Design for Cell-Specific Targeting of Proteins Utilizing Sugar-Recognition Mechanism: Effect of Molecular Weight of Proteins on Targeting Efficiency

Hepatic targeting of proteins utilizing the sugar-recognition mechanism was investigated in mice after intravenous injection. Five proteins with different molecular weights, i.e., bovine -globulins (IgG), bovine serum albumin (BSA), recombinant human superoxide dismutase (SOD), soybean trypsin inhibitor (STI), and chicken egg white lysozyme (LZM), were modified with 2-imino-2-methoxyethyl 1-thiogalactoside to obtain galactosylated proteins (Gal-IgG, Gal-BSA, Gal-SOD, Gal-STI, and Gal-LZM). The numbers of galactose residues were 38, 20, 11, 6, and 5 for Gal-IgG, Gal-BSA, Gal-SOD, Gal-STI, and Gal-LZM, respectively. All galactosylated proteins were dose-dependently taken up by the liver and the relative amount accumulated in the liver was decreased with an increase of the administered dose. At low doses (0.05 and 0.1 mg/kg), Gal-IgG, Gal-BSA, and Gal-SOD could be taken up by the liver up to more than 70–80% of dose within 10 min after intravenous injection, but the maximum amounts accumulated in the liver were approximately 40 and 30% of the dose for Gal-STI and Gal-LZM, respectively. Pharmacokinetic analysis revealed that the hepatic uptake clearance (CL_liver) was quite different around the molecular weight of 32 kDa and correlated with the amount delivered to the liver; Gal-IgG, Gal-BSA, and Gal-SOD has a large CL_liver that is close to the hepatic plasma flow rate (85 ml/hr), whereas those of Gal-STI and Gal-LZM were approximately 10 ml/hr at low doses. As for the total amount accumulated in the liver, high glomerular filtration rate of Gal-STI and Gal-LZM was also shown to cause insufficient delivery to the liver apart from being caused by their low CL_liver.     

Propofol combined with epidural anesthesia for a patient complicated with myelodysplastic syndrome (MDS)

A 55 year-old-female with myelodysplastic syndrome (MDS) underwent hemi-colectomy. We planned to avoid the use of nitrous oxide, because of its myelo suppressive effects. Therefore, we maintained the anesthesia with propofol combined with epidural anesthesia. After the surgical operation, the patient developed no hematological complications.     

Tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) stimulate osteoclastic bone resorption

As both tissue inhibitor of metalloproteinases-1 (TIMP-1) and TIMP-2 have been reported to inhibit bone resorption, we examined whether TIMP-1 or TIMP-2 in fetal calf serum (FCS), with which culture media were supplemented, affected osteoclastic bone resorption in vitro. Contrary to our expectation, almost complete suppression of osteoclastic bone resorption was observed when both TIMP-1 and TIMP-2 were removed from the FCS. Bone resorption was, however, almost fully restored by the addition of recombinant TIMPs. TIMPs stimulate bone resorption at significantly lower concentrations (∼ng/ml) than those (∼μg/ml) required to inhibit bone resorption. To understand the mechanism of TIMP-dependent bone resorption, we counted and compared the number of tartrate-resistant acid phosphatase-(TRAP-) positive and multinuclear cells in cultures containing either 10% FCS or TIMP-1-free and/or TIMP-2-free FCS. There was essentially no difference in number among these, suggesting that the TIMP role seems to be related to the functional expression of osteoclasts. Metallo-proteinase inhibitors, either BE16627B[l-N-(N-hydroxy-2-isobutylsuccinynamoyl)-seryl-l-valine] or R94138 {N-methyl-(3S)-2-[(2R)-2-hydroxycarbamoylmethylundecanoyl] hexahydropyridazine-3-carboxamide}, could not replace TIMPs, suggesting that the osteoclast-stimulating activity of TIMPs cannot be ascribed to merely their inhibitory effect on matrix metalloproteinases. Received: Oct. 15, 1998 / Accepted: April 5, 1999     

Significance of platelet-derived microparticles and activated platelets in diabetic nephropathy

