Oral Administration of PLGA Nanoparticles to Deliver Antisense Oligonucleotides to Inflammatory Lesions in the Gastrointestinal Tract.

In this study, we prepared antisense oligonucleotide (ASO)-encapsulated nanoparticles (NPs) with a suitable profile for oral administration for the treatment of inflammatory bowel disease (IBD). We chose a water-in-oil-in-water (w/o/w) method to prepare the NPs using poly(lactide-co-glycolide) as a matrix and Pluronic as a stabilizer. The obtained NPs had a suitable diameter (158 nm) for the penetration of the mucus layer, endocytic uptake by enterocytes, and accumulation in inflammatory lesions in the intestine. The amount of ASOs in the NPs was relatively large (6.41% (w/w)). When the NPs were stably dispersed in solutions that mimicked gastrointestinal (GI) juice, minimal leakage of ASOs was demonstrated over the required period. The NPs were administered orally to mice with colitis induced by dextran sodium sulfate, which reduced target gene expression in the colons and rectums of the mice, whereas naked ASO administration caused no reduction in gene expression. Thus, the NPs have the potential of promising oral carriers of ASOs for the treatment of IBD that specifically target inflammatory lesions in the GI tract, thereby reducing the non-specific toxic effects of ASOs.

[Evaluation of a Bone Coordinate System Constructed Using MR Image Composing].

PURPOSE: To evaluate the accuracy of a bone coordinate system constructed using MR image composing. METHOD: A femoral coordinate system constructed using image composing of MR images of a whole bovine femur was evaluated using CT images. The MR images were acquired by moving the table and were processed with 3D distortion correction and composing. To evaluate the reproducibility of the measurements, the same operator repeated the construction of the femoral coordinate system. In addition, distortions in the MR images were evaluated in comparison with those in the CT images. RESULT: The center position of the femoral coordinate system constructed using the MR image composing was 1.6±0.9 mm on the X-axis, 1.5±0.8 mm on the Y-axis, and 0.2±0.3 mm on the Z-axis, and the rotation of each axis was 1° or less. The distortion of the composed MR image was about 0.3%. CONCLUSION: The femoral coordinate system constructed using MR image composing had the same accuracy as a system constructed with CT images. The effect of MR image composing on the construction of the femoral coordinate system was small.

[A Method for Evaluating the T2∗-weighting Effect in MRI].

PURPOSE: To propose a method for evaluating the T2*-weighting effect in MRI. METHODS: Multiple solutions with different concentrations of a superparamagnetic iron oxide contrast agent were made and their signal intensities on T2*-weighted images were measured. The relationship between iron concentration and signal intensity was determined, and we simulated an iron concentration map representing a simplified model of a brain microbleed and converted the pixel values in the map to signal intensity based on the determined relationship, generating a simulated T2*-weighted image. An 'S-value' parameter was defined to evaluate the low-intensity regions in the simulated image. S-values were obtained using T2*-weighted sequences acquired with different echo time (TE) values on three MRI scanners (Philips 1.5 T, GE 3.0 T, and Siemens 3.0 T). Another parameter (A-value) defined by the American Society for Testing and Materials (ASTM-F2119) for assessing artifacts was applied to evaluate the weighting effect in the T2*-weighted image of a laboratory-made susceptibility-effect phantom. RESULTS: With all three scanners, the S-values increased as the TE increased, indicating enhancement of the T2*-weighting effect. For every TE, the S-values obtained for the Philips scanner were the largest, followed by those for the GE and Siemens scanners. The results of this comparative evaluation were similar to those obtained using A-values. CONCLUSION: Comparisons with the established A-value parameter showed our proposed method for the quantitative evaluation of the T2*-weighting effect using S-values to be valid. The proposed method has the advantage that the S-values do not depend on a specific susceptibility-effect phantom.

Effects of trunk lean and foot lift exercises in sitting position on abdominal muscle activity and the contribution rate of transversus abdominis.

PURPOSE: Abdominal hollowing exercise has been recommended to improve trunk stability. Trunk lean and foot lift exercises while sitting may easily promote abdominal muscle activity even in people who cannot perform abdominal hollowing consciously. The purpose of the present study was to examine the changes in abdominal muscle activity and contribution rate of the transversus abdominis muscle (TrA) when leaning the trunk and lifting the foot during sitting. METHODS: The muscle stiffnesses (indicators of muscle activity) of the right rectus abdominis, external oblique, internal oblique, and TrA of 14 healthy men were measured during abdominal hollowing and the following nine sitting tasks: reference posture, 15° and maximal posterior trunk lean, 20° and maximal ipsilateral and contralateral trunk lean, and ipsilateral and contralateral foot lift. The TrA contribution rate was calculated by dividing the TrA stiffness by the sum of the abdominal muscles' stiffnesses. RESULTS: The TrA stiffness was significantly higher in abdominal hollowing than in reference posture, posterior and ipsilateral trunk lean, and ipsilateral foot lift, but not higher than in contralateral trunk lean and contralateral foot lift. There was no significant difference in the TrA contribution rates between abdominal hollowing and ipsilateral or contralateral foot lift. CONCLUSION: The contralateral trunk lean or contralateral foot lift could enhance TrA activity for people who cannot perform abdominal hollowing consciously. The contralateral foot lift could particularly be beneficial to obtain selective activity of TrA.

