Macrophage depletion prevents anti-graft antibody production and results in long-term survival in xenotransplantation.

Liposome-encapsulated dichloromethylene diphosphonate (clodronate) is known to deplete macrophages. We examined the effect of clodronate on xenoreactive antibody production and xenograft rejection. Hamster cardiac grafts were transplanted into Lewis rats. Clodronate (4 mL/kg) was injected intravenously on the day before transplantation. In some groups, cyclosporine A (CsA) at a dose of 15 mg/kg was given daily intramuscularly until the end of each experiment. Untreated Lewis rats rejected the grafts at 2 and 3 days after transplantation. Neither CsA treatment alone nor clodronate treatment alone prolonged graft survival. Five of 7 Lewis recipients treated with clodronate and CsA did not reject hamster hearts for 100 days. Antibody production in the CsA plus clodronate-treated group was suppressed compared with control groups.

Excessive portal flow causes graft nonfunction in small size liver transplantation: an experimental study in pigs.

We investigated the effects of portocaval shunt (PCS) on excessive portal flow in producing sinusoidal microcirculatory injury in small-for-size liver transplants in pigs. The posterior segment of a whole liver (25%) was transplanted orthotopically. The pigs were divided two groups: group A, graft with PCS (n = 11), and group B, graft without PCS (n = 11). The PCS was a side-to-side anastomosis of the portal vein and the inferior vena cava. In group A, eight pigs survived for more than 4 days; all pigs except for one died of graft nonfunction within 24 hours in group B. The portal flow after reperfusion decreased in group A, but increased about three times greater in group B than that before the operation (P < .01). In group B, destruction of the sinusoidal lining and bleeding in the periportal areas were observed after reperfusion, findings that were not recognized in group A. These results suggest that graft nonfunction after small-for-size liver transplantation may be attributable to excessive portal flow producing sinusoidal microcirculatory injury.

Age-related effects of heat stress on protective enzymes for peroxides and microsomal monooxygenase in rat liver.

To evaluate the age-related response of essential cell functions against peroxidative damage in hyperthermia, we studied the biochemical response to heat stress in both young and aged rats. Passive hyperthermia was immediately observed in rats after exposure to hot environments. In aged rats, the rectal temperature maintained thermal homeostasis and increased to the same degree as in young rats. In these aged animals, the damage from heat stress was more serious than in young animals. In aged rats under normal environmental conditions, hepatic cytosolic glutathione peroxidase (GSH peroxidase) activities were markedly higher than those activities in younger rats. Hepatic cytosolic GSH peroxidase activities were induced by heat stress in young rats but were decreased by hot environments in aged rats. Hepatic catalase activities in young rats were not affected by hot environments, whereas in aged rats, hepatic catalase activities were seriously decreased. Catalase activities in the kidney of aged rats were also reduced by hot environments. Lipid peroxidation in the liver was markedly induced in both young and aged rats. Because the protective enzymes for oxygen radicals in aged rats were decreased by hot environments, lipid peroxidation in the liver was highly induced. In aged rats, lipid peroxidation in intracellular structures such as mitochondria and microsomes was also markedly induced by hot environments. In both young and aged rats, hyperthermia greatly increased the development of hypertrophy and vacuolated degeneration in hepatic cells. In aged rats, both mitochondria and endoplasmic reticulum of the hepatic cells showed serious distortion in shape as a result of exposures to hot environments. Microsomal electron transport systems, such as cytochrome P450 monooxygenase activities, were seriously decreased by heat stress in aged rats but not in young rats. Although the mitochondrial electron transport systems were not affected by acute heat stress in young rats, their activities were simultaneously inhibited after long-lasting heat exposure. In isolated hepatic cells and polymorphonuclear leukocytes in animals, the 70-kDa heat shock-induced proteins were markedly increased by heat stress. In conclusion, the heat stress-inducible oxygen radical damage becomes more severe according to the age of rats. Because aging and hyperthermia have a synergistic effect on lipid peroxidation, protective enzyme activities for oxygen radicals may be essential for surviving and recovering from thermal injury in aged animals and also in humans.

Residuals of beta-hexachlorocyclohexane, dichlorodiphenyltrichloroethane, and hexachlorobenzene in serum, and relations with consumption of dietary components in rural residents in Japan.

To estimate levels of organochlorine residuals in the Japanese population and the contribution of dietary factors to these levels, we determined serum levels of beta-hexachlorocyclohexane (beta-HCH), hexachlorobenzene (HCB), p,p'-dichlorodiphenyldichloroethane (p,p'-DDD), 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (p,p'-DDE) and p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT) in 41 volunteers (14 men and 27 women) in a rural area of Northern Japan. These organochlorine levels were measured using gas-chromatography mass-spectrometry. By a self-administered dietary history questionnaire, the usual dietary intake was estimated. Their median levels (range) were as follows: beta-HCH, 0.50 (0.05-1.50); HCB, 0.20 (0.02-0.70); and total DDT (p,p'-DDE + p,p'-DDT), 5.0 (0.9-31.0) ng/ml serum. Levels of p,p'-DDD were detected in only seven subjects (0.05-0.6 ng/ml serum). The beta-HCH levels were increased with rice and milk intakes, but the least squares means were not simply increased according to the quartile of the intakes. Concerning HCB, fish intake showed a borderline significant correlation (0.20, P = 0.052). In terms of total DDT, intakes of meat, fish, vegetable and milk showed a positive relationship, although none of them provided statistically significant results. No other statistically significant relation between any organochlorines and any food intakes examined was observed in this study. The present study suggests that organochlorine compounds are transported into the human body via foods in the Japanese population. Their effects on health should thus be investigated and monitored.

