Structure-function analyses of a stereotypic rheumatoid factor unravel the structural basis for germline-encoded antibody autoreactivity.

Rheumatoid factors (RFs) are autoantibodies against the fragment-crystallizable (Fc) region of IgG. In individuals with hematological diseases such as cryoglobulinemia and certain B cell lymphoma forms, the RFs derived from specific heavy- and light-chain germline pairs, so-called "stereotypic RFs," are frequently produced in copious amounts and form immune complexes with IgG in serum. Of note, many structural details of the antigen recognition mechanisms in RFs are unclear. Here we report the crystal structure of the RF YES8c derived from the IGHV1-69/IGKV3-20 germline pair, the most common of the stereotypic RFs, in complex with human IgG1-Fc at 2.8 Å resolution. We observed that YES8c binds to the CH2-CH3 elbow in the canonical antigen-binding manner involving a large antigen-antibody interface. On the basis of this observation, combined with mutational analyses, we propose a recognition mechanism common to IGHV1-69/IGKV3-20 RFs: (1) the interaction of the Leu432-His435 region of Fc enables the highly variable complementarity-determining region (CDR)-H3 to participate in the binding, (2) the hydrophobic tip in the CDR-H2 typical of IGHV1-69 antibodies recognizes the hydrophobic patch on Fc, and (3) the interaction of the highly conserved RF light chain with Fc is important for RF activity. These features may determine the putative epitope common to the IGHV1-69/IGKV3-20 RFs. We also showed that some mutations in the binding site of RF increase the affinity to Fc, which may aggravate hematological diseases. Our findings unravel the structural basis for germline-encoded antibody autoreactivity.

Effect of Metabolic Inhibitors on the Hepatic Disposition of 5-Fluorouracil after Application to the Rat Liver Surface.

We evaluated the effects of 5-fluorouracil (5-FU) metabolic inhibitors, gimeracil or uridine, on the hepatic disposition of 5-FU after application to the liver surface in rats, aiming to enhance the availability of 5-FU in the liver. 5-FU solution with or without metabolic inhibitors was applied to the rat liver surface using a cylindrical diffusion cell. The liver, blood and the remaining solution in the diffusion cell were collected at specified times, and assayed for 5-FU content. 5-FU absorption properties were not altered by addition of gimeracil and uridine. The 5-FU concentration in the diffusion cell attachment site of the rat liver (site 1) at 0.1-0.4 M ratios of gimeracil to 5-FU was significantly higher than that of the control. On the contrary, the addition of uridine did not increase the 5-FU concentration at site 1. At a 0.1 M ratio of gimeracil to 5-FU, the maximum 5-FU plasma concentration was the lowest, and the area under the 5-FU concentration-time curve at site 1 was 3.4 times greater than that of the control. We demonstrated that applying 5-FU with gimeracil to the rat liver surface could increase the availability of 5-FU in the liver.

Evaluation of hypothermia on the in vitro metabolism and binding and in vivo disposition of midazolam in rats.

The effect of hypothermia on the in vivo pharmacokinetics of midazolam was evaluated, with a focus on altered metabolism in the liver and binding to serum proteins. Rat primary hepatocytes were incubated with midazolam (which is metabolized mainly by CYP3A2) at 37, 32 or 28 °C. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of midazolam were estimated using the Michaelis-Menten equation. The Km of CYP3A2 midazolam remained unchanged, but the Vmax decreased at 28 °C. In rats, whose temperature was maintained at 37, 32 or 28 °C by a heat lamp or ice pack, the plasma concentrations of midazolam were higher, whereas those in the brain and liver were unchanged at 28 °C. The tissue/plasma concentration ratios were, however, increased significantly. The unbound fraction of midazolam in serum at 28 °C was half that at 37 °C. These pharmacokinetic changes associated with hypothermic conditions were due to reductions in CYP3A2 activity and protein binding.

Novel diagnostic method of peritoneal injury using dual macromolecular markers.

Long-term peritoneal dialysis (PD) frequently produces morphological and functional changes of the peritoneum, which makes continuation of PD difficult. Moreover, the progression of peritoneal injury causes complications and poor prognosis. Since therapeutic treatments for peritoneal injury during PD have yet to be established, it is important to diagnose peritoneal injury as early as possible. The aim of this study was to develop a method of monitoring peritoneal function to diagnose peritoneal injury. Model rats of peritoneal injury were prepared by intraperitoneal injection of methylglyoxal (MGO) for five consecutive days. Then, marker substances of various molecular weights (phenolsulfonphthalein, fluorescein isothiocyanate-dextran (FD)-10, FD-40, FD-70, FD-2000 or tetramethylrhodamine-dextran (RD)-10) were injected into the peritoneal cavity. At 120 min after injection, the remaining amounts of all marker substances were significantly decreased in the MGO-treated rats compared with those in the vehicle-treated rats. Molecular weight dependence of the peritoneal permeability was observed. A substance with a molecular weight of approximately 10000 was found to be suitable to diagnose peritoneal injury. Moreover, coadministration of RD-10 with FD-2000 enabled us to monitor enhanced peritoneal permeability and the transfer of water simultaneously, without the recovery of whole PD fluid, even in the case of different ultrafiltration volumes. We demonstrated the usefulness of administering substances to evaluate peritoneal permeability and the transfer of water simultaneously to diagnose peritoneal injury. This study should be valuable for safe and effective PD.

A radiogallium-DOTA-based bivalent peptidic ligand targeting a chemokine receptor, CXCR4, for tumor imaging.

