Role of the blood–cerebrospinal fluid barrier transporter as a cerebral clearance system for prostaglandin E2 produced in the brain

An increasing level of prostaglandin (PG) E₂ is involved in the progression of neuroinflammation induced by ischemia and bacterial infection. Although an imbalance in the rates of production and clearance of PGE₂ under these pathological conditions appears to affect the concentration of PGE₂ in the cerebrospinal fluid (CSF), the regulatory system remains incompletely understood. The purpose of this study was to investigate the cellular system of PGE₂ production via microsomal PGE synthetase‐1 (mPGES‐1), the inducible PGE₂‐generating enzyme, and PGE₂ elimination from the CSF via the blood–CSF barrier (BCSFB). Immunohistochemical analysis revealed that mPGES‐1 was expressed in the soma and perivascular sheets of astrocytes, pia mater, and brain blood vessel endothelial cells, suggesting that these cells are local production sites of PGE₂ in the CSF. The in vivo PGE₂ elimination clearance from the CSF was eightfold greater than that of d ‐mannitol, which is considered to reflect CSF bulk flow. This process was inhibited by the simultaneous injection of unlabeled PGE₂ and β‐lactam antibiotics, such as benzylpenicillin, cefazolin, and ceftriaxone, which are substrates and/or inhibitors of organic anion transporter 3 (OAT3). The characteristics of PGE₂ uptake by the isolated choroid plexus were at least partially consistent with those of OAT3. OAT3 was able to mediate PGE₂ transport with a Michaelis–Menten constant of 4.24 μM. These findings indicate that a system regulating the PGE₂ level in the CSF involves OAT3‐mediated PGE₂ uptake by choroid plexus epithelial cells, acting as a cerebral clearance pathway via the BCSFB of locally produced PGE₂.     

Effects of algal canopy clearance on plant, fish and macroinvertebrate communities on eastern Tasmanian reefs

Changes in assemblages of plants, macroinvertebrates and fishes on three eastern Tasmanian reefs were monitored over 12 months in replicated control blocks and adjacent 10×12-m blocks cleared of fucoid, laminarian and dictyotalean algae. Removal of canopy-forming plants produced less change to biotic assemblages than reported in studies elsewhere, with the magnitude of change for fish and invertebrate taxa lower than variation between sites and comparable to variation between months.The introduced annual kelp Undaria pinnatifida exhibited the only pronounced response to canopy removal amongst algal taxa, with a fivefold increase in cleared blocks compared to control blocks. Marine reserves are suggested to assist reef communities resist invasion by U. pinnatifida, through an indirect mechanism involving increased predation pressure on sea urchins and reduced formation of urchin barrens that are amenable to U. pinnatifida propagation.Large invertebrates were more associated with turfing algae or the reef substratum than the macroalgal canopy. The herbivorous sea urchin Heliocidaris erythrogramma and abalone Haliotis ruber showed the strongest response to clearing amongst common macroinvertebrate species, with a halving of population numbers. Observed densities of the common monacanthid fish Acanthaluteres vittiger also declined by about 50%. The relatively high level of resistance shown by eastern Tasmanian reef biota to patch disturbance was attributed largely to high diversity and biomass of turfing macroalgae damping effects of canopy clearance.     

Detection of Minute Virus of Mice Using Real Time Quantitative PCR in Assessment of Virus Clearance During the Purification Of Mammalian Cell Substrate Derived Biotherapeutics

A real time quantitative PCR assay has been developed for detecting minute virus of mice (MVM). This assay directly quantifies PCR product by monitoring the increase of fluorescence intensity emitted during enzymatic hydrolysis of an oligonucleotide probe labelled covalently with fluorescent reporting and quenching dyes via Taq polymerase 5'-->3' exonuclease activity. The quantity of MVM DNA molecules in the samples was determined using a known amount of MVM standard control DNA fragment cloned into a plasmid (pCR-MVM). We have demonstrated that MVM TaqMan PCR assay is approximately 1000-fold more sensitive than the microplate infectivity assay with the lowest detection limit of approximately one particle per reaction. The reliable detection range is within 100 to 10(9) molecules per reaction with high reproducibility. The intra assay variation is <2.5%, and the inter assays variation is <6.5% when samples contain >100 particles/assay. When we applied the TaqMan PCR to MVM clearance studies done by column chromatography or normal flow viral filtration, we found that the virus removal factors were similar to that of virus infectivity assay. It takes about a day to complete entire assay processes, thus, the TaqMan PCR assay is at least 10-fold faster than the infectivity assay. Therefore, we concluded that this fast, specific, sensitive, and robust assay could replace the infectivity assay for virus clearance evaluation.     

