Starting with 41.5 l of plasma from anti-HBe positive carriers of HBs antigen, 11,400 doses of a hepatitis B vaccine with 42 micrograms HBsAg-protein and 11,300 national units HBsAg activity per dose were obtained. After purification, HBsAg is obtained in 99% purity with a yield of more than 90% protein. A possible residual infectivity was inactivated by a diluted formalin solution. The infectivity test in chimpanzees confirmed the absence of infectious hepatitis viruses (HBV and nonA-nonB). In guinea pigs the immunogenicity of the vaccine was comparable to that of the reference preparation from the U.S. National Institute of Health. The presence of Al(OH)3 in the vaccine increased the anti-HBs titre by factors of 30-50. After vaccination with two doses 41 of 45 persons became anti-HBs positive, with three doses 42 of 45 persons developed anti-HBs. Median anti-HBs titre after the third doses: schedule I (three doses in intervals of 6 weeks) 427 mWHO-U/ml; schedule II (two doses at an interval of 4 weeks, third doses 4 months after the first doses) 1535 mWHO-U/ml. The vaccine was well tolerated. There were minor local reactions only.

The new thrombin inhibitor CRC 220 was characterized in vivo for its antithrombotic effects. CRC 220 led to a dose-dependent prolongation of clotting parameters as determined in rats, rabbits, dogs, sheeps, pigs and monkeys. We evaluated the efficacy of CRC 220 to prevent thrombus formation in arteries and in the microcirculation in different animal models. In a rabbit model of tissue factor-induced coagulation activation, infusion of 0.5 mg/kg x h CRC 220 (3 hours) led to a significant prevention of fibrinogen decrease. In a rat model of lethal LPS-induced DIC CRC 220 significantly prevented the mortality rate after a 4h-infusion of 0.75 mg/kg x h. Thrombin-induced platelet aggregation in rat lungs could be prevented by the i.v. bolus injection of CRC 220. A dose of 0.3 mg/kg leads to a reduction of more than 80% of platelet deposition in the lung, significant inhibition was still observed 90 minutes after CRC 220 administration; at this time the inhibitor had already been cleared from plasma. Arterial thrombosis was induced in rabbits by squeezing and stenosis of the A. carotis. The i.v. bolus administration of CRC 220 dose-dependently prevented thrombus formation, an ED50 of 0.03 mg/kg was calculated. This dose was associated with only a minor prolongation of aPTT.

Recently, we showed in a cynomolgus monkey model that the combination of interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factors (GM-CSF) demonstrates synergistic effects, if administered sequentially. Not only were progenitor cells of the myelocytic lineage stimulated cooperatively, but platelet release was also observed. These effects could not be found using these factors alone. In this study, GM-CSF was administered with erythropoietin (EPO) in a sequential manner. This treatment of GM-CSF and EPO resulted in a GM-CSF-stimulated increase in cells of myelomonocytic differentiation, and enhanced stimulation of erythroid and megakaryocytic lineages. The observed effects of growth factor combination were seen to the same extent with intravenous or subcutaneous administration. The results obtained may predict the potential usefulness of GM-CSF treatment together with EPO. This could improve the effectiveness of single growth factors in only limited indications, e.g., reduced need for transfusion (platelets, blood), correction of iatrogenic anemia, and recovery from myelosuppression due to chemo- and radiotherapeutic agents.

We tested the ability of recombinant human (rhu) mast cell growth factor (MGF), also known as c-kit ligand, to stimulate the colony formation of human bone marrow cells in semisolid medium alone and in combination with rhu erythropoietin (EPO), rhu Interleukin 3 (IL-3), rhu granulocyte colony stimulating factor (G-CSF) and rhu granulocyte-macrophage colony stimulating factor (GM-CSF). The addition of MGF to cultures containing EPO or EPO + IL-3, GM-CSF and G-CSF, resp., resulted in macroscopic erythroid burst-forming units (BFU-E). Multipotential (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]) progenitors were stimulated by MGF in the presence of EPO. Colony-forming unit granulocyte-macrophage (CFU-GM) were activated by MGF only in combination with GM-CSF. The combination of MGF with EPO was used for synergism studies in healthy cynomolgus monkeys. In the chosen concentration MGF alone had no effect on white blood cell (WBC) counts and on platelets, but a slight effect on reticulocytes. EPO by itself increased reticulocyte counts with no effects on WBC or platelets. The combination of both factors resulted in a significant increase of reticulocytes. No other effects were seen. These studies demonstrate the potent synergistic interaction of MGF and other hematopoietic growth factors.