We measured levels of platelet-derived microparticles (PMP), which have coagulative activity and are produced by platelet activation or physical stimulation, and CD62P/CD63-positive platelets in patients with diabetes mellitus to determine their clinical significance and effects on complications of diabetes including diabetic nephropathy. We also compared these levels before and after administration of the antiplatelet drug cilostazol. Plasma PMP and CD62P/CD63-positive platelet levels were significantly higher in patients with diabetes mellitus than normal controls. CD62P-positive platelet levels were significantly higher in patients with nephropathy than in patients without complications. After administration of cilostazol, PMP and CD62P/CD63-positive platelet levels were significantly decreased. The increases in platelet activity and its related procoagulant activity appear to account in part for the hypercoagulability observed in diabetes mellitus. Our findings suggest that activated platelets might play a role in the development of diabetic nephropathy. Furthermore, antiplatelet therapy with cilostazol for diabetic patients may be useful as antithrombin therapy including antiplatelet therapy, since it suppresses the production of intrinsic coagulants produced by platelet activation.     

Effects of hemodialysis membrane on serum lipid profile of maintenance hemodialysis patients

Atherosclerosis and lipid abnormalities are still insuperable complications for maintenance hemodialysis patients. We observed the serum lipid profile of 27 maintenance hemodialysis patients (M : F; 20 : 7, age; 54.9 +/- 6.2 y. o., hemodialysis duration; 10.8 +/- 4.9 years, body weight; 53.6 +/- 4.4 kg) using a low flux cellulose membrane, cellulose (1.5 m2), a vitamin-E-modified dialysis membrane, CL-15E (CL- 15E 1.5 m2, Terumo), and polysulfon, PS (PS-1.3UW 1.3 m2, Fresenius) dialysers. Each membrane dialyzer was used for 3 months. The blood flow rate was 200 ml/min, and hemodialysis time, 4 hours. When the dialyzers were replaced, fasting blood was collected at the beginning of hemodialysis and serum lipid parameters were measured. Seven additional maintenance hemodialysis patients were selected and TC, TG, HDL-C were measured as controls, because their dialyzers (low flux cellulose 1.5 m2) and hemodialysis conditions were not changed during the study. TC was decreased by PS and there were significant differences between cellulose and PS, and between CL-15E and PS. However, these changes were conducted within the normal range of TC. TG was not significantly changed during the study. HDL-C was decreased by CL-15E and PS as well as TC. There were significant differences in HDL-C between cellulose and CL-15E, and between cellulose and PS. Apo B, Apo B/A-I were decreased by PS and there were significant differences between cellulose and PS, respectively, LP(a) was not changed during the study. RLP-C (Cellulose vs. PS, CL-15E vs. PS), VLDL-C (Cellulose vs PS), and LDL-C (cellulose vs. PS, CL-15E vs. PS) were significantly decreased between membranes, respectively. Although the precise mechanism is yet unknown, the uptake of LDL and remnant into receptors of the liver might be improved by PS hemodialysis. In conclusion, these data suggest that PS decreased the serum levels of the lipid profile in maintenance hemodialysis patients and may be effective in improving their lipid abnormality.     

Impaired glucose tolerance, diabetes mellitus, and gallstone disease: An extended study of male self-defense officials in Japan

Few studies have investigated the relation between glucose tolerance status and ultrasonographically determined gallstone disease. Using a 75-g oral glucose tolerance test, we examined the association of impaired glucose tolerance (IGT) and non-insulin-dependent diabetes mellitus (NIDDM) with gallstone disease in Japanese men. Subjects were men aged 48 to 59 of the Japan Self-Defense Forces who received a preretirement health examination between October 1986 to December 1994. After exclusion of 12 men under insulin treatment in the consecutive series of 7637 men, 174 were found to have gallstones; 103 were at the state of postcholecystectomy, and 6899 had normal gallbladder. IGT and NIDDM were associated with a modestly increased risk of gallstone disease; adjusted odds ratios were 1.3 (95% confidence interval [CI]: 0.9–1.8) for IGT and 1.3 (95% CI: 0.8–2.0) for NIDDM after adjustment for hospital, rank, smoking, alcohol use, and body mass index. Adjusted odds ratio for IGT and NIDDM combined was 1.3 (95% CI: 1.0–1.7, p=0.08). When prevalent gallstones and postcholecystectomy were considered separately, NIDDM showed a significant, positive association with postcholecystectomy, but not with prevalent gallstones. The findings add to evidence that glucose intolerance is associated with a modest increase in the risk of gallstone disease.     

