Gene therapy with bilirubin-UDP-glucuronosyltransferase in the Gunn rat model of Crigler-Najjar syndrome type 1.

Crigler-Najjar syndrome type 1 (CN type 1) is an autosomal recessive disorder characterized by nonhemolytic jaundice resulting from mutations to the gene encoding bilirubin-UDP-glucuronosyltransferase (UDPGT). The Gunn rat is an accurate animal model of this disease because the bilirubin-UDPGT gene in this strain carries a premature stop codon. The primary objective of this study was to complement this deficiency in vivo using liver-directed gene therapy. The efficiency of adenovirus type 5 (Ad5)-mediated gene transfer to the neonatal rat liver was first assessed by intravenous (i.v.) injection of an Ad5 vector carrying a nuclear-localized LacZ gene. An Ad5 vector expressing the cDNA encoding human bilirubin-UDPGT (Ad5/CMV/hUG-Br1) was then generated and injected i.v. into neonatal Gunn rats. Plasma samples were collected and bilirubin levels were determined at regular intervals. Although the mean level of bilirubin in homozygous Gunn rats 1-2 days after birth was already 14.5-fold higher than that of heterozygous siblings, treatment with Ad5/CMV/hUG-Br1 reduced plasma bilirubin to normal levels within 1 week. Plasma bilirubin in the treated homozygous rats remained normal for 4 weeks before gradually climbing to intermediate levels that were approximately half that of untreated homozygotes by 12 weeks. Administration of Ad5-mediated gene therapy to neonatal Gunn rats effectively complemented the deficiency in bilirubin-UDPGT, resulting in substantial reductions in plasma bilirubin over a 3-month period. The efficacy of Ad5-mediated gene therapy in neonates suggests that this approach might be effective against other hepatic disorders, including autosomal recessive deficiencies in lipid metabolism and vascular homeostasis.

Corneal endothelial repair. Regulation of prostaglandin E2 synthesis.

PURPOSE: The authors have investigated the hypothesis that prostaglandin E2 (PGE2) synthesis is regulated during corneal endothelial wound healing. Previous studies have shown that PGE2 is an important mediator of endothelial mitosis, migration, and differentiation. METHODS: Biosynthesis of PGE2 was investigated in a wound closure model of the cultured rabbit corneal endothelium and in cultures treated with experimental agents. Prostaglandin E2 synthesis was measured by enzyme-linked immunosorbent assay. Pharmacologic experiments were designed to evaluate the contributions of protein kinases, phospholipase A2, and cyclooxygenase to endogenous PGE2 synthesis. RESULTS: Prostaglandin E2 synthesis is increased markedly in response to injury and is proportional to the extent of wounding. Biosynthesis of PGE2 returns to basal levels concurrently with recovery of the injury. Synthesis is dependent on the activities of protein kinase C (PKC), phospholipase A (PLA), and cyclooxygenase. Two forms of cyclooxygenase are present in corneal endothelial cells, and pharmacologic studies indicate that the activity of the COX 2 contributes to injury-dependent PGE2 synthesis. CONCLUSIONS: Prostaglandin E2 synthesis is increased in injured corneal endothelial cells. This synthesis is dependent on the coordinated regulation of PKC, PLA, and cyclooxygenase. Prostaglandin E2 synthesis presents an attractive target for pharmacologic manipulation of endothelial recovery from injury.

[Effect of dl-alpha-tocopherol on incorporation of selenium in "selenium-indicating" organs and on glutathione peroxidase activity in rat and rabbit erythrocytes following application of therapeutic doses of sodium selenite]. / Einfluss von dl-alpha-Tokopherol auf die Seleneinlagerung in "selenanzeigende" Organe und die Aktivität der Glutathionperoxydase in den Erythrozyten von Kaninchen und Ratten nach der Aplikation von Natriumselenit in therapeutischer Dosis.

