Improved formulations of antisense oligodeoxynucleotides using wrapped liposomes.

Previously, we demonstrated that wrapping dextran fluorescein anionic/cationic lipid complexes with neutral lipids produced a stable formulation that markedly increased the duration of the compound in plasma after intravenous administration to rats. The improved drug-delivery properties of the wrapped liposomes (WL) relative to other formulations suggested that this technology could offer important advantages for the administration of other polyanionic drugs, including antisense oligodeoxynucleotides (ODN). In the present study, we investigated the value of WL for formulating fluorescence-labeled phosphorothioated ODN (F-ODN). WL encapsulating F-ODN/cationic lipid complexes were prepared efficiently using similar methodology to that used in our earlier study. Studies confirmed that these WL were stable in vitro. Following intravenous administration to mice, free F-ODN and naked F-ODN/cationic lipid complexes were rapidly eliminated whereas administration of the WL resulted in high blood concentrations of drug that were maintained for several hours. Additional studies were conducted in mice that were inoculated with tumor cells (Caki-1 xenograft model, human kidney); in these experiments, intravenous administration of WL delivered 13 times more F-ODN to the tumor site than achieved after injection of free F-ODN.     

Measurement of intrahepatic shunt using Rutland method

Intrahepatic shunt ratio was calculated from the fraction of the uptake constant and the blood background, analyzing the data of 99mTc-(Sn)-N-pyridoxyl-5-methyltryptophan (99mTc-PMT) hepatobiliary imaging by Rutland method. Shunt flow and total hepatic blood flow (THBF) were also calculated from the values for the effective hepatic blood flow (EHBF) estimated from the blood clearance technique. In 15 cases of normal volunteer, the average of the shunt ratio, shunt flow, and THBF were 18.4 +/- 5.4 (S.D.)%, 137.8 +/- 49 ml/min, and 753 +/- 83.2 ml/min, respectively. In 8 cases of chronic hepatitis, the values were 35.2 +/- 2.6%, 276 +/- 55.4 ml/min, and 794.1 +/- 119.4 ml/min, respectively. In 12 cases of liver cirrhosis, the values were 51.4 +/- 12.6%, 353.9 +/- 141.3 ml/min, and 685.6 +/- 174.8 ml/min, respectively. In cases of chronic hepatitis and liver cirrhosis, in comparison with normal cases, the values for the shunt ratio and shunt flow were significantly increased (p less than 0.001) and those for EHBF were significantly decreased (p less than 0.001), but those for THBF were not significantly altered. There was a significant inverse correlation between the shunt ratio and EHBF (r = -0.842) in all 35 cases. This method is considered to be useful for differential diagnosis, estimation of the grade of the diseases, and evaluation of pathophysiology of various diffuse liver diseases.     

Graft Versus Host Disease with Mixed Chimerism

We report a patient with graft versus host disease (GVHD) with mixed chimerism (MC). The patient had chronic myelogenous leukemia and received bone marrow transplantation (BMT) from his elder sister. Eighty days after BMT, erythematous lesions appeared on his chest. Histological examination from the skin lesion revealed lymphocytic infiltration into the upper dermis. Eosinophilic necrotic keratinocytes were scattered through the epidermis. Liquefaction degeneration was also recognized. Sicca syndrome appeared from 110 days after BMT. Detection of host origin Y-chromosome-specific DNA by polymerase chain reaction (PCR) method in bone marrow and peripheral blood showed that all bone marrow samples obtained 6 months from BMT were positive for Y-specific DNA, while peripheral blood became positive in the 60th month after BMT. The host origin normal karyotype (46,XY) in the bone marrow samples was identified for the first time in the 60th month after BMT. These results indicate that host-origin hematopoietic cells survived after BMT.     

Strain Difference of Susceptibility to 4-Nitroquinoline 1-Oxide-induced Tongue Carcinoma in Rats

Strain difference of susceptibility to 4-nitroquinoline 1-oxide (4NQO)-induced squamous cell carcinomas of the tongue among Dark-Agouti, Long-Evans, Sprague-Dawley, ACI/Ms, Fischer 344, Donryu and Wistar/Furth rats was surveyed by evaluating the survival times, incidences and sizes of developed tumors as markers of susceptibility. Administration of 4NQO dissolved in drinking water induced squamous cell carcinomas in various sites of the upper digestive tract mucosa of all the experimental male and female rats of the seven strains. Regarding the mean survival times, Wistar/Furth rats survived much longer than any other strain of rats, and Dark-Agouti showed the shortest survival. The incidence of large, mass-type carcinomas of the tongue of Dark-Agouti rats was higher than in any other strain of rats, while that of Wistar/Furth rats was the lowest. Subsequently the mitotic activity and bromodeoxyuridine incorporation in the tongue epithelium of Dark-Agouti and Wistar/Furth rats were estimated after a short-term administration of 4NQO. There was a pronounced difference between the two strains of rats, because the proliferative responses of the tongue epithelium of Dark-Agouti rats to the 4NQO stimulation were much higher than those of Wistar/Furth rats. These results indicated that there are marked differences in the susceptibility to 4NQO-induced tongue carcinoma among the seven strains of rats, and that Dark-Agouti and Wistar/Furth rats could be useful as models of highly and poorly susceptible strains, respectively, for further genetic analysis.     

