Blood-brain barrier (BBB) characteristics are induced and maintained by cross-talk between brain microvessel endothelial cells and neighbouring elements of the neurovascular unit. While pericytes are the cells situated closest to brain endothelial cells morphologically and share a common basement membrane, they have not been used in co-culture BBB models for testing drug permeability. We have developed and characterized a new syngeneic BBB model using primary cultures of the three main cell types of cerebral microvessels. The co-culture of endothelial cells, pericytes and astrocytes mimick the anatomical situation in vivo. In the presence of both pericytes and astrocytes rat brain endothelial cells expressed enhanced levels of tight junction (TJ) proteins occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. Further morphological evidence of the presence of interendothelial TJs was provided by electron microscopy. The transendothelial electrical resistance (TEER) of brain endothelial monolayers in triple co-culture, indicating the tightness of TJs reached 400Omegacm(2) on average, while the endothelial permeability coefficients (P(e)) for fluorescein was in the range of 3x10(-6)cm/s. Brain endothelial cells in the new model expressed glucose transporter-1, efflux transporters P-glycoprotein and multidrug resistance protein-1, and showed a polarized transport of rhodamine 123, a ligand for P-glycoprotein. To further characterize the model, drug permeability assays were performed using a set of 19 compounds with known in vivo BBB permeability. Good correlation (R(2)=0.89) was found between in vitroP(e) values obtained from measurements on the BBB model and in vivo BBB permeability data. The new BBB model, which is the first model to incorporate pericytes in a triple co-culture setting, can be a useful tool for research on BBB physiology and pathology and to test candidate compounds for centrally acting drugs.

Organic anion transporters (OAT) play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We reported previously (Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519 -1524) that tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited organic anion transport in COS-7 cells expressing a mouse organic anion transporter (mOAT1), suggesting an important role of glycosylation in mOAT1 function. In the present study, we investigated the effect of disrupting putative glycosylation sites in mOAT1 as well as its human counterpart, hOAT1, by mutating asparagine to glutamine and assessing mutant transporters in HeLa cells. We showed that the putative glycosylation site Asp-39 in mOAT1 was not glycosylated but the corresponding site (Asp-39) in hOAT1 was glycosylated. Disrupting Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 without affecting their cell surface expression, suggesting that the loss of function is not because of deglycosylation of Asp-39 per se but rather is likely because of the change of this important amino acid critically involved in the substrate binding. Single replacement of asparagines at other sites had no effect on transport activity indicating that glycosylation at individual sites is not essential for OAT function. In contrast, a simultaneous replacement of all asparagines in both mOAT1 and hOAT1 impaired the trafficking of the transporters to the plasma membrane. In summary, we provided the evidence that 1) Asp-39 is crucially involved in substrate recognition of OAT1, 2) glycosylation at individual sites is not required for OAT1 function, and 3) glycosylation plays an important role in the targeting of OAT1 onto the plasma membrane. This study is the first molecular identification and characterization of glycosylation of OAT1 and may provide important insights into the structure-function relationships of the organic anion transporter family. Organic anion transporters (OAT)1 play essential roles in the body disposition of clinically important anionic drugs including anti-human immunodeficiency virus therapeutics, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories (1). Several OAT isoforms have been identified by us and others (2). OAT1 and -3 are predominantly expressed in the kidney and brain. In the kidney, these transporters utilize a tertiary transport mechanism to move organic anions across the basolateral membrane into the proximal tubule cells for subsequent exit/elimination across the apical membrane into urine. Through this tertiary transport mechanism, Na ϩ -K ϩ -ATPase maintains an inwardly directed (blood to cell) Na ϩ gradient. The Na ϩ gradient then drives a Na ϩ -dicarboxylate cotransporter, sustaining an outwardly directed dicarboxylate gradient that is utilized by a dicarboxylate/organic anion (OA) exchanger to move th...