An explanation of the functions of both vitamin E and selenium in metabolism and an account of the correlations between them is followed by reference to the results obtained by the authors of this paper from studies into the effects of dl-alpha-tocopherol on selenium levels in the M. longissimus dorsi, blood, and liver as well as on the activity of glutathione-peroxidase (EC 1.11.1.9) in erythrocytes of rabbit and rat, following application of therapeutic doses of selenium (0.5 mg/kg live weight). In both species selenium application increased the glutathione-peroxidase activity in erythrocytes. Vitamin E had no additional effect. Application of selenium was followed by rise in intraorganic selenium concentrations. In rabbit, the effect of vitamin T on intraorganic distribution of selenium caused an increased of the selenium level in the liver, but not in the muscles. No vitamin E effect was recordable in the rat. The findings are discussed, with conclusions being suggested for the treatment of metabolic disorders in the context of selenium and vitamin E and for non-invasive liver therapy.

[Glutathione peroxidase activity in erythrocytes and selenium concentration in blood of untreated and selenium-treated rabbits]. / Aktivität der Glutathionperoxidase in den Erythrozyten und Selenkonzentration im Blut unbehandelter und selensubstituierter Kaninchen.

White New Zealander rabbits were tested for erythrocyte-borne reference values of glutathion peroxidase Px activity, with correlations being established between that activity and selenium content of the blood. The average glutathion peroxidase Px activity in untreated clinically intact rabbits was 11.8 K/g Hb. That value doubled following five selenium applications in therapeutic dosage. The values empirically determined were well adapted to normal distribution. Selenium concentrations recorded from organs of control animals were in fair agreement with values established in earlier studies (Wiesner et al., 1978). The correlation coefficient was r = 0.7117 (n = 44, alpha less than 0.001). The equation of regressive straight line Y oder X (y) was y = -5.3 + 59.94x, and that of X over Y (y) was y = 0,27 + 0,0095y, when Y defined the activity of glutathion peroxidase Px and X the selenium level in the blood.

[Distribution pattern, statistical analysis and correlation of selenium levels in swine selenium-indicating organs]. / Verteilungstyp, statistische Msszahlen und Korrelationen des Selengehalts in "selenanzeigenden" Organen von Schweinen.

There is a potential risk of excessive selenium levels in organs of swine, resulting in toxicity and residues in pork, or selenium deficit. Therefore, random selenium mean values in "selenium-indicating" organs of pigs selected from suspicious populations were compared with mean and limiting values (reference or normal values) recorded from animals with intact metabolism. Prerequisites required for such comparative assessment included the availability of estimated variance values and knowledge of the presence of abscence of agreement between normal distribution and empirical frequency distribution for the population concerned. Knowledge must be available also on the informative value of measured selenium data in blood plasma and their relevance to the general selenium situation in the organism and muscle at large. These were some of the problems studied by determining selenium levels in the liver, kidneys, blood plasma, and M. longissimus dorsi. Organic selenium concentrations were found to be distributed with right axis deviation but almost normal. The parameters established were typical of the majority of data known from literature. Those date, however, are quite variable, so that the need for independently prepared reference values cannot be abandoned. Correlation analysis showed reciprocal relationships between selenium levels in blooc plasma, liver, and muscles but much less correlation between these, on the one hand, and selenium in kidneys, on the other. The correlations between blood plasma and muscle selenium were close enough to take blood plasma values recorded from the living animal as reference from which to draw conclusions as to the muscular selenium state.

[Studies of the tolerance and toxicity of Luzern-green-meal pellets after selenium fertilization]. / Untersuchungen über die Verträglichkeit und Toxizität von Luzernegrünmehl-Pellets nach Selendüngung.

In a feeding trial, rabbits allotted in 3 experimental groups were fed rations containing 2.09, 9.83 and 19.5 mg selenium/kg feed in the form of selenium-enriched alfalfa green meal pellets. The selenium enrichment was done by foliar application (spraying) of the 20--25 cm high plant stand with 2.5 kg SeO2 per hectare in watery solution. The control animals were given normal alfalfa green meal pellets of 0.16 ppm selenium content. Toxicity and lethality, tolerance limit and nutritive effect of the pellets were studied. Plant-assimilated selenium was found to be converted more efficiently by the animal organism than was selenium from inorganic compounds (higher retention rate, better gain in body weight and lower feed expenditure). Therefore, the selenium supply to farm animals should be improved by feeding crops that were given selenium dressings.

[Long-term feeding of acetyl urea to dairy cows]. / Langfristige Fütterung von Azetylharnstoff an Milchkühe.

