 integrase-transfected cells had less genomic DNA damage caused by integrase expression.

Discussion and conclusion: These results demonstrated that the ?C31 integrase system could produce long-term (2 months) in vivo gene expression in mouse kidney.     

Prevalence and viral DNA loads of three novel human polyomaviruses in skin cancers from Japanese patients

Merkel cell polyomavirus (MCPyV), human polyomaviruses 6 (HPyV6) and 7 (HPyV7) are novel human polyomaviruses. This study investigated their detection rates and DNA loads in various skin cancers from Japanese patients. MCPyV, HPyV6 and HPyV7 were detected in 22.2%, 3.2% and 1.6% of squamous cell carcinomas, 18.0%, 2.0% and 4.0% of basal cell carcinomas, and 19.1%, 4.3% and 4.3% of melanomas, respectively. Quantitative real‐time polymerase chain reaction showed that their DNA loads were low. These findings provide the first evidence of the prevalence of HPyV6 and HPyV7 in skin cancers in Asia. Nucleotide differences were found in the large T‐sequenced region between Japanese and North American isolates: a nucleotide substitution of A to G for HPyV6; and a nucleotide substitution of T to C and the insertion of a gap for HPyV7. This suggested that two genotypes of HPyV6 and HPyV7 would be present and associated with geographical origin.     

Comparison of dose accuracy between 2D array detectors for pre-treatment IMRT QA

The dosimetric properties between various 2D array detectors were compared and were evaluated with regard to the accuracy in absolute dose and dose distributions for clinical treatment fields. We used to check the dose accuracy: 2D array detectors; MapCHECK (Sun Nuclear), EPID (Varian Medical Systems), EPID-based dosimetry (EPIDose, Sun Nuclear), COMPASS (IBA) and conventional system; EDR2 film (Eastman Kodak), Exradin A-14SL ion chamber (0.016 cc, Standard Imaging). First, we compared the dose linearity, dose rate dependence, and output factor between the 2D array detectors. Next, the accuracy of the absolute dose and dose distributions were evaluated for clinical fields. All detector responses for the dose linear were in agreement within 1%, and the dose rate dependence and output factor agreed within a standard deviation of ±1.2%, except for EPID. This is because EPID is fluence distributions. In all the 2D array detectors, the point dose agreed within 5% with treatment planning system (TPS). Pass rates of each detector for TPS were more than 97% in the gamma analysis (3 mm/3%). EPIDose was in a good agreement with TPS. All 2D array detectors used in this study showed almost the same accuracy for clinical fields. EPIDose has better resolution than other 2D array detectors and thus this is expected for dose distributions with a small field.     

Hepatocyte-targeted gene transfer by combination of vascularly delivered plasmid DNA and in vivo electroporation

To increase transgene expression in the liver, electric pulses were applied to the left lateral lobe after intravenous injection of naked plasmid DNA (pDNA) or pDNA/liver targeting vector complex prepared with galactosylated poly(L-lysine) or galactosylated polyethyleneimine. Electroporation (250 V/cm, 5 ms/pulse, 12 pulses, 4 Hz) after naked pDNA injection dramatically increased the expression up to 200,000-fold; the expression level obtained was significantly greater than that achieved by the combination of pDNA/vector complex and electroporation. We clearly demonstrated that the expression was dependent on the plasma concentration of pDNA at the time when the electric pulses were applied. Separation of liver cells revealed that the distribution of naked pDNA as well as transgene expression was largely selective to hepatocytes in the electroporated lobe. The number of cells expressing transgene product using vascularly administered naked pDNA followed by electroporation was significantly (P<0.01) greater and more widespread than that obtained by local injection of naked pDNA. These results indicate that the application of in vivo electroporation to vascularly administered naked pDNA is a useful gene transfer approach to a large number of hepatocytes.     

Aortic arch thrombosis in the neonate

Aortic arch thrombosis (AAT) of the neonate is rare but life-threatening by fatal compromise associated with thrombotic obstruction of the ascending aorta. We report a neonate with AAT who demonstrated a severe coarctation of the aorta and cerebral hypo-perfusion immediately after birth. Echocardiography confirmed the diagnosis of AAT on the findings of a large thrombus located on the transverse arch and blocking the cervical arterial branches. Low-molecular-weight heparin reduced the size of the thrombus and improved the hemodynamics of coarctation and cerebral perfusion. Echocardiography is a powerful tool to make a diagnosis and to monitor the size and regression of AAT.     

Immunostimulatory characteristics induced by linear polyethyleneimine-plasmid DNA complexes in cultured macrophages

Intravenously injected plasmid DNA (pDNA) complexed with cationic liposome (lipoplexes) caused NF-kappaB-mediated cytokine production from macrophages, induced by CpG sequence in the pDNA. We have reported that cytokine production caused by linear polyethyleneimine (PEI)-pDNA complexes (PEI polyplexes) was much lower than that caused by lipoplexes (Kawakami, S., Ito, Y., Charoensit, P., Yamashita, F., and Hashida, M. [2006]. J. Pharmacol. Exp. Ther. 317, 1382-1390). As Toll-like receptor-9 recognizing CpG sequence is expressed in the endosomal compartment, we hypothesized that the buffering capacity of PEI enhanced the escape of PEI polyplexes from endosomes, and that consequently cytokine production was decreased. In this study, the mechanism of lower cytokine production induced by PEI polyplexes, compared with lipoplexes, was investigated using the murine macrophage-like cell line RAW 264.7. Although transfection efficacy and cellular association were similar for PEI polyplexes and lipoplexes, tumor necrosis factor-alpha and interleukin-6 production and NF-kappaB activation caused by polyplexes were significantly lower than with lipoplexes. As for intracellular distribution, PEI polyplexes spread into cytosol whereas lipoplexes accumulated in vesicles, suggesting enhancement of escape from endosomes by PEI. Bafilomycin A1, an inhibitor of early endosomes, enhanced cytokine production and NF-kappaB activation by PEI polyplexes but not by lipoplexes; however, chloroquine, an inhibitor of late endosomes, inhibited PEI polyplex-induced cytokine production and NF-kappaB activation, suggesting that the buffering effect of PEI on early endosomes decreases NF-kappaB-mediated cytokine production. In conclusion, we demonstrate that cytokine production and NF-kappaB activation induced by PEI polyplexes are significantly lower than with lipoplexes in cultured macrophages. The significantly low cytokine response of PEI polyplexes may be due to effective transition of PEI polyplexes from endosomes to cytosol.