Development and validation of a bioassay for interleukin-2.

A reliable and precise method for the determination of IL-2 activity, based on stimulation of CTLL cell proliferation, was developed. Cells were incubated with different concentrations of IL-2 for 24 h in microtiter plates. The stimulatory effect was measured on a plate-reading spectrophotometer by reading the optical density of formazan, which is produced by viable cells from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The bioassay was designed as a four-dose parallel line test, fulfilling pharmacopoeial requirements for assay validity, and the inter-assay relative standard deviation (RSD) for a group of four experiments was 2.6%. The International Standard for human IL-2 and the Reference Reagent for Recombinant DNA-derived IL-2 were employed for potency determinations. The method was found suitable for potency assessments of pharmaceutical formulations of IL-2.

Autoradiography of 3H-alpha-fluoromethyl histidine in mice: correlation with the kidney histidine decarboxylase activity.

Tritium-alpha-fluoromethyl histidine (3H-alpha-FMH), designed as a kcat-inhibitor of mammalian histidine decarboxylase (EC 4.1.1.22), was administered intravenously in male and pregnant female mice of the NMRI strain and the distribution of tritium in the body recorded by whole-body and microautoradiography. The results showed penetration of radioactivity into most tissues within 5 min. after the injection. After 4 hrs the highest levels of radioactivity were present in the intestinal content and in the kidneys. In the pregnant animal there was also a high labelling of the foetal tissues. When whole-body sections were washed in TCA prior to the autoradiographic exposure to retain only protein-bound radioactivity, a distinct labelling pattern was seen in the kidneys of the pregnant female mice but not in those of the male mice. Microautoradiography of the kidneys showed that the cells involved were located within the proximal convoluted tubuli. In several mouse strains, including the NMRI, the activity of kidney histidine decarboxylase is low in the males but high in females during a transient period of pregnancy. Incorporation of tritium into kidney protein after treatment with 3H-alpha-FMH, was correlated to a loss in histidine decarboxylase activity. The isotopic labelling was confined mainly to a component which cofractionated with histidine decarboxylase in polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions. Our data indicate that the cells described above represent the location of kidney histidine decarboxylase.

Quantitation of human chorionic gonadotrophin (hCG) by radioreceptor assay.

A sensitive radioreceptor assay was developed for pharmaceutical preparations of human chorionic gonadotrophin with the use of rat testicular membranes as receptor preparation and human 125I-chorionic gonadotrophin as tracer. The addition of unlabelled human chorionic gonadotrophin or luteinizing hormone inhibited the binding of 125I-chorionic gonadotrophin to the receptors in a concentration dependent way. Concentrations of human chorionic gonadotrophin between 30-300 mIU ml(-1) were normally used for a three-dose assay fulfilling pharmacopoeial statistical requirements for assay validity. The relative standard deviation for five assays was 7%. Estimates of potency of commercial preparations of human chorionic gonadotrophin obtained with the radioreceptor assay correlated well with corresponding estimates from in vivo assays. The proposed radioreceptor assay, however, provides a considerable saving in the number of animals required, requires less technical support, and is more precise than the in vivo method.

Hyperplasia and hypertrophia in the denervated and distended rat urinary bladder.

The parasympathetically denervated and distended rat urinary bladder was found to have increased fourfold in weight when examined 3 weeks postoperatively. Both in muscularis and mucosa of such a bladder the synthesis of proteins, RNA and DNA was increased severalfold. An increase in the polyamines putrescine, spermidine and spermine was also found; these polyamines are usually linked to protein synthesis. The results suggest that the cells of the two layers increase both in size and number. Hyperplasia was, in a previous study, suggested as a possible explanation for a right-ward shift of the active length-tension curve of muscle strips in the denervated rat urinary bladder.

In vitro metabolism of cadaverine in the pregnant rat.

The in vitro metabolism of 14C-cadaverine by diamine oxidase was investigated in various tissues of pregnant and non-pregnant rats. The metabolites formed were: delta 1-piperideine, delta-aminovaleric acid, carbon dioxide and some unidentified compound(s). In most of the tissues investigated delta 1-piperideine was the predominant metabolite, but considerable amounts of delta-aminovaleric acid and the unidentified compound(s) were also formed. The oxidative products of cadaverine might be of importance in various physiological and pathophysiological connections associated with elevated diamine oxidase levels.

Effect of His-Phe, a competitive inhibitor of histidine decarboxylase, on gastric acid secretion in chronic gastric fistula rats: delay in acid secretion response to pentagastrin.

The dipeptide His-Phe, earlier shown to inhibit mammalian histidine decarboxylase, was analysed concerning its effect in vivo on pentagastrin-induced gastric acid secretion. Chronic gastric fistula rats were used and the effectors in saline were given as continuous i.v. infusions while acid was collected from the fistula. Addition of pentagastrin to the infusion solution resulted in an immediate increase in the acid output of the control runs. In the His-Phe experiments the dipeptide was introduced one hour before pentagastrin. A significant decrease in the acid output was obtained. This effect was optimal at a dose of about 6 mg/h and during the first few hours of the experiments. In spite of the continuous His-Phe infusion the acid secretion increased with time to the control values. These results are discussed in relation to preliminary observations on effects of alpha-fluoromethyl histidine on gastric acid secretion and the effect of this and His-Phe on gastric histamine content and histidine decarboxylase activity.

Polyamine metabolism as related to growth and hormones.

Previous work had revealed a connection between high histamine formation and certain types of rapid growth. Since then evidences have accumulated that diamines abd polyamines affect an impressive number of reactions pertinent to cell growth and proliferation. In studies on polyamine metabolism as related to growth and hormones we have preferentially been working with three types of biological model systems, i.e. the pregnant rat, the gonadotrophin stimulated rat ovary and the mouse kidney induced to growth by androgenic or anabolic steroids.