Outlines of the immunological research in the Institute of Microbiology; Semmelweis Medical University.

The working group on immunology under the leadership of Pál Földes began its activity with poliovirus studies during the severe epidemics of 1957. It was he who first in Hungary isolated poliovirus strain from patients [1]. His colleague was Ilona, Szeri who had gained her first experiences in virology at József Sinkovics's virus laboratory. Then Zsuzsanna Bános and Piroska Anderlik joined them and became permanent members of the working group on immunology. Since 1965 with the leadership of Ilona Szeri, they have been conducting basic researches into immunology for over three decades at the Institute, with a wide sphere of collaborators. Research has been intended to acquire more thorough and precise knowledge of the role in immunobiology of the thymus and lymphoid system and of pathogenesis of the wasting syndrome as well as of interactions of the microorganisms and the organism. The most significant results are going to be summed up in the following.

Biological effects of microorganisms and their substances in mice.

In our experiments we evidence from several aspects that the normal microbial flora has a permanent and life-long immune modulating role in conventional organisms and a stimulating effect both on specific and non-specific defense. However, in case of artificial interventions (stress, drugs) affecting the organism, existence of the normal flora may have an adverse effect on it (endotoxin effect, bacterial translocation). The immunomodulants show a stimulating effect mainly in organisms with undeveloped immune system, and their effects are independent from the presence or absence of the microbial flora. With ageing, effect of immunomodulants can change and become indifferent or even suppressive. Dose-dependence of stimulating or suppressing effect of immunomodulants may be related to their non-immunological effects (endotoxin effect, bacterial translocation). Finally, on the basis of our experiments, we consider the Gf mouse suitable for examining the effect of a given agent in the practice, on the one hand, and for observing the host organism's reactions, free from the influence of the normal microbial flora, on the other. Along with the known physiological and pathological events, our results draw also attention to as distant fields as drug sensitivity, drug interactions influencing drug sensitivity. The authors put emphasis on importance of germfree environment during immunosuppressive treatments in humans and when making special examinations under experimental conditions.

Modulation of the cellular immune response to intracerebral lymphocytic choriomeningitis virus (LCMV) infection in mice.

Certain viruses do not kill the cells they infect. The immunological response of the host in such situations may be involved in the development of pathologic changes and clinical illness. Pioneering work by Rowe has shown that death associated with acute LCMV infection in the mouse is resulted from the immune response. Many investigators using a variety of techniques including neonatal thymectomy, irradiation, or treatment of adult mice with antilymphoid drugs or antithymocyte sera have confirmed and extended Rowe's observations. The study of LCMV and the disease it causes in its natural murine host has provided the initial findings that open new fields in viral immunobiology, viral immunopathology, and cell-cell recognition.

[Interaction between Bordetella pertussis vaccine and chlorpromazine in mice]. / Bordetella pertussis vakcina és chlorpromazin kölcsönhatása egérkísérletekben.

In acute toxicity experiments authors observed increased mortality on the first day following 75 mg/kg chlorpromazine (CPZ) treatment in mice pretreated with Bordetella pertussis vaccine (9 x 10(9) killed bacterium/mouse) compared to control animals treated with CPZ alone. Initially, the increased drug sensitivity observed after combined treatment was attributed to summation of the toxic effects. However, the cumulation of mortality did not cease on the following days; furthermore, an increase of bacterial translocation (translocation index of P-CPZ group: 4.5) was observed on days 6 and 7, i.e. when the lymphocytosis, splenic hypertrophy and shrinkage of thymus--changes consequent to the vaccination--were at their maximum levels. On the basis of all these and on literary data it is supposed that the early cumulation of deaths after combined treatment may be in connection with an interaction between the two agents and that the side-effects following vaccination of humans may be induced by undesirable pharmacological interactions.

