Positive effects of dialyzable leukocyte extract (DLE) on recovery of mouse haemopoiesis suppressed by ionizing radiation and on proliferation of haemopoietic progenitor cells in vitro.

Dialyzed leukocyte extract (DLE) (Immodin SEVAC, Czech Republic) was shown to enhance the recovery of the pools of hemopoietic stem cells (CFUs) and of granulocyte-macrophage hemopoietic progenitor cells (GM-CFC) in the bone marrow in vivo, as well as to increase the numbers of leukocytes and thrombocytes in the peripheral blood of mice exposed to a sublethal dose of gamma-rays, with an ensuing increase in the numbers of mice surviving the lethal radiation dose. In experiments performed in vitro, DLE or sera of mice administered with DLE were added to cultures of intact mouse bone marrow cells containing suboptimal concentrations of hemopoietic stimulatory cytokines, namely recombinant mouse interleukin-3 (rmIL-3) or recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF); under these experimental conditions, both DLE and sera of mice administered DLE were found to increase the counts of GM-CFC colonies in the cultures. It can be hypothesized on the basis of the findings obtained in vitro that the described co-stimulating activity (CoSA) of DLE may play a role also under in vivo conditions; the enhancement of the recovery of hemopoiesis suppressed by ionizing radiation may be due to a co-operation of the stimulatory effects of DLE with the action of cytokines endogenously produced in irradiated tissues.

Stimulation of hemopoietic colony formation from mouse marrow cells in vitro using human dialyzable leukocyte extracts-IMMODIN-SEVAC.

The influence of dialyzable extract from human leukocytes (DLE) on the in vitro growth of the granulocyte-macrophage colony-forming cell (GM-CFC) colonies from progenitors of mouse bone marrow cells was studied. DLE alone did not induce the colony growth but it modulated the number of colonies if administered together with a colony-stimulating factor (CSF). The costimulatory effect prevailed in a broad range of DLE dilution and the index of increase was enhanced with the lowering of the CSF concentration. The costimulatory augmentation of clonal proliferation of GM-CFC with DLE was further strengthened by addition of indomethacin, thus indicating an intervening role of prostaglandins in the modulatory influence of DLE.

The influence of age on transfer factor treatment of cellular immunodeficiency, chronic fatigue syndrome and/or chronic viral infections.

A group of 222 patients suffering from cellular immunodeficiency (CID), frequently combined with chronic fatigue syndrome (CFS) and/or chronic viral infections by Epstein-Barr virus (EBV) and/or cytomegalovirus (CMV), were immunologically investigated and treated with transfer factor (TF). The age range was 17-77 years. In order to elucidate the influence of aging on the course of the disease and on treatment, 3 subgroups were formed: 17-43 years, 44-53 years, and 54-77 years. Six injections of Immodin (commercial preparation of TF by SEVAC, Prague) were given in the course of 8 weeks. When active viral infection was present, IgG injections and vitamins were added. Immunological investigation was performed before the start of therapy, and subsequently according to need, but not later than after 3 months. The percentages of failures to improve clinical status of patients were in the individual subgroups, respectively: 10.6%, 11.5% and 28.9%. The influence of increasing age on the percentage of failures to normalize low numbers of T cells was very evident: 10.6%, 21.2% and 59.6%. In individuals uneffected by therapy, persistent absolute lymphocyte numbers below 1,200 cells were found in 23.1%, 54.5% and 89.3% in the oldest group. Statistical analysis by Pearson's Chi-square test, and the test for linear trend proved that the differences among the individual age groups were significant. Neither sex, nor other factors seemed to influence the results. The results of this pilot study show that age substantially influences the failure rate of CID treatment using TF. In older people, it is easier to improve the clinical condition than CID: this may be related to the diminished number of lymphocytes, however, a placebo effect cannot be totally excluded.

The effect of DLE fractions on GM-progenitors of haematopoietic stem cells in vitro.

Dialysable leucocyte extract (DLE) prepared from buffy coats of human blood, potentiates the effect of Colony-stimulating factor (CSF) on the growth of granulocyte-macrophage colony forming cell (GM-CFC) colonies in vitro. This relative increase of the number of colonies is apparent when diluted CSF (present in lung conditioning medium) as a control, and DLE, in a wide range of concentrations are added to the culture of mouse bone marrow cells. Fractionation of DLE on Amicon membranes revealed that the activity resides in molecules of 0-5 kD. Molecules 5-10 kD have no potentiating effect. DLE and its fractions (0-5 kD, 0-1 kD), except fractions 0-500 D and 5-10 kD, when added undiluted i.e. at the initial concentration, exerted a suppressive effect: colonies are not formed despite the presence of CSF. In a pilot experiment, it was shown that DLE is able to stimulate colony-forming activity of earlier progenitors of erythroid cells (BFUe), under the influence of erythropoietin.

Course of liver regeneration after partial hepatectomy in rats treated with dialysates obtained 17 hours after partial hepatectomy.