Haemodynamic changes associated with thermodilution cardiac output determination during metabolic acidosis or hypoxic hypoxia in dogs

Haemodynamic alterations elicited by iced injectate during thermodilution cardiac output measurements were evaluated in the presence of metabolic acidosis or hypoxic hypoxia in 14 instrumented anaesthetized dogs. The alterations in some haemodynamic variables during slowing of the heart rate following injection of 3 ml iced injectate were slightly greater in metabolic acidosis and hypoxic hypoxia as compared to animals without metabolic acidosis or hypoxic hypoxia (P < 0.05), but the changes were clinically insignificant. No serious haemodynamic changes were found during any cardiac output measurement by thermodilution in the presence of metabolic acidosis or hypoxic hypoxia. The values of cardiac output measured by thermodilution correlated closely with those of pulmonary blood flow measured by an electromagnetic flowmeter in the metabolic acidosis and hypoxic hypoxia groups (r > 0.9). It is concluded that thermodilution using iced injectate will estimate right ventricular output accurately in conditions of metabolic acidosis and hypoxic hypoxia.     

Chronological changes in nonhaemorrhagic brain Infarcts with short T1 in the cerebellum and basal ganglia

Our purpose was to investigate nonhaemorrhagic infarcts with a short T1 in the cerebellum and basal ganglia. We carried out repeat MRI on 12 patients with infarcts in the cerebellum or basal ganglia with a short T1. Cerebellar cortical lesions showed high signal on T1-weighted spin-echo images beginning at 2 weeks, which became prominent from 3 weeks to 2 months, and persisted for as long as 14 months after the ictus. The basal ganglia lesions demonstrated slightly high signal from a week after the ictus, which became more intense thereafter. Signal intensity began to fade gradually after 2 months. High signal could be seen at the periphery until 5 months, and then disappeared, while low or isointense signal, seen in the central portion from day 20, persisted thereafter. Received: 1 February 1999 Accepted: 13 September 1999     

Efficacy and safety evaluation of human reovirus type 3 in immunocompetent animals: racine and nonhuman primates.

PURPOSE: Human reovirus type 3 has been proposed to kill cancer cells with an activated Ras signaling pathway. The purpose of this study was to investigate the efficacy of reovirus in immunocompetent glioma animal models and safety/toxicity in immunocompetent animals, including nonhuman primates. EXPERIMENTAL DESIGN: Racine glioma cells 9L and RG2 were implanted s.c. or intracranially in Fisher 344 rats with or without reovirus antibodies, followed by treatment of reovirus. To study whether reovirus kills contralateral tumors in the brain and to determine viral distribution, we established an in situ dual tumor model followed by reovirus intratumoral inoculation only into the ipsilateral tumor. To evaluate neurotoxicity/safety of reovirus, Cynomolgus monkeys and immunocompetent rats were given intracranially with reovirus, and pathological examination and/or behavioral studies were done. Viral shedding and clinical biochemistry were systematically studied in monkeys. RESULTS: Intratumorally given reovirus significantly suppressed the growth of both s.c. and intracranially tumors and significantly prolonged survival. The presence of reovirus-neutralizing antibodies did not abort the reovirus' antitumor effect. Reovirus inhibited glioma growth intracranially in the ipsilateral but not the contralateral tumors; viral load in ipsilateral tumors was 15 to 330-fold higher than the contralateral tumors. No encephalitis or behavioral abnormalities were found in monkeys and rats given reovirus intracranially. No treatment-related clinical biochemistry changes or diffuse histopathological abnormality were found in monkeys inoculated intracranially with Good Manufacturing Practice prepared reovirus. Microscopic changes were confined to the region of viral inoculation and were dose related, suggesting reovirus intracranially was well tolerated in nonhuman primates. CONCLUSIONS: These data show the efficacy and safety of reovirus when it is used in the treatment of gliomas in immunocompetent hosts. Inoculation of reovirus into the brain of nonhuman primates did not produce significant toxicities.