After an introductory survey of investigations dealing with the conversion of acetyl urea in the ruminant organism, a feeding trial using dairy cattle is described in which the effects of long-term acetyl urea feeding on the clinical picture and various performance parameters have been studied. Five Black-Pied cows of medium milk yield were fed a natural diet and received, over at least 14 months, a daily acetyl urea supplementation of 430 g (= 40 to 44% of the digestible crude protein). The clinical control, including the analysis of blood parameters, did not produce any deviation from normal. As to milk and butterfat yields, the experimental group reached results comparable to those of the control group. Over the entire experimental period the experimental group and the control averaged per cow and day of milking 11.1 and 11.3 kg FCM, respectively. The reproductive behaviour proved normal for all the cows under study. Postmortem findings of 4 cows revealed, among other things, chronic kidney alterations that cannot be safely exluded to be caused by the experiments. A stress over several months of two cows fed 850 g acetyl urea per day was not found to affect animal performance and health.

[LD50 values and selenium concentration in rabbit organs after parenteral administration of sodium selenite and determination of toxicity of urososelevit pro inj]. / LD50 und Selenkonzentration in den Organen von Kaninchen nach intramuskulärer Applikation von Natriumselenit und Prüfung der Toxizität von Ursoselevit pro inj

The LD50-values of sodium selenite after i/m injection to rabbits was determined by means of probit-regression straight line. Simultaneously a combination of vitamin E and sodium selenite was tested for toxicity in order to examine the effect of vitamin E on the selenium toxicity. The LD50/24h was 2.53 mg sodium selenite/kg body mass and in combination with vitamin E (30 IU vitamin E/5 mg NaSeO3) 2.73 mg/kg. Furthermore the selenium concentration was determined in blood, liver, kidney and muscles of animals which died or were killed after 24 h respectively. Not only chemical analysis but also relations of time-dose-efficiency and significantly increased LD50-values show a certain protective effect of vitamin E on selenium intoxication.

[LD 50 and selenium concentration in the organs of rabbits following oral administration of sodium selenite and testing of the toxicity of Ursoselevit-Prämix]. / LD50 und Selenkonzentration in den Organen von Kaninchen nach oraler Applikation von Natriumselenit und Prüfung der Toxizität von Ursoselevit-Prämix

LD50/24hr was established in the first of a series of experiments on 72 rabbits for orally applied sodium selenite. The dosage was 8.62 mg/kg live weight, the confidence interval being (1 - alpha = 0.95) +/- 0.13 mg/kg. The value was four times as high following intravenous application. Complete lethality was recorded from 15 mg Na2SeO3/kg live weight within 21 hours. Thirty-six animals were involved in the second experiment of the series. They had 50 or 100 per cent Ursoselevit-Prämix (30 ppm Se) in their rations. Body mass development of the test animals was superior to that recorded from the controls in the first 50 days, after which limit the former declined strongly in a few days. Their general condition worsened. Postmortem findings, following slaughter, included catarrhal enteritis, toxic liver dystrophy, scattered pulpous tumours in the spleen, and interstitial nephritis. In the third experiment (50 per cent Ursoselevit-Prämix with 60 ppm Se in the rations), the test animals developed better than the controls during the first two months, after which point they exhibited the same clinical symptoms as those observed in the second experiment, stopped to put on weight, and eventually turned cachectic. The pathomorphological findings were identical with those obtained from the second experiment. The selenium concentrations in the organs of the test animals all were much higher than those of the controls. Their amounts in excess to base values were up to eleven times in the blood, nine times in the liver, twelve times in the kidneys, and 13 times in the muscles.

[LD 50 and selenium concentration in organs following intravenous administration of sodium selenite in the rabbit]. / LD50 und Selenkonzentration in den Organen nach intravenöser Verabfolgung von Natriumselenit an Kaninchen

In experiments on 72 rabbits, the LD50 of sodium selenite by intravenous injection was found to be 2.24 mg/kg body weight. The clinical picture and pathological changes associated with acute selenium poisoning are described. The increase in selenium concentration was up to nine times the normal in liver and up to six times in kidney and muscle. There was no sign of a return to normal values 24 hours after administration.