[Interaction between immunomodulatory drugs in mice with special regard to changes of drug sensitivity and rate of bacterial translocation]. / Immunmoduláns anyagok kölcsönhatása egérkísérletekben különös tekintettel gyógyszerérzékenység változásra és a bakteriális transzlokáció alakulására.

In acute toxicity experiments the changes in drug sensitivity and in the rate of bacterial translocation (BT) were investigated in mice treated with immunomodulatory drugs: dianhydrogalactitol (DAG) in doses 20 and 30 mg/kg, chlorpromazine (CPZ) in doses 60 and 75 mg/kg and Mannozym (M) in dose 40 mg/kg for zymosan content. The drugs were used separately or in combination. The sensitivity of mice to immunosuppressive DAG or CPZ was higher in the case of combined treatment, than that of separately treated ones. The rate of BT was also higher in combined treated mice. The pretreatment with M that has immunostimulatory effect, influenced neither the sensitivity of mice to DAG or CPZ, nor the normal very low rate of BT. The present results reinforced the authors' earlier observations, that the effects of immunosuppressive drugs could cumulate and cause more serious damage of the organism. The authors suggest that the increase in drug sensitivity to immunosuppressive agents is in connection with increased rate of BT and effect of endotoxin.

Interaction between Bordetella pertussis vaccine and chlorpromazine in mouse experiments.

In acute toxicity experiments the mortality of mice pretreated with Bordetella pertussis vaccine increased on the first day following chlorpromazine (CPZ) treatment, compared to control animals treated with CPZ alone. Initially, the increased drug sensitivity observed after combined treatment was attributed to summation of the toxic effects. However, the cumulation of mortality did not cease on the following days, furthermore, an increase of bacterial translocation was observed on days 6 and 7, i.e. when the lymphocytosis, splenic hypertrophy and shrinkage of thymus-changes consequent to the vaccination-were at their maximum levels. On the basis of all these and on literary data it is supposed that the early cumulation of deaths after combined treatment may be in connection with an interaction between the two agents and that the side-effects following vaccination of humans may be induced by undesirable pharmacological interactions.

Interaction of immunomodulant substances in mouse experiments with special regard to drug sensitivity and bacterial translocation.

In acute toxicity experiments changes in drug sensitivity and in the rate of bacterial translocation (BT) were investigated in mice treated with immunomodulatory drugs: dianhydrogalactitol (DAG) in doses 20 and 30 mg/kg, chlorpromazine (CPZ) in doses 60 and 75 mg/kg and Mannozym (M) in dose equivalent to 40 mg per kg zymosan. The drugs were used separately or in combination. The sensitivity of mice to immunosuppressive DAG or CPZ was higher in the case of combined treatment than that of separately treated ones. The rate of BT was also higher in mice receiving combined treatment. Pretreatment with M exerting an immunostimulatory effect, influenced neither the sensitivity of mice to DAG or CPZ nor the very low normal rate of BT. The present results reinforced the authors' earlier observations that the effects of immunosuppressive drugs cumulated in and caused more serious damage of the organism. The increase in drug sensitivity to immunosuppressive agents may be connected with an increased rate of BT and effect of endotoxin.

Influence of the thymus and the normal microflora on the plasma fibronectin concentration in mice.

The plasma fibronectin (pFN) concentration (cc) of untreated genetically or artificially athymic mice, or treated with TP-4 (thymus hormone sequence analog synthetic preparation) showed no significant difference from their euthymic or untreated controls. In contrast, the pFN cc in mice with different microbiological state showed significant alterations; the highest level occurred in conventional mice and the lower level in germfree mice was increased by bacterial monocontamination. The alternation from SPF into conventional state in nude mice also resulted in the increase of the pFN cc. Based on these and earlier results, it was assumed that the pFN cc is independent from the presence or absence of the thymus, but it depends on the actual microbiological state of the macroorganism.

[Effect of pretreatment with immunomodulating agents on the outcome of lymphocytic choriomeningitis virus infection in athymic (nude) and euthymic mice]. / Lymphocytas choriomeningitis (LCM) vírusfertözés lefolyása immunmodulánsokkal elökezelt genetikailag thymus-hiányos (nude) és normál immunrendszerü egerekben.

