A novel ß-glucan-oligonucleotide complex selectively delivers siRNA to APCs via Dectin-1.

Delivering therapeutic nucleic acids to targeted cells and organs has been a challenge for decades. A novel technology to deliver oligonucleotide therapeutics to immune cells is here described. In this approach, a macromolecular complex of oligonucleotides and the ß-1,3-glucan schizophyllan (SPG) is selectively delivered to cells expressing a lectin receptor, Dectin-1, via SPG-Dectin-1 interaction. Detailed investigation of Dectin-1-expressing cells revealed that Dectin-1 is expressed in all subsets of monocytes as well as dendritic cell (DC) populations, including conventional DCs (cDCs) and plasmacytoid DCs (pDCs), in humans. The expression patterns in mice and humans are comparable, except for the expression in pDCs. The results indicate that Dectin-1 is expressed on cells capable of professional antigen presentation, except for B cells. We chose CD40 as a target gene for small interfering RNA (siRNA) as CD40 expression in antigen-presenting cells (APCs), particularly in DCs, plays critical roles in regulating immune responses. Dose-dependent cellular uptake of siCD40-SPG complexes was confirmed in cells expressing Dectin-1. Gene silencing activity was confirmed in vitro by the reduction of CD40 mRNA and by the site-specific cleavage of CD40 mRNA as determined by the 5' RNA ligase-mediated rapid amplification of cDNA ends (5'RLM-RACE) technique. In vivo activity of siCD40-SPG complexes was demonstrated as the reduced CD40 protein expression in monocytes and DCs in mice. Furthermore, the in vivo activity of siCD40-SPG targeting human CD40 was confirmed in cynomolgus monkeys by the 5'RLM-RACE technique. In conclusion, we have demonstrated the receptor-ligand binding-mediated delivery of siRNA targeting immune-regulating monocytes and DCs via the interaction of SPG and its receptor, Dectin-1. As monocytes and DCs play central roles in inducing and controlling immune responses, Dectin-1-targeted delivery of nucleic acids should provide a useful tool for developing drugs to treat a wide range of diseases, including autoimmune diseases, allergy, and cancer, as well as transplantation.

Refraction artifact on abdominal sonogram.

Ultrasonography (US) is the first-line diagnostic tool for observing the whole abdomen. Unfortunately, a wide spectrum of refraction-related artefactual images is very frequently encountered in routine US examinations. In addition, most practitioners currently perform abdominal US examinations without sufficient knowledge of refraction artifacts (RAs). This review article was designed to present many representative RA images seen in the clinical setting, with a brief explanation of the mechanism of these images, in certain cases through an analyzed and reconstructed method using computer simulation that supports clinical observations. RAs are encountered not only with B-mode US but also with Doppler US, contrast-enhanced US, and shear wave elastography. RAs change their appearance according to the situation, but they always have a significant effect on detailed interpretation of abdominal US images. Correct diagnosis of abdominal US relies on a deep understanding of each characteristic artifactual finding, which necessitates knowledge of basic US physics. When analyzing mass lesions, computer simulation analysis helps to reveal the global images of RAs around a lesion.

Diagnostic problems in two-dimensional shear wave elastography of the liver.

Two-dimensional shear wave elastography (2D-SWE) is used in the clinical setting for observation of the liver. Unfortunately, a wide spectrum of artifactual images are frequently encountered in 2D-SWE, the precise mechanisms of which remain incompletely understood. This review was designed to present many of the artifactual images seen in 2D-SWE of the liver and to analyze them by computer simulation models that support clinical observations. Our computer simulations yielded the following suggestions: (1) When performing 2D-SWE in patients with chronic hepatic disease, especially liver cirrhosis, it is recommended to measure shear wave values through the least irregular hepatic surface; (2) The most useful 2D-SWE in patients with focal lesion will detect lesions that are poorly visible on B-mode ultrasound and will differentiate true tumors from pseudo-tumors (e.g., irregular fatty change); and (3) Measurement of shear wave values in the area posterior to a focal lesion must be avoided.

Long-term prognosis of hearing loss in patients with unilateral Ménière's disease.

CONCLUSION: The finding of deteriorated hearing loss at the initial visit at middle to high frequencies is a factor of poor hearing prognosis in Ménière's disease. Early intervention with instructions for lifestyle changes may lead to good outcomes in hearing. OBJECTIVE: An attempt was made to examine long-term changes in hearing loss in unilateral Ménière's disease and factors associated with prognosis of hearing loss retrospectively. METHODS: Based on their last hearing level of the affected ear, 36 patients were subdivided into two groups: the poor prognosis of hearing (PPH) group and the good prognosis of hearing (GPH) group. RESULTS: In the PPH group, the hearing levels at the initial visit at middle and high frequencies were significantly worse than those in the GPH group. Moreover, the hearing loss progressed during the first 2 years of the disease, and stayed flat to approximately 50 dB at the later stage. Conversely, the hearing loss at the onset in the GPH group showed no further progression over the first 2 years, and remained constant to approximately 35 dB at the later stage. In addition, the mean intervals from the onset to the initial visit in the PPH group were significantly longer than those in the GPH group.

HVJ-E/importin-ß hybrid vector for overcoming cytoplasmic and nuclear membranes as double barrier for non-viral gene delivery.

In order to enhance the nuclear import of the transgene, we prepared plasmid DNA/importin-ß conjugates consisting of biotinylated poly(ethylenimine)s and recombinant streptavidin-fused importin-ß. Hemagglutinating virus of Japan-envelope vector containing the PEI polyplex/importin-ß conjugate showed high transfection efficiency not only in vitro but also in vivo. We showed that novel HVJ-E/importin-ß-conjugated PEI polyplex hybrid vector could overcome plasma and nuclear membrane barriers to achieve effective transfection.

Long-term expression with a cationic polymer derived from a natural polysaccharide: schizophyllan.

Among the various synthetic gene carriers based on biomaterials, cationic polymers with polysaccharide backbones have long been studied as nonviral vectors due to their low immunogenicity and high water solubility. Schizophyllan, a beta-(1,3)-glucan, is one of the various polysaccharides that are clinically administered. Furthermore, its safety in the human body has already been confirmed. Various functional groups can be selectively introduced into the side chain, not into the main chain of schizophyllan. Therefore, we have synthesized various oligoamine conjugates from schizophyllan. It was confirmed that their in vitro transfection efficiencies are superior to that of polyethylenimine by adjusting the molecular weight and the degree of amination of cationic schizophyllan. While it was possible to reduce cytotoxicity by adjusting the amount of DNA complex per cell, as seen with poly-L-lysine, polyethylenimine, and chitosan, PEGylation was the most effective means of reducing toxicity. Furthermore, using cationic schizophyllan carriers, it was also possible to express a reporter protein for a long period of time due to a long residence time of plasmid DNA in cells.

Errors in measuring blood flow velocity behind hepatic mass lesions using color doppler sonography.

The authors designed microcomputer simulation models to investigate the combined effect of a distorted grayscale ultrasound image and an inaccurately measured Doppler angle on the accuracy of measurement of blood-flow velocity in vessels located behind mass lesions in the liver. Error decreased when flow was measured at the midpoint of the posttumoral segment of the vessel and increased with distance between the mass and the vessel, with inclination of the vessel, and with decrease in the diameter of the mass. Error was least with the sector probe, intermediate with the convex probe, and greatest with the linear probe. These findings suggest that the error can be minimized when measuring blood flow velocity in a vessel located behind a mass, if the Doppler images are acquired using a sector probe from the midpoint of the posttumoral segment of the vessel.