Initial treatment of systemic lupus erythematosus with a new artificial reticuloendothelial system.

Artificial reticuloendothelial system, which was made on an immobilized phenylalanine column, has been developed to remove denatured protein from serum. This system was used to treat systemic lupus erythematosus effectively. Lupus angiitis has been markedly improved with 12 treatments.

[Whole body heat production as a quantitative marker of anesthetic management under general anesthesia].

In order to evaluate the effects of the rapid induction and the slow induction of general anesthesia on the management of anesthesia and the circadian rhythm, the time-dependent whole body heat production in mice (ICR, male, 6 to 10 weeks after birth) was measured by calorimeter. In the single administration of either an i.v. anesthetic (thiamylal) or a volatile anesthetic (isoflurane), the minimum heat production and its duration time were similar. In the co-administration of i.v. and volatile anesthetics, the minimum heat production was significantly lower (P < 0.01) and the duration of this minimum production was significantly more sustained (P < 0.01) compared to those seen in the single administration of each anesthetic. Moreover, by measurement of the whole body heat production, the slow induction of general anesthesia markedly affected the circadian rhythm on the next postoperative day. These results indicate that the measurement of the whole body heat production will be a marker for the recovery from general anesthesia and the return to the usual life style (such as the QOL).

[Removal of small molecular proteins by push/pull HDF and alleviation of shoulder joint pain].

The purpose of the present study was to compare hemodialysis (HD) and push/pull HDF in terms of uremic substance removal and clinical improvement. The treatment of patients complaining of shoulder joint pain was changed from conventional HD to HD or push/pull HDF using a hemodiafilter with large membrane pores. Push/pull HDF showed significantly greater removal of beta 2-microglobulin (beta 2-m) and other small molecular proteins than HD, and serum beta 2-m was significantly lower in concentration with push/pull HDF than HD. There was a decrease in the shoulder joint pain from push/pull HDF, and the range of upper arm movement thereby increased. However, neither this decrease in pain nor the increase in upper arm movement resulted with HD treatment. Hence, it was concluded that push/pull HDF is a more effective form of therapy.

[Cardiovascular effects of, and catecholamine response to, high dose fentanyl or NLA in patients for valve replacement].

We measured the cardiovascular effect of, and catecholamine and other hormonal responses to, anesthetic doses of fentanyl and original NLA in 25 patients for open heart surgery. The patients were randomly divided into three groups (group N, F30, F75). During induction, in group N; droperidol 0.25 mg.kg-1 and fentanyl 5 micrograms.kg-1, in group F30; fentanyl 30 micrograms.kg-1, and in group F75; fentanyl 75 micrograms.kg-1 were administered intravenously. Additional fentanyl was administered at a rate of 100 to 200 micrograms.h-1. Droperidol 0.25 mg.kg-1 was administered in group N when cardiopulmonary bypass (CPB) was disconnected. Plasma samples were assayed for norepinephrine, epinephrine, ACTH and cortisol before and after induction, during sternotomy, 60 minutes after institution of CPB, after weaning from CPB, and before as well as after extubation. Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP) and rate pressure product (RPP) were calculated simultaneously at the blood samplings. In all groups, no remarkable change in cardiovascular dynamics was observed. CPB was associated with marked increases in catecholamines, but high dose fentanyl in dose of 75 micrograms.kg-1 was able to suppress epinephrine level more than in group N. In high dose fentanyl group (F30, F75) ACTH was within normal ranges, even during CPB. The results suggest that high dose fentanyl is a complete anesthetic in patients for cardiac surgery. But a large dose of fentanyl causes small decreases in heart rate and arterial blood pressure. Our data indicate that group F30 is an attractive anesthetic technique for patients with valvular disease.

[Changes in liver tissue amino acids during hemorrhagic shock].

Changes in amino acids in liver tissue during hemorrhagic shock were examined in eight mature mongrel dogs using the microdialysis method. The mean blood pressure was maintained at 50 mmHg by exsanguination, and the values of liver tissue amino acids after 30 minutes showed significant decreases of aspartate, glutamate, histidine, glutamine, glycine and taurine. The decrease of taurine was especially marked. The values dropped further 30 minutes after returning the blood into circulation with some exceptions. These results suggest the possibility that hemorrhagic shock causes reduction in uptake of amino acids by the liver or consumption of amino acids in the liver, and that bile acid metabolism appears to be disturbed by the marked drop in the taurine level. It is necessary to give careful consideration to the possible existence of reduced amino acid metabolism in the liver in the treatment of hemorrhagic shock.