The primary growth factor for erythropoiesis is the glycoprotein hormone erythropoietin (EPO) [1], which acts through high-affinity cell surface receptors [2, 3]. However, EPO is not the sole regulatory erythropoietic hormone, and other factors such as interleukin-3 (IL-3) [4], erythroid-potentiating activity (EPA) [5], and murine granulocyte-macrophage colony-stimulating factor (GM-CSF) [6] appear to have an interactive role with EPO in mediating erythropoiesis. The mechanisms involved in these interactions are as yet unclear, although it has been demonstrated that EPA induces increased activity of EPO receptors, whereas IL-3 induces a rapid down-modulation of EPO receptors [7]. GM-CSF has been shown in the murine system to have no effect on EPO-binding [8].

The potency of purified chick embryo cell rabies vaccine Rabipur was evaluated by four different in vivo test systems: (1) NIH test with homologous strain LEP challenge; (2) intramuscular challenge of vaccinated laboratory animals; (3) postexposure vaccine treatment trials in laboratory animals; and (4) antibody induction tests in laboratory animals. These different test systems were compared with the Standard NIH test and an in vitro test, the modified ABT. Each of the four in vivo methods chosen demonstrated potency values superior to that of the Standard NIH test in which challenge was with CVS strain. Potency relations were: Standard NIH test versus alternative methods versus modified ABT 1.0-2.2-1.85 respectively. There was good correlation between alternative in vivo methods and the ABT. In addition examples of antibody induction in man following administration of various postexposure treatment regimens are presented which lead to the conclusion that postexposure vaccination method and composition of the study group play a more important role in achieving optimum treatment results than vaccine potency, provided that it corresponds to WHO requirements (minimum 2.5 IU per dose).

The regulation of blood cell formation is mediated by a group of polypeptides classified as hematopoietic growth and differentiation factors. Overlapping as well as distinct functions have been described for three of these cytokines: interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). Furthermore, interleukin 1 (IL-1) has been shown to promote hematopoietic regeneration after cytoreductive drug treatment. Evidence has been provided by in vitro studies that combinations of these factors exert a highly synergistic action on the proliferation and differentiation of committed hematopoietic progenitor cells. Additionally, these findings have been supported by studies of in vivo blood cell formation in nonhuman primates. We report here that IL-3 acts synergistically with GM-CSF or G-CSF on myelocytic cell development only if an administration time of eight days of IL-3 is followed by GM-CSF or G-CSF. Short-time IL-3 application of two and four days only resulted in platelet production. The reverse administration schedule did not show synergistically enhanced stimulation of myelocytic cells. However, G-CSF treatment followed by IL-3 did induce a two-fold increase in platelet numbers. This would appear to confirm previously reported in vitro findings that G-CSF shortens the G0 period of human hematopoietic stem cells, which subsequently proliferate in the presence of IL-3. The effects of IL-3 on myelocytic and megakaryocytic development seems to be differently regulated. Whereas, IL-1 failed to display synergistic activity with GM-CSF or G-CSF is sequentially applied. Only simultaneous application either in combination with GM-CSF or with G-CSF demonstrated enhanced efficacy.

This article gives a survey on the pharmacological action of the new polyamine compound 15-Deoxyspergualin (15-DS). 15-DS is a derivative of the antitumor antibiotic spergualin which has been isolated by Prof. Umezawa's group in Japan. Besides its antitumor activity 15-DS was shown to possess immunosuppressive properties making the substance suitable for the application in organ transplantation and autoimmune disorders. Due to its immunopharmacological mode of action known hitherto 15-DS clearly differs from other immunosuppressants as e.g. Cyclosporin A.

In protection tests on white mice vaccinated with BCG vaccine and challenged with a pathogenic strain of Mycobacterium bovis, the survival times are considerably altered by several variables. In the strains of mice used mainly in this study (NMRI and Albany), the median survival time of a group was roughly doubled in the sensitive range of the test system either by a twofold increase in the immunization period, a threefold decrease in the challenge dose or a 100-fold or less increase in the vaccine dose. The shape of the survival curve of an animal group depends on the median survival time achieved. The Gaussian distributions (sum curves) of the logarithms of the individual survival times are near linearity and parallelity in groups of animals which either survive for short or very long periods. In an intermediate range, however, the survival curves show a flatter and sometimes S-shaped course. This intermediate range of survival corresponds to the time at which the lung findings shift from acute to chronic. The occurrence of acute or chronic findings depends on the individual survival time after challenge. The autopsies show that both findings are equally frequent ≈35 days after challenge. Individual survival times should be evaluated by non-parametric methods due to their non-normal (bimodal) distribution. Evaluation of the gross lung findings supports these results but is, however, less efficient. The discriminating power of the test system can be altered by changes in any of the variables and is best when animal groups attaining <20 days median survival time are compared with groups attaining >30 days. A twofold increase in the median survival time generally provides evidence of significance that may already be obtained 30 days after challenge. With a vaccination-challenge interval of 21 days or more, a 50 μl vaccine dose generally induces a significant increase in the survival times of the vaccinated animals versus non-vaccinated controls. With increasing immunization periods (vaccination-challenge interval), however, a difference in the efficacy of several vaccines or vaccine doses will be evened out.