Altered clearance of human α2-macroglobulin complexes following reaction with cis-dichlorodiamineplatinum(II)

Clearance studies were performed in mice using α₂-macroglobulin (α₂M), α₂M-trypsin comlex and α₂M-CH₃NH₂ complex. All three species were incubated with cis-dichlorodiamine platinum(II) (cis-DDPt) at concentrations between 9.0 μM and 1.67 mM for 4 h and then dialyzed. The clearance rate of native α₂M was unchanged following incubation with cis-DDPt. α₂M-trypsin and α₂M-CH₃NH₂ cleared rapidly from the ciruculation; however, reaction with cis-DDPt significantly decreased the plasma elimination rate of both complexes. Non-denaturing gel electrophoresis and α₂M activity assays demonstrated relative stability following incubations with cis-DDPt which markedly altered clearance. Evidence for cis-DDPt crosslinking of α₂M subunits was obtained: however, whether this crosslinking is involved in altered clearance remains undetermined. Iodoacetamide treatment of α₂M did not duplicate the effect of cis-DDPton α₂M clearance, nor did it inhibit the effect of cis-DDPt on α₂M clearance. Plasma elimination of α₂M complex was also unaltered by pretreatment of mice with intravenous free cis-DDPt.     

Carbohydrate compositional effects on tissue distribution of chicken riboflavin-binding protein

Riboflavin-binding proteins (RBP) purified from chicken egg white, yolk and the serum of laying hens differ in their carbohydrate compositions reflecting tissue-specific modifications of a single gene product. All three are complex glycoproteins having more than twice as many N-acetylglucosamine residues (>12) as mannose residues (approx. 6). Egg white RBP is distinctive in having only one sialic acid and two galactose residues. Serum RBP contains approx. five sialic acid and seven galactose residues. In addition there is one residue of fucose. The carbohydrate composition of yolk RBP indicates the hydrolysis, respectively, of one, one, two and 3 residues of sialic acid, fucose, galactose, and N-acetylglucosamine from its precursor, serum RBP. The effect of these differing levels of glycosylation on plasma clearance, ovarian uptake and tissue distribution of ¹²⁵I-labeled riboflavin-binding proteins in laying hens were compared. 2 h after intravenous injection, 19% of the egg white RBP, 29% of the yolk RBP, and 37% of the serum RBP remained in circulation. The kinetics of plasma clearance was distinctly biphasic for each of the radioiodinated proteins. The initial rapid-turnover component ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=13}\text{min}$) ranged from 27% of the serum RBP sample to 48% of the egg white RBP sample. The remaining slow-turnover components were cleared with half-lives fo 81 min (egg white RBP), 101 min (yokl RBP), and 121 min (serum RBP). 16 h after injection, only 4% of the egg white RBP was deposited in the yolk of developing oocytes while about 12% of the serum RBP and yolk RBP was deposited. This higly significant difference is apparently due to preferential, carbohydrate-dependent clearance of egg white RBP by the liver rather than preferential uptake of serum and yolk RBP by the ovarian follicle. We find no evidence for carbohydrate-directed uptake of riboflavin-binding protein by the ovarian follicle.     

The TMEM16A chloride channel as an alternative therapeutic target in cystic fibrosis

Cystic fibrosis (CF), a multiorgan genetic disease, is caused by loss of function of CFTR, a cAMP-regulated anion channel. In CF airway epithelia, defective Cl⁻ and bicarbonate secretion impairs mucociliary clearance and other innate defense mechanisms, favoring the colonization of the lungs by highly virulent bacteria. The airway epithelium expresses TMEM16A, a second type of Cl⁻ channel that is activated by cytosolic Ca²⁺. TMEM16A is particularly expressed in goblet cells. This specific localization could be important in the release and hydration of mucins. Activation of TMEM16A with pharmacological agents could circumvent the primary defect in CF. This strategy needs to be carefully designed and tested to avoid possible undesired effects due to the expression of TMEM16A in other cell types such as bronchial smooth muscle cells.This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.     

Effect of antigen-dependent clearance on pharmacokinetics of anti-heparin-binding EGF-like growth factor (HB-EGF) monoclonal antibody

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family and is an important therapeutic target in some types of human cancers. KM3566 is a mouse anti-HB-EGF monoclonal antibody that neutralizes HB-EGF activity by inhibiting the binding of HB-EGF to its receptors. Based on the results of our pharmacokinetics study, a humanized derivative antibody, KHK2866, is rapidly cleared from serum and shows nonlinear pharmacokinetics in cynomolgus monkeys. In this study, we examined the antigen-dependent clearance of an anti-HB-EGF monoclonal antibody in vivo and in vitro in order to pharmacokinetically explain the rapid elimination of KHK2866. We revealed tumor size-dependent clearance of KM3566 in in vivo studies and obtained good fits between the observed and simulated concentrations of KM3566 based on the two-compartment with a saturable route of clearance model. Furthermore, in vivo imaging analyses demonstrated tumor-specific distribution of KM3566. We then confirmed rapid internalization and distribution to lysosome of KM3566 at a cellular level. Moreover, we revealed that the amounts of HB-EGF on cell surface membrane were maintained even while HB-EGF was internalized with KM3566. Recycled or newly synthesized HB-EGF, therefore, may contribute to a consecutive clearance of KM3566, which could explain a rapid clearance from serum. These data suggested that the rapid elimination in pharmacokinetics of KM3566 is due to antigen-dependent clearance. Given that its antigen is expressed in a wide range of normal tissue, it is estimated that the rapid elimination of KHK2866 from cynomolgus monkey serum is caused by antigen-dependent clearance.