Humans are generally in standing or sitting positions on Earth during the day. The musculoskeletal system supports these positions and also allows motion. Gravity acting in the longitudinal direction of the body generates a hydrostatic pressure difference and induces footward fluid shift. The vestibular system senses the gravity of the body and reflexively controls the organs. During spaceflight or exposure to microgravity, the load on the musculoskeletal system and hydrostatic pressure difference is diminished. Thus, the skeletal muscle, particularly in the lower limbs, is atrophied, and bone minerals are lost via urinary excretion. In addition, the heart is atrophied, and the plasma volume is decreased, which may induce orthostatic intolerance. Vestibular-related control also declines; in particular, the otolith organs are more susceptible to exposure to microgravity than the semicircular canals. Using an advanced resistive exercise device with administration of bisphosphonate is an effective countermeasure against bone deconditioning. However, atrophy of skeletal muscle and the heart has not been completely prevented. Further ingenuity is needed in designing countermeasures for muscular, cardiovascular, and vestibular dysfunctions.

ObjectivesTo evaluate the safety and effectiveness of tocilizumab (TCZ) in patients with systemic juvenile idiopathic arthritis (sJIA) in real-world clinical settings in Japan.MethodsPaediatric patients with sJIA initiating TCZ between April 2008 and February 2012 and those previously enrolled in clinical trials who initiated TCZ before April 2008 were enrolled in a Japanese registry surveillance programme. Safety and effectiveness parameters were collected for 52 weeks.ResultsOf 417 patients enrolled, mean age was 11.2 years and 48.0% were female. TCZ exposure was 407.0 patient-years (PYs). Baseline corticosteroid use was higher than in clinical trials. Rates of total adverse events (AEs) and serious AEs (SAEs) were 224.3/100 PYs and 54.5/100 PYs, respectively, with SAEs higher than previously reported. The most frequent AEs and SAEs were infections and infestations (69.8/100 PYs and 18.2/100 PYs, respectively). 74 serious infections occurred in 55 patients (18.2/100 PYs); higher than previously reported. 26 macrophage activation syndrome events were reported in 24 patients (6.4/100 PYs). Fever and rash symptoms improved from baseline to week 52 (54.6% to 5.6% and 43.0% to 5.6%, respectively). At 4 weeks, 8 weeks and 52 weeks, 90.5%, 96.2% and 99.0% of patients achieved normal C reactive protein levels (<0.3 mg/dL), respectively.ConclusionsThese first real-world data demonstrated that TCZ was well tolerated, with acceptable safety and effectiveness in patients with sJIA. Higher incidences of SAEs and serious infections may be due to differences, such as corticosteroid use and concomitant diseases, between patient populations enrolled in previously reported clinical trials and this study.

Thyrotropin (TSH) receptor (TSHR) A and B subunits are formed by intramolecular cleavage of the single chain receptor at two separate sites. The region involved in cleavage at Site 2 has been identified, but previous mutagenesis studies failed to identify Site 1. We now report fortuitous observations on the effect of trypsin on the TSHR that localizes a small region harboring Site 1. Thus, as detected by immunoblotting and by 125I-TSH cross-linking to TSHR expressed on the surface of intact CHO cells, trypsin clipped a small polypeptide fragment bearing a glycan moiety from the C terminus of the A subunit. Based on the TSHR primary structure, this small fragment (1-2 kDa) contains Asn-302. This information, together with estimation of the size of the deglycosylated A subunit relative to a series of C-terminal truncated TSHR ectodomain variants, places cleavage Site 1 in the vicinity of, or closely upstream to, residue 317. Remarkably, mutagenesis of every amino acid residue between residues 298-316 (present study) and 317-362 (previous data) did not prevent cleavage at Site 1. However, cleavage at this site was abrogated by deletion of a 50-amino acid segment (residues 317-366) unique to the TSHR in the glycoprotein hormone receptor family. In summary, these data provide novel insight into TSHR intramolecular cleavage. Cleavage at Site 1 does not depend on a specific amino acid motif and differs from cleavage at Site 2 by involvement of a mechanism requiring the presence of the enigmatic TSHR 50-amino acid "insertion."