Old pulmonary embolism causing intrabronchial bleeding

A 51-year-old man was admitted because of hemoptysis. Physical examinations and chest XP revealed no abnormal findings. Fiberoptic bronchoscopy showed pulsatile bleeding at the orifice of right B6. Right bronchial arteriography showed a markedly dilated and tortous bronchial artery and shunting to the pulmonary arterial system in the middle and lower lobes. Pulmonary arteriography showed complete obstruction of the right middle lobar and lower lobe segmental arteries (A6, 9, 10). The hemoptysis was thought to be due to increased blood flow of the right bronchial artery, which compensated for reduced right pulmonary arterial flow. Right middle and lower lobe resection was done to prevent further hemoptysis. The resected specimen revealed old thromboemboli in the right middle and lower lobe pulmonary arteries. In this case old pulmonary embolism should be considered as a cause of intrabronchial bleeding.     

Cyclosporine overcomes cold preservation/reperfusion injury of liver graft: Chemokine release and liver ultrastructure

There is a growing body of evidence that the cytokine, tumor necrosis factor-α (TNF-ga), plays an important role in the development of hepatic ischemia/reperfusion injury. We found that the immunosuppressants, cyclosporine-A (CsA), azathioprine, and FK506, have protective effects on such injury. The purpose of the present study was to elucidate mechanisms involved in these beneficial effects of the immunosuppressant, CsA, on liver injury following cold preservation and transplantation, with special reference to the suppression of TNF-α release. Rat livers were stored in Euro-Collins solution (EC) at 4°C for 6h and orthotopically transplanted. The animals allotted to two groups: group A (untreated controls) and group B (CsA pretreatment of recipients). CsA (10 mg/kg, p.o.) was given for 3 consecutive days preoperatively. CsA pretreatment of the recipients significantly improved the 2-week survival rate (0/6 for group A, 3/6 for group B;P<0.05) and this was associated with a significant decrease in serum TNF-α levels 2h posttransplantation (group A, 69.8±15.7 pg/ml; group B, 22.8±6.8; mean±SEM;n=12 each;P<0.05) and amelioration of sinusoidal endothelial injury, assessed by electron microscopy. Plasma endotoxin levels following reperfusion of the grafts were not altered by the CsA therapy. Morphologically, CsA pretreatment of the recipients did not alter activation of Kupffer cells. CsA pretreatment of the recipient aids in preventing cold preservation/reperfusion injury of the liver graft, possibly by modulating effects of TNF-α.     

Coagulation changes during liver resection]

Thrombin-antithrombin III complex (TAT) and plasminogen activator inhibitor (PAI) were measured during liver resection surgery in 8 patients. TAT and PAI activities of patients under liver resection were compared with those of 11 patients under resection of esophageal carcinoma. TAT activity increased during liver resection (P < 0.001) and reached 14 times (P < 0.001) of its control value in the recovery room. PAI activity was very stable during operation, but increased to twice (P < 0.01) of its control value in the recovery room. TAT activity of patients after liver surgery in the recovery room was (P < 0.05) more than twice of that of patients after esophageal surgery. We conclude that hypercoagulable state occurred during liver resection to a greater degree compared with that observed with esophageal surgery, and that its cause might be liver resection itself.     

Ultrasonic duplex system to facilitate assessment of portal blood flow velocity

The usefulness of an ultrasonic duplex system to assess portal blood flow was investigated. In a model involving a steady flow through a vinyl tube in agar, there was a significant linear correlation between the maximum blood flow velocity measured by this system (V-max) and the mean blood flow velocity calculated from the actually measured blood flow volume (V-mean), that is, V-mean = 0.53 X V-max was obtained (r = 0.994; n = 47). This equation was used to calculate the mean portal blood flow velocity by this system (V-dopp) in 10 patients with liver disease, and the findings were compared with data simultaneously obtained by cineangiographic mapping of Lipiodol droplets released into the portal vein through a catheter placed in situ at the time of surgery (V-cine). A linear correlation between V-dopp and V-cine was statistically significant (r = 0.970; n = 13), and the regression line was V-cine = 1.29 X V-dopp -2.11. The ultrasonic duplex system proved reliable for a quantitative assessment of portal hemodynamics.