In vitro analysis of the mode of action of the immuno-suppressive drug 15-deoxyspergualin.

The purpose of the study was to analyse the action of the immunosuppressive drug 15-deoxyspergualin (DOS) in vitro. We studied: a) the influence of DOS alone and DOS in combination with various monoclonal antibodies on alloantigen stimulation in the mixed lymphocyte culture (MLC), b) the influence of DOS treatment on the MHC class I and II expression of splenocytes, lymph node cells and peritoneal macrophages, c) the influence of DOS treatment on a suppressor cell population. Our study showed that: a) DOS inhibits interleukin 1 (IL-1) secretion by macrophages, leading to reduction of immune response to alloantigens. This effect was neutralized by addition of IL-1; b) DOS treatment has no influence on MHC class II antigen expression, but induces changes of MHC class I expression. After DOS application in a population of spleen macrophages a subpopulation of cells with reduced MHC class I antigen expression appeared. Down-regulation of these molecules was also observed in immunomorphological studies of kidney graft sections of rats treated with DOS after transplantation; c) after DOS treatment suppressor cells were detected in "suppressor" MLC, 16-33 days after kidney transplantation. Their activity was confirmed 137 days after treatment with DOS, but were inactive in the case of third party cells. These results suggest that DOS action is based on a blockade of antigen presentation by reducing IL-1 production, down-regulation of MHC class I antigen and by inducing suppressor cell population.

Human monoclonal antibody Ha6D3, a candidate for treatment of leukaemia? In vitro reactivity of Ha6D3 with leukaemic cells and in vivo applications in a chimpanzee.

The human monoclonal antibody Ha6D3 of the IgM type was used to stain malignant lymphoma cells from peripheral blood in flow cytometry and from cryosections of lymph nodes using the immunoperoxidase technique. It was found to react with peripheral white blood cells of all 12 cases of leukaemia and with lymph node cells of seven out of 11 B cell lymphomas and with the one T cell lymphoma tested so far. For in vivo experiments a batch of 70 mg Ha6D3 was purified and 6 mg Ha6D3 was injected intravenously into a chimpanzee with time intervals of 10 months and 1 month. The side effects observed were shivering, some muscular spasms and variations in the heart frequency. A decrease of lymphocytes of more than 50% was documented by haematogram analysis. The flow cytometry data showed that the Ha6D3 antigen does not modulate. Even after three repeated injections applied in a time interval of several months no immune response to Ha6D3 could be detected in vivo or in vitro. Based on these data we suggest that Ha6D3 may become a candidate for the treatment of certain leukaemias in vivo.

Investigations on the interferon-induced 2'-5' oligoadenylate system using analytical capillary isotachophoresis.

The activity of the interferon inducible enzyme 2'-5' oligoadenylate synthetase (2-5A synthetase, E.C. 2.7.7) which converts ATP into a series of 2'-5' oligoadenylates was measured using analytical capillary isotachophoresis. The turnover rate of ATP during the reaction was monitored by determination of its concentration at the beginning and the end of the 2-5A synthetase reaction. The enzyme was analysed in extracts of peripheral blood mononuclear cells either pretreated or not with interferon.

[Bipolar temperature controlled diathermy of the sclera for controlled refractive change of the cornea]. / Bipolare temperaturgesteuerte Diathermie der Sklera zur kontrollierten refraktiven Veränderung der Hornhaut.

A bipolar scleral diathermy unit measuring tissue temperature beneath the electrodes was used in 27 autopsy eyes to induce corneal curvature changes in order to modify corneal refraction. The optimal coagulation temperature was found to be between 65 degrees and 70 degrees C. The refractive effect was influenced by the distance of the electrodes (1 mm, 2 mm, 3 mm), their location ("limbal distance") and their position in reference to the limbus ("radial", "parallel"). The highest corneal curvature changes were observed with diathermy applied directly at the limbus or 1 mm distance from the limbus. The corneal meridian corresponding to the treated sclera regularly became significantly steeper and the untreated 90 degrees meridian flatter. Limbus parallel diathermy administered directly at the limbus showed a 0.91 mm increase in the corneal curvature with a 2 mm distance or a 0.34 mm increase with a 3 mm distance of both electrodes. Placing the electrodes radially or parallel of the limbus revealed greatest changes in corneal refraction using diathermy directly at the limbus or 1 mm distant from the limbus. The refractive effect decreased as the distance from the limbus increased. Corneal astigmatism could be decreased and increased in a quasi-controlled manner. Over-lapping diathermy offered the potential of gradually changing the corneal power. Histological sections showed that scleral collagen had only a superficial coagulation effect. Scleral diathermy offers great advantages in comparison to corneal incisions for the therapy of corneal astigmatism. Further investigations are in progress.

Plasmodium falciparum: inhibition in vitro with lactoferrin, desferriferrithiocin, and desferricrocin.

The microbial iron chelators desferriferrithiocin and desferricrocin as well as human lactoferrin were tested in vitro against Plasmodium falciparum. The microbial chelators inhibit the growth of P. falciparum in a dose dependent way. Parasite multiplication is stopped at 25-30 microM desferriferrithiocin, whereas 60-90 microM desferricrocin are needed to exhibit the same effect. After iron saturation, the microbial chelators are ineffective. Human lactoferrin (30 microM), both iron free and iron saturated, inhibits P. falciparum. A 3-day preincubation of host erythrocytes with iron free and iron saturated lactoferrin prior to infection enhances this effect, which is therefore attributed to lactoferrin bound iron. It has been suggested that the lactoferrin/iron complex generates oxygen free radicals, which may cause membrane damage of both erythrocyte and parasite. This process can be considered to lead to growth inhibition of the parasite.

Flow-cytometric determination of glutathione alterations in vital cells by o-phthaldialdehyde (OPT) staining.

A flow-cytometric method for the detection of changes of cellular glutathione content in vital cells is described. The reaction is based on formation of a fluorescent product between o-phthaldialdehyde (OPT) and reduced glutathione (GSH). OPT is a more GSH-specific dye than other thiol-specific dyes (e.g., bromobimanes), because it forms a cyclic compound with GSH. Changes of GSH induced by oxidation or thiol-blocking agents are visualized in vital cells after a 5-min staining at room temperature.

Plasmodium vinckei: suppression of mouse infections with desferrioxamine B.

Plasmodium vinckei kills NMRI mice within 6 days after infection. Treatment of infected animals with desferrioxamine B for 5 days was found to suppress the parasitemia in a dose-dependent manner. The desferrioxamine B-iron complex (DFO/Fe3+) was ineffective, which suggests that the iron-chelating capacity of free desferrioxamine B is the antimalarial principle. All mice survived when they were given 0.3 mg desferrioxamine B/g every 12 hr for 14 days after infection. In addition, they were resistant to reinfection for at least 8 weeks. Eight months after desferrioxamine B treatment, all mice had lost their induced immunity and were as susceptible to malaria as controls. These results illustrate the dependence of the malarial parasite on ionic iron and suggests new methods for the therapy of parasitic diseases.