We have developed a novel radiogallium (Ga)-DOTA-based bivalent peptidic ligand targeting a chemokine receptor, CXCR4, for tumor imaging. A CXCR4 imaging probe with two CXCR4 antagonists (Ac-TZ14011) on Ga-DOTA core, Ga-DOTA-TZ2, was synthesized, and the affinity and binding to CXCR4 was evaluated in CXCR4 expressing cells in vitro. The affinity of Ga-DOTA-TZ2 for CXCR4 was 20-fold greater than the corresponding monovalent probe, Ga-DOTA-TZ1. (67)Ga-DOTA-TZ2 showed the significantly higher accumulation in CXCR4-expressing tumor cells compared with (67)Ga-DOTA-TZ1, suggesting the bivalent effect enhances its binding to CXCR4. The incorporation of two CXCR4 antagonists to Ga-DOTA could be effective in detecting CXCR4-expressing tumors.

The role of fibronectin in pulmonary gene transfer following intravenous administration of lipoplex in mice.

We analyzed the effect of serum and fibronectin on pulmonary transgene expression after intravenous injection of cationic liposome-plasmid DNA (pDNA) complex (lipoplex) in mice. 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) methyl sulfate salt/cholesterol lipoplex was incubated with several serum components for 5 min at 37°C prior to injection. We analyzed pulmonary transgene expression and pulmonary accumulation of lipoplex. While interaction with serum did not decrease pulmonary transgene expression, interaction with heat-inactivated serum did decrease it. Moreover, interaction with fibronectin enhanced pulmonary transgene expression. Inhibition of the binding of fibronectin to integrin decreased pulmonary transgene expression after injection of untreated lipoplex. We found that pulmonary accumulation of lipoplex changed depending on the kind of interacting serum components after injection. Furthermore, interaction with fibronectin increased pulmonary accumulation of lipoplex. Interaction with serum was required for pulmonary gene transfer following intravenous injection of lipoplex. Fibronectin appears to be a particularly critical component. Furthermore, the binding of fibronectin interacting with lipoplex to integrin was an important mechanism for pulmonary transgene expression.

Evaluation for effect of hypothermia on the disposition of 4-nitrophenol in rats by in-vitro metabolism study and rat liver perfusion system.

OBJECTIVES: The aim of this study was to evaluate the effect of hypothermia on the in-vivo pharmacokinetics of 4-nitrophenol (4NP) using rat liver homogenate and rat liver perfusion system. METHODS: Rat liver homogenate was incubated with 4NP, which is mainly metabolized by cytochrome P450 2E1, at 37, 34, 32 or 28°C. The Michaelis constant (Km ) and maximum elimination velocity (Vmax ) of 4NP were calculated by a Hanes-Woolf plot. The hepatic extraction ratio (Eh ) of 4NP was evaluated in a rat liver perfusion study at 37, 34, 32 or 28°C. Moreover, the plasma concentration profiles of 4NP after its intravenous (i.v.) administration to rats were analysed by the moment theory and were compared with in-vitro parameters. KEY FINDINGS: While the Km of 4NP was not changed, the Vmax and Eh were reduced at low temperatures. The plasma concentrations of 4NP after its i.v. administration to rats were significantly increased at 28°C. CONCLUSION: Changes in the pharmacokinetics of 4NP under hypothermic conditions were caused by alterations in Vmax and Eh . We may be able to predict the disposition of a drug by in-vitro studies.

In vivo evaluation of a radiogallium-labeled bifunctional radiopharmaceutical, Ga-DOTA-MN2, for hypoxic tumor imaging.

On the basis of the findings obtained by X-ray crystallography of Ga-DOTA chelates and the drug design concept of bifunctional radiopharmaceuticals, we previously designed and synthesized a radiogallium-labeled DOTA chelate containing two metronidazole moieties, (67)Ga-DOTA-MN2, for hypoxic tumor imaging. As expected, (67)Ga-DOTA-MN2 exhibited high in vivo stability, although two carboxyl groups in the DOTA skeleton were conjugated with metronidazole moieties. In this study, we evaluated (67/68)Ga-DOTA-MN2 as a nuclear imaging agent for hypoxic tumors. (67)Ga-labeling of DOTA-MN2 with (67)GaCl(3) was achieved with high radiochemical yield (>85%) by 1-min of microwave irradiation (50 W). The pharmacokinetics of (67)Ga-DOTA-MN2 were examined in FM3A tumor-bearing mice, and compared with those of (67)Ga-DOTA-MN1 containing one metronidazole unit and (67)Ga-DOTA. Upon administration, (67)Ga-DOTA-MN2 exhibited higher accumulation in the implanted tumors than (67)Ga-DOTA. Tumor-to-blood ratios of (67)Ga-DOTA-MN2 were about two-fold higher than those of (67)Ga-DOTA-MN1. Autoradiographic analysis showed the heterogeneous localization of (67)Ga-DOTA-MN2 in the tumors, which corresponds to hypoxic regions suggested by well-established hypoxia marker drug, pimonidazole. Furthermore, in positron emission tomography (PET) study, the tumors of mice administered (68)Ga-labeled DOTA-MN2 were clearly imaged by small-animal PET at 1 h after administration. This study demonstrates the potential usefulness of (67/68)Ga-DOTA-MN2 as a nuclear imaging agent for hypoxic tumors and suggests that two functional moieties, such as metronidazole, can be conjugated to radiogallium-DOTA chelate without reducing the complex stability. The present findings provide useful information about the chemical design of radiogallium-labeled radiopharmaceuticals for PET and single photon emission computed tomography (SPECT) studies.