Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF͞MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-purified GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91. Cysteinylated GIF, but not the wild-type GIF͞MIF, possessed immunosuppressive effects on the in vitro IgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells. In vitro treatment of wild-type recombinant human GIF͞MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in ␣2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wildtype GIF͞MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF͞MIF molecules are responsible for the generation of GIF bioactivity.

Objectives This study aimed to investigate the effects of renal ischaemia/reperfusion (I/R)-induced acute kidney injury (AKI) on the distribution of midazolam (MDZ), a probe drug for cytochrome P450 3A (CYP3A) activity. Methods We established an AKI model inducing ischaemia of both renal pedicles for 60 min followed by 24-h reperfusion. MDZ was administered intravenously (i.v.) to the rats via the jugular vein, and then, blood samples were collected to determine the plasma concentration of MDZ. Key findings While the plasma concentration of MDZ after i.v. administration was decreased in the I/R rats, the tissue concentration was not altered. In addition, the tissue-to-plasma (T/P) ratio of MDZ was increased in the I/R rats. The unbound fraction of MDZ and the level of indoxyl sulphate (IS) in plasma were elevated in the I/R rats. Furthermore, the unbound fraction of MDZ was significantly increased by the addition of IS. Conclusions These results indicated that the displacement of albumin-bound MDZ by IS changed the unbound fraction of MDZ and elevated the T/P ratio of MDZ in I/R rats.

We developed a biocompatible splenic vector for a DNA vaccine against melanoma. The splenic vector is a ternary complex composed of plasmid DNA (pDNA), biodegradable dendrigraft poly-L-lysine (DGL), and c-polyglutamic acid (c-PGA), the selective uptake of which by the spleen has already been demonstrated. The ternary complex containing pDNA encoding luciferase (pCMV-Luc) exhibited stronger luciferase activity for RAW264.7 mouse macrophage-like cells than naked pCMV-Luc. Although the ternary complex exhibited strong luciferase activity in the spleen after its tail vein injection, luciferase activity in the liver and spleen was significantly decreased by a pretreatment with clodronate liposomes, which depleted macrophages in the liver and spleen. These results indicate that the ternary complex is mainly transfected in macrophages and is a suitable formulation for DNA vaccination. We applied the ternary complex to a pUb-M melanoma DNA vaccine. The ternary complex containing pUb-M suppressed the growth of melanoma and lung metastasis by B16-F10 mouse melanoma cells. We also examined the acute and liver toxicities of the pUb-M ternary complex at an excess pDNA dose in mice. All mice survived the injection of the excess amount of the ternary complex. Liver toxicity was negligible in mice injected with the excess amount of the ternary complex. In conclusion, we herein confirmed that the ternary complex was mainly transfected into macrophages in the spleen after its tail vein injection. We also showed the prevention of melanoma metastasis by the DNA vaccine and the safety of the ternary complex.

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We examined the influence of liver disease on the absorption from the liver surface of fluorescein isothiocyanate (FITC)-dextran 10 (FD-10, MW: 11000) and several marker compounds with different molecular weights. The purpose of this study was to determine the feasibility of liver surface application of macromolecular compounds in the disease state. We used male Wistar rats treated with carbon tetrachloride (CCl 4) or D-galactosamine (GAL). FD-10 and other marker compounds were applied to the liver surface using a cylindrical diffusion cell in liver-intoxicated rats. The blood, bile, urine, and the remaining solution in the diffusion cell were collected for assay. FD-10 was absorbed by first-order kinetics from the liver surface in the liver-intoxicated rat models. The calculated rate constant k a values in the normal, CCl 4 and GAL groups were 0.000965, 0.00125 and 0.00104 min 1 , respectively. Increased absorption of FITC-dextrans in the liverintoxicated rats was observed. In both CCl 4 and GAL groups, an inverse relationship was observed between the molecular weight and k a from the rat liver surface of the marker compounds. The limits of the molecular weight absorbed from the liver surface were extrapolated to be 71200, 135000, and 105000 in the normal, CCl 4 , and GAL groups, respectively. In conclusion, increased absorbability from the rat liver surface indicates that liver surface application for liver targeting of macromolecules in the diseased state is indeed feasible. Therefore, our findings can support further research on liver surface application of drugs under liver disease.