Addition of polyethylene glycol to protein (PEGylation) to improve stability and other characteristics is mostly nonspecific and may occur at all lysine residues, some of which may be within or near an active site. Resultant PEGylated proteins are heterogeneous and can show markedly lower bioactivity. We attempted to develop a strategy for site-specific mono-PEGylation using tumor necrosis factor-alpha (TNF-alpha). We prepared phage libraries expressing TNF-alpha mutants in which all the lysine residues were replaced with other amino acids. A fully bioactive lysine-deficient mutant TNF-alpha (mTNF-alpha-Lys(-)) was isolated by panning against TNF-alpha-neutralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-alpha-Lys(-) was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-alpha-Lys(-), with superior molecular uniformity, showed higher bioactivity in vitro and greater antitumor therapeutic potency than randomly mono-PEGylated wild-type TNF-alpha. These results suggest the usefulness of the phage display system for creating functional mutant proteins and of our site-specific PEGylation approach.

No abstract

Microglia are the principal phagocytes that clear cell debris in the central nervous system (CNS). This raises the question, which cells remove cell debris when microglial phagocytic activity is impaired. We addressed this question using Siglech dtr mice, which enable highly specific ablation of microglia. Non-microglial mononuclear phagocytes, such as CNS-associated macrophages and circulating inflammatory monocytes, did not clear microglial debris. Instead, astrocytes were activated, exhibited a proinflammatory gene expression profile, and extended their processes to engulf microglial debris. This astrocytic phagocytosis was also observed in Irf8-deficient mice, in which microglia were present but dysfunctional. RNA-seq demonstrated that even in a healthy CNS, astrocytes express TAM phagocytic receptors, which were the main astrocytic phagocytic receptors for cell debris in the above experiments, indicating that astrocytes stand by in case of microglial impairment. This compensatory mechanism may be important for the maintenance or prolongation of a healthy CNS.

Intestinal tract is the boundary that prevents harmful molecules from invading into the mucosal tissue, followed by systemic circulation. Intestinal permeability is an index for intestinal barrier integrity. Intestinal permeability has been shown to increase in various diseases—not only intestinal inflammatory diseases, but also systemic diseases, including diabetes, chronic kidney dysfunction, cancer, and cardiovascular diseases. Chronic increase of intestinal permeability is termed ‘leaky gut’ which is observed in the patients and animal models of these diseases. This state often correlates with the disease state. In addition, recent studies have revealed that gut microbiota affects intestinal and systemic heath conditions via their metabolite, especially short-chain fatty acids and lipopolysaccharides, which can trigger leaky gut. The etiology of leaky gut is still unknown; however, recent studies have uncovered exogenous factors that can modulate intestinal permeability. Nutrients are closely related to intestinal health and permeability that are actively investigated as a hot topic of scientific research. Here, we will review the effect of nutrients on intestinal permeability and microbiome for a better understanding of leaky gut and a possible mechanism of increase in intestinal permeability.

We have synthesized a polymeric drug carrier, polyvinylpyrrolidone-co-dimethyl maleic anhydride [poly(VP-co-DMMAn)], for use in renal drug delivery. About 80% of the 10-kDa poly(VP-co-DMMAn) selectively accumulated in the kidneys 24 h after intravenous administration to mice. Although this accumulated poly(VP-co-DMMAn) was gradually excreted in the urine, about 40% remained in the kidneys 96 h after treatment. Poly(VP-co-DMMAn) was taken up by the renal proximal tubular epithelial cells and no cytotoxicity was noted. Higher doses did not produce toxicity in the kidneys or other tissues. In contrast, polyvinylpyrrolidone of the same molecular weight did not show any tissue-specific distribution. Poly(VP-co-DMMAn)-modified superoxide dismutase accumulated in the kidneys after intravenous administration and accelerated recovery from acute renal failure in a mouse model. In contrast, polyvinylpyrrolidone-modified superoxide dismutase and native superoxide dismutase were not as effective. Thus, poly(VP-co-DMMAn) is a useful candidate as a targeting carrier for renal drug delivery systems.