Comparison of Absolute Protein Abundances of Transporters and Receptors among Blood-Brain Barriers at Different Cerebral Regions and the Blood-Spinal Cord Barrier in Humans and Rats.

This work was designed to clarify the absolute abundances of transporters and receptors at different cerebral regions of the blood-brain barriers (BBB) and blood-spinal cord barrier (BSCB) in humans and rats, using physiologically relevant units (pmol/g tissue and fmol/cm2); 39 and 29 proteins including tight-junction proteins and markers were quantified in human and rat capillary samples, respectively. Protein expression levels of almost all proteins were identical within a 2-fold range between BBB and BSCB in rats, while many proteins showed >2-fold smaller expression levels in BSCB than BBB in humans. Protein expression levels of transporters and receptors in humans were remarkably smaller than those in rats in both BBB and BSCB in units of pmol/g tissue and fmol/cm2. Protein expression levels (fmol/cm2) of MDR1 and BCRP at the BBB in humans were 9.88-fold and 5.23-fold smaller than those in rats, respectively. GLUT1 expression (pmol/g tissue) at cortical BBB in a human was 2.49- and 3.76-fold greater than that at white matter BBB and BSCB, respectively. INSR and LRP1 proteins were detected at cortical BBB, but not at white matter BBB or BSCB in humans. These findings throw light on regional differences and species differences in pharmacokinetics and physiological functions in the central nervous system.

Involvement of Claudin-11 in Disruption of Blood-Brain, -Spinal Cord, and -Arachnoid Barriers in Multiple Sclerosis.

It is important to understand the molecular mechanisms of barrier disruption in the central nervous system (CNS) of patients with multiple sclerosis (MS). The purpose of the present study was to clarify whether claudin-11 is involved in the disruption of two endothelial barriers (blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB)) and two epithelial barriers (blood-arachnoid barrier (BAB) and blood-CSF barrier (BCSFB)) in the CNS in MS. Immunohistochemical analysis revealed that, in both normal human and mouse, claudin-11 is co-localized with claudin-5 in the brain and spinal cord capillaries. The absolute protein expression level of claudin-11 was nearly equal to that of claudin-5 in rat brain capillaries, but was 2.81-fold greater in human brain capillaries. The protein expressions of claudin-11 were significantly downregulated in the brain and spinal cord capillaries of an MS patient and experimental autoimmune encephalomyelitis (EAE) mice. Specific downregulation of claudin-11 with siRNA significantly increased the transfer of membrane-impermeable FITC-dextran across human brain capillary endothelial cell (hCMEC/D3) monolayer. As for the epithelial barrier, claudin-11 protein expression was not decreased in choroid plexus epithelial cells forming the BCSFB in EAE mice, whereas it was decreased in brain and spinal cord meninges that form the BAB. Specific downregulation of claudin-11 with siRNA in a rat choroid plexus epithelial cell (TR-CSFB) monolayer significantly increased the permeability of FITC-dextran. In conclusion, our present findings indicate that claudin-11 expression at the BBB, BSCB, and BAB, but not the BCSFB, is downregulated in multiple sclerosis, impairing the functional integrity of these barriers.

Cluster of Differentiation 46 Is the Major Receptor in Human Blood-Brain Barrier Endothelial Cells for Uptake of Exosomes Derived from Brain-Metastatic Melanoma Cells (SK-Mel-28).

Brain metastasis is a frequent complication of cancer and may be mediated, at least in part, by the internalization of cancer-cell-derived exosomes into brain capillary endothelial cells. Clarifying the mechanism(s) of this internalization is of interest because it could help us to develop ways to block brain metastasis, as well as affording a potential new route for drug delivery into the brain. Therefore, the purpose of the present study was to address this issue by identifying the receptors involved in the internalization of exosomes derived from a brain-metastatic cancer cell line (SK-Mel-28) into human blood-brain barrier endothelial cells (hCMEC/D3 cells). The combination of sulfo-SBED-based cross-linking and comprehensive proteomics yielded 20 proteins as exosome receptor candidates in hCMEC/D3 cells. The uptake of PKH67-labeled exosomes by hCMEC/D3 cells measured at 37 °C was significantly reduced by 95.6% at 4 °C and by 15.3% in the presence of 1 mM RGD peptide, an integrin ligand. Therefore, we focused on the identified RGD receptors, integrin α5 and integrin αV, and CD46, which is reported to act as an adenovirus receptor, together with integrin αV. A mixture of neutralizing antibodies against integrin α5 and integrin αV significantly decreased the exosome uptake by 11.8%, while application of CD46 siRNA reduced it by 39.0%. Immunohistochemical analysis confirmed the presence of CD46 in human brain capillary endothelial cells. These results suggest that CD46 is a major receptor for the uptake of SK-Mel-28-derived exosomes by human blood-brain barrier endothelial cells (hCMEC/D3 cells).