A critical review of the NIH test is given. The results of four studies are presented which show that in the case of purified chick embryo cell inactivated rabies vaccine there is a profound difference between the result after heterologous intracerebral challenge with the prescribed CVS strain and homologous challenge with the LEP-C 26 strain. CVS strains from various laboratories produced relatively uniform results following intracerebral administration. Two in vitro tests, the single radial immunodiffusion test and the modified antibody binding test were employed in the studies. The results are discussed and alternative procedures to the NIH test are recommended for future consideration.

Recent progress in molecular cloning has provided access to several major human colony-stimulating factors - GM-CSF, IL-3, G-CSF and M-CSF. Now they are available highly purified from different expression systems (e.g. yeast, E. coli, CHO cells). These molecules, acting multifunctionally in the hematopoietic system, are responsible for proliferation and differentiation of bone marrow-derived progenitor cells in vitro. First clinical studies performed with colony-stimulating factors have shown that neutropenia caused by drug-induced immunosuppression in patients with refractory cancer or autologous bone marrow transplantation was reversed using rh GM-CSF or rh G-CSF. We have investigated the effect of the consecutive administration of rh IL-3 and GM-CSF on hematopoiesis in normal cynomolgus monkeys. Whereas administration of rh IL-3 alone did not result in an increase of WBC counts, the combination therapy of rh IL-3 followed by rh GM-CSF exhibited significant synergistic effects and raised WBC numbers. Furthermore, application of both factors resulted in a platelet rise not seen when one of the factors was used alone. The response was dose dependent and implicated that the therapeutical potential of rh GM-CSF could be expanded if used in combination with rh IL-3.

The antiallergic effect of Fc fragments prepared from human polyclonal IgG was investigated in several experimental models. In an in vitro assay, isolated ileum of cynomolgus monkeys was sensitized with serum from atopic patients. In six of fifteen monkeys the subsequent addition of specific allergens reproducibly resulted in an ileum contraction measured in the Schultz-Dale apparatus. In all six positive monkeys pre-treatment of the isolated ileum with Fc (papain) before sensitization inhibited ileum contraction. Fc (plasmin) or Fc (pepsin) was less or not effective, aggregated IgG, monomeric IgG, sulfonated IgG, the Fc-free F(ab')2 moiety, or albumin had no significant or no reproducible inhibitory effect. In an in vivo assay passive cutaneous anaphylaxis was studied in 28 cynomolgus monkeys. Different dilutions in PBS of the particular specific allergens were subsequently administered to sensitized skin sites, simultaneously to i.v. injection of Evans blue. The degree of the local allergic (passive cutaneous anaphylactia) reaction was evaluated by measuring the diameter of the blue area at the dermal injection sites. About 50% of sera induced positive reaction in monkeys. Compared to the control (application of PBS), the degree of the positive reactions could be reduced by about 50% if Fc (papain) (optimal dose 25 mg/kg body weight) was injected i.v. either 2 h before or after local sensitization. No significant inhibitory effect could be found with the Fab moiety of IgG. Follow-up studies showed that the inhibitory effect of Fc lasted for about 10 days. The pharmacological basis of the observed antiallergic action of Fc is not yet understood.

The modified antibody binding test, an in vitro method, was validated by a number of studies and was used as a reliable method for in process controls in the manufacture of rabies vaccines for man and animals over the last 6 years. A calculation was made showing the number of laboratory mice which were not needed for animal experiments because these were substituted by the in vitro method mentioned above.

In order to overcome the difficulties and risks involved in production of HDC rabies vaccine for human use, a new tissue culture rabies vaccine for use in man has been developed. The Flury LEP-C25 strain was propagated in primary chick embryo cells (CEC), the tissue culture fluid was purified and concentrated by continuous density gradient centrifugation and inactivated by betapropiolactone. The antigen concentrates were tested by the modified antibody binding test and the vaccine adjusted to contain >2.5IU per dose. It was imposed that this vaccine should match the high standards of potency and tolerability achieved in the field by the HDC-vaccine. Therefore, this vaccine had to undergo numerous laboratory trials before clinical tests in man were considered.

A new inactivated rabies vaccine (purified chick embryo cell vaccine) has been developed using the Flury LEP-C 25 strain of rabies virus propagated in primary chick embryo cell cultures. The antigen was purified and concentrated by continuous density gradient centrifugation and inactivated by betapropiolactone. This vaccine was tested for innocuity, tolerability and protective capacity in a series of laboratory tests and compared with human diploid cell strain (HDC)-vaccines of similar antigenicity. The results indicated that this new vaccine was excellently tolerated and that its protective activity met the high standard of HDC-vaccine, conditions which were imposed on this vaccine before entering clinical trials in man.