Histological observations on some of the endocrine glands in the ironfish, with special regard to the hypophysis.

Endocrine glands of the ironfish were studied light microscopically to elucidate the relationship between the races of the genus Carassius. The rostral pars distalis of the adenohypophysis consists mainly of acidophils corresponding to prolactin cells and lead-hematoxylin positive cells corresponding to corticotrophs (ACTH cells). The prolactin cells are obviously arranged in the form of follicles. The proximal pars distalis contains orangenophils (STH cells) and two types of basophils (TSH and GTH cells). In the pars intermedia, three types of cells are identified: periodic acid Schiff positive cells, lead-hematoxylin stainable cells and orangenophils containing PAS positive coarse granules. In addition, small agranular cells are seldom demonstrated in the entire adenohypophysis. A considerable amount of aldehyde fuchsin positive neurosecretory material is laden in the cells of the nucleus preopticus and in the pars nervosa. The nucleus lateralis tuberis is composed of three portions: pars anterior, pars posterior, and pars lateralis. The thyroid follicles found in the pharyngeal region and head kidney show a mild state of activity. The interrenal cells intermingled with the chromaffin cells have a considerable amount of eosinophil cytoplasm. Judging from the gonadal condition, the breeding season of the ironfish may extend to the end of July.

Application of the chemiluminescent assay to cytotoxicity test: detection of menadione-catalyzed H2O2 production by viable cells.

Menadione-catalyzed H2O2 production by viable cells is proportional to viable cell number. The correlations between the viable cell number and the concentration of H2O2 produced are determined with the rapid chemiluminescent assay (S. Yamashoji, T. Ikeda, and K. Yamashoji, 1989, Anal. Biochem. 181, 149-152). This chemiluminescent assay of viable cells requires only 10 min and is much faster than NR (neutral red) inclusion and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assays, which require 3-5 h. When viable cells are incubated with antitumor drugs, detergents, mycotoxins, and glycoalkaloids for 24-48 h, a decrease in menadione-catalyzed H2O2 production in a dose- or incubation time-dependent manner is observed. In general, the 50% inhibition concentration determined by the chemiluminescent assay is lower than that determined by NR inclusion and MTT reduction assays, and the order of relative cytotoxic effects of agents is the same among these assays. Furthermore, clear cytotoxic effects are observed by the chemiluminescent assay after 1 h exposure of trypsinized cells to toxic compounds. Therefore, the chemiluminescent assay is expected to be more useful for the rapid detection of cytotoxic compounds than NR inclusion and MTT reduction assays.

Aberrant CYP1A1 induction: discrepancy of CYP1A1 mRNA and aryl hydrocarbon hydroxylase activity in mutant cells of mouse hepatoma line, Hepa-1.

We have isolated new benzo[a]pyrene-resistant clones, cl-21 and cl-32, of the mouse hepatoma line, Hepa-1. CYP1A1-dependent aryl hydrocarbon hydroxylase activity is not inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3-methylcholanthrene in these two cell lines. However, mRNA of CYP1A1 is inducible in cl-21 and cl-32 cells, as in the wild-type cells, in spite of an undetectable level of cytosolic Ah receptor. The cl-21 cDNA of Cyp1a-1 was found to have a single mutation leading to an amino acid substitution from Leu (118) to Arg (118). However, the CYP1A1 protein band was not detected on Western immunoblots. The cDNA of cl-32 was found to have a single mutation leading to an amino acid change from Arg (359) to Trp (359). The presence of the mature protein in cl-32 was confirmed by Western blot analysis. Somatic cell hybridization experiments demonstrated that the phenotype of cl-21 and cl-32 is recessive and that these clones belong to the same complementation group. These data suggest that there may be a non-Ah receptor-mediated mechanism of CYP1A1 induction.

Interaction of antimalarial agent artemisinin with cyclodextrins.

To obtain an effective solution of the poorly water soluble antimalarial agent artemisinin, the use of several kinds of cyclodextrins (CDs) as solubilizers was examined. The following CDs were used in this study: alpha-CD, beta-CD, gamma-CD as parent CDs, 2-hydroxypropyl-beta-CD (HP-beta-CD), sulfobutyl ether beta-CD (SBE7-beta-CD), heptakis (2,6-di-O-methyl)-beta-CD (DM-beta-CD), 2,3,6-partially methylated-beta-CD (PM-beta-CD) as modified CDs, and glucosyl-beta-CD (G1-beta-CD), and maltosyl-beta-CD (G2-beta-CD) as branched CDs. The solubility curves of artemisinin with CDs can all be classified as type AL. The apparent stability constants for artemisinin-parent CD complexes increased in the order of alpha- < gamma- < or = beta-CD. The constants for artemisinin-beta-CD derivative (and beta-CD) complexes increased in the order of G2-beta-CD approximately equal to G1-beta-CD approximately equal to PM-beta-CD approximately equal to beta-CD < HP-beta-CD < SBE7-beta-CD < DM-beta-CD. These results suggest that the addition of CDs enables the solubilization of artemisinin.