Balb/c (euthymic) and nu/nu (athymic) mice were treated intraperitoneally with TP-4 (a synthetic tetrapeptide, thymopoietin sequence analog, Pharmaceutical Product's Factory Gedeon Richter, Budapest, Hungary) or with Mannozym (0.1% zymosan suspension, Institute for Serobacterological Production and Research, HUMAN, Budapest, Hungary), and were infected intracerebrally with LCM virus. Both of the agents contributed to the development of fatal choriomeningitis, consequently they stimulated the cellular immune response in euthymic mice, but the athymic mice, either treated or not, survived the infection, consequently the agents had no effect on the course of LCM virus infection. The results showed that the cellular immune response stimulating effect by both agents was thymus-dependent. Using these agents immunostimulatory effect can be realized only in the presence of the thymus or the T-dependent lymphoid system, respectively.

Effect of microbial immunomodulants on the course of LCMV infection in old mice with thymus involution.

Old mice with thymus involution were treated intraperitoneally with a live vaccine containing a mesogenic strain of attenuated Newcastle Disease Virus or with Mannozym (M, 1% zymosan suspension). One day after the treatments mice were infected with lymphocytic choriomeningitis virus (LCMV) intracerebrally. The fatal course of the consequent LCMV infection was stimulated by each of the pretreatments, indicating that the cellular immune response was stimulated. The results are compared with results of experiments carried out on suckling, young adult and old mice in similar experimental systems. The authors' previous publication suggesting that the direction and degree of the immunomodulant effect may be influenced by the actual age-dependent condition of the lymphoid system, have been confirmed.

Effect of Mannozym on the course of LCMV infection in mice with undeveloped and normal immune system.

Adult germfree (Gf) mice with undeveloped immune system due to antigen deficient environment, conventional (Cv) mice with normal immune system and Cv suckling mice with undeveloped immune system due to age were treated intraperitoneally with Mannozym (M, 0.1% zymosan suspension) 4 days or 4 days and 1 day before the intracerebral inoculation with lymphocytic choriomeningitis virus (LCMV). One dose of M was equal to 40 mg/kg of zymosan. In suckling mice, both applied doses of M contributed the development of fatal lymphocytic choriomeningitis after infection with 100 LD50 dose of LCMV, thus M pretreatment increased the cellular immune response to LCMV infection. M pretreatments had no influence on the course of LCMV infection either in adult Gf or in Cv mice. Spleen hypertrophy was caused by applied doses of M both in adult (Gf and Cv) and Cv suckling mice, but modulating effect on the cellular immune response manifested simultaneously only in Cv sucklings.

The course of LCMV infection in euthymic and athymic mice pretreated with immunomodulatory agents.

Balb/c (euthymic) and nu/nu (athymic) mice were treated intraperitoneally with TP-4 (a synthetic tetrapeptide, thymopoietin sequence analog) or with Mannozym (1% zymosan suspension), and were infected intracerebrally with LCM virus. Both of the agents contributed to the development of fatal choriomeningitis, consequently stimulated the cellular immune response in euthymic mice, but the athymic mice either treated or not, survived the infection, consequently the agents had no effect on the course of LCM virus infection. Both agents exerted a thymus-dependent cellular immune response stimulating effect. That is, an immunostimulatory effect can be realized only in the presence of the thymus or the T-dependent lymphoid system.

[Effect of microbial immunomodulators on the course of lymphocytic choriomeningitis virus infection in old mice with physiological thymus involution]. / Mikrobiális immunmodulánsok hatása lymphocytas choriomeningitis vírusfertözés lefolyására fiziológiás thymus-involutio állapotában lévö öreg egerekben.

Aged mice with physiological thymus involution were treated intraperitoneally with 0.2 ml of live Newcastle Disease Virus (NDV) containing attenuated NDV vaccine (HA titre: log 2(16)/ml) one day before or with Mannozym (0.1% zymosan suspension) four days and one day before intracerebral inoculation with 100 LD50 dose of LCMV. One dose of M was equal to 40 mg/kg zymosan. Both, NDV vaccine and M pretreatments contributed to the development of fatal lymphocytic choriomeningitis, thus enhanced the cellular immune response to LCMV infection. Present results are compared with earlier results which were attained on young adult suckling mice in similar system with pretreatment with different microbial immunomodulators. Present results reinforced the authors' earlier observations that the direction and degree of immunomodulatory effects can be influenced by the actual conditions, like age, of the immune system.

The course of LCMV infection in gnotobiotic and conventional adult mice pretreated with attenuated NDV vaccine.

A single intraperitoneal treatment with two different doses of live Newcastle Disease Virus (NDV) containing attenuated NDV vaccine one day before intracerebral inoculation with lymphocytic choriomeningitis virus (LCMV) had no influence on the ratio and time of deaths after infection with a 100 LD50 dose of LCMV either in gnotobiotic or in conventional mice. There was no difference either in the LD50 values determined on the basis of three parallel LCMV titration performed on mice pretreated with two different doses of vaccine or untreated. NDV vaccine pretreatment thus did not influence the cellular immune response to LCMV infection either in gnotobiotic or in conventional adult mice. As the NDV vaccine increased the cellular immune response to LCMV infection in suckling mice according to earlier results, the present results reinforce our earlier statement that the direction of immunomodulatory effects can be influenced by age.

Effect of chlorpromazine (CPZ) on the course of LCM virus infection in mice with developed and undeveloped immune system.

The cellular immune response to lymphocytic choriomeningitis (LCM) virus in germfree adult and conventional (Cv) suckling mice with undeveloped immune systems and in Cv adult mice with developed immune systems was suppressed by a single large, sublethal dose of the calmodulin antagonistic chlorpromazine and stimulated by a 100-times smaller dose administered intraperitoneally one day before the intracerebral virus infection. CPZ thus exerted a two-directional dose-dependent immunomodulatory effect in mice with both undeveloped and developed immune system.

Bacterial translocation after cold stress in young and old mice.

Spontaneous translocation of the normal intestinal flora was observed in higher rate in old mice being in a state of thymus involution than in young ones. The proportion of bacterial translocation 24 and 48 h after cold stress increased in both young and old mice, the increase of translocation as compared to controls was larger in case of young mice than in old ones. The distribution of isolated bacterial strains according to Gram stain also differed in young and old groups.

Effect of mannozym treatment on plasma fibronectin concentration in germfree and conventional mice.

As a result of a single intraperitoneal treatment with 1 mg/ml of Mannozym (M) the plasma fibronectin (FN) level was significantly increased both in germfree (Gf) and conventional (Cv) mice, and though it started from a lower value in Gf mice, it rose to a similar level to that of Cv animals. The maximum of FN level was observed on the first day after treatment both in Gf and Cv mice. It had returned to normal by the 7th day in Cv mice, but it was higher in Gf mice as compared to untreated controls even on the 14th day. Thus the increase of FN level was of higher degree and longer duration following M treatment in Gf mice than in Cv animals. In treated Gf mice the plasma FN concentration was in the same range as in untreated Cv mice even at the termination of experiment. Both in Gf and Cv mice, there was a relative spleen weight increase, the degree of which was similar, but the duration was longer in Gf mice than in Cv ones.

Plasma fibronectin concentration in germfree and conventional mice of various age.

Lower plasma fibronectin (FN) levels were detected in C3H germfree (Gf) mice as compared to conventional (Cv) ones. FN levels increased with aging both in Gf and Cv mice. Stimulation of the immune system with live Newcastle disease virus (NDV) vaccine increased the FN levels in young germfree mice, but had no effect in conventionals.