Various theories have been put forward to explain the regenerative capacity of liver tissue induce by partial hepatectomy (PH). One of them presumes the existence of humoral factors stimulating proliferation of the liver tissue. We evaluated the course of liver regeneration after 65-70% PH as influenced by dialysates (DIA) of the organs of a rat killed 17 h after PH. In addition to kidney DIA, we were particularly interested in the effect of liver and spleen DIA. The experiments were carried out on rats weighting 310-370 g. Kidney, liver or spleen dialysate was administered subcutaneously and the rats were killed 12 or 24 h later by exsanguination from the abdominal aorta. In further rats, PH was performed 24 h after administering DIA and the rat were killed 18, 24, 30, 48 and 72 h after the operation. The initiation of liver regeneration was stimulated by all the given DIA, but especially by liver DIA. The faster onset of liver regeneration 18 h after PH in rats given spleen DIA is interesting. DIA did not greatly affect the hepatocytes of intact liver, but accelerated the initiation of liver regeneration after PH by synchronizing the cell cycle of proliferating hepatocytes. DIA obtained 17 h after PH contained substances which primarily stimulated liver DNA synthesis. From the changes in inhibition of the migration of spleen macrophages in the medium containing liver antigens, and from the circulating immunocomplex values, we conclude that DIA activation of the immune system, a well as the hepatic stimulator substance contained in the DIA, participates in acceleration of the liver regeneration process.

The patterns of complex and partially purified mycobacterial antigens in macrophage migration inhibition testing.

In rabbits immunized intratarsally by M. tuberculosis, M. kansasii and M. avium the responses to homologous and heterologous antigens were assessed by direct and indirect macrophage migration inhibition tests. Complex cytoplasmic antigens were obtained by disruption of bacterial mass and by ultracentrifugation of the supernatants. The partially purified antigens were prepared by gel chromatography of the complex antigens on a Sephadex G 150 column. The middle fraction (260/280 ratio approx. 1, molecular weight approx. 32 KD) was employed as partially purified antigen. In the direct tests the migration activity of immune spleen macrophages was significantly reduced by homologous complex and partially purified antigens (MI = 0.63 to 0.72) and it differed significantly from responses obtained with heterologous antigens (MI = 0.75 to 0.92); however, these were still lower than those in nonimmunized control animals where MI ranged from 0.89 to 1.01. In the indirect tests, the strongest responses were recorded again with homologous complex and partially purified antigens (MI = 0.43 to 0.53). The responses in heterologous systems differed even more markedly than in direct tests (MI = 0.65 to 0.81); and, these were again still significantly lower than in control animals (MI = 0.89 to 0.98). In both direct and indirect tests, the complex and partially purified antigens did not vary substantially in their immunogenic capacity. The presence of cross-reacting responses in heterologous systems can be explained by a close relatedness of mycobacteria used in the immunization schedule and by the presence of common epitopes in complex and purified testing antigens.

In vitro cellular immune response to homologous and heterologous antigens in rabbits sensitized by five species of Mycobacterium and Nocardia asteroides.

Rabbits were sensitized with killed Mycobacterium tuberculosis, M. kansasii, M. avium, M. scrofulaceum, M. fortuitum, and Nocardia asteroides, and their response to homologous and heterologous antigens was assessed in vitro by direct and indirect macrophage migration inhibition tests. The antigens were obtained by disintegration of bacterial mass and by purification of the supernatants by ultracentrifugation. In the direct test, hypersensitivity to homologous antigen was most marked with M. tuberculosis, M. kansasii, and M. avium (migration indices [MI] = 0.42 to 0.50), but was significantly weaker with organisms possessing a lower degree of pathogenic activity (MI for M. fortuitum and N. asteroides = 0.70 and 0.72, respectively). Reactivity to heterologous antigens was also highest in animals sensitized with strongly pathogenic species, approximating normal values in rabbits sensitized with weak pathogens. In the indirect test, the strongest responses were obtained again to homologous antigens (MI = 0.42 to 0.67), and they differed more markedly from reactions to heterologous antigens than in the direct test. The weakest activity of heterologous antigens was again found with M. fortuitum and N. asteroides, where MIs were 0.82 to 0.93.

Experimental skin sensitization in guinea pigs verified by the skin test and the lymphocyte migration inhibition test.

The sensitization properties of a number of chemicals were estimated on experimental models, using white female guinea pigs. Sensitization as effected in four ways a) intradermal application jointly with Freund's complete adjuvants (FCA), b) repeated epicutaneous applications together with FCA intradermally, c) a single intradermal application of the tested substance to the ear lobe, d) a single injection of the substance together with FCA into the paws. The results were evaluated with the skin test (ST) and in vitro with the macrophage migration inhibition test (MIT). The suitability of these methodical procedures was verified and agreement in the positivity of the ST and MIT confirmed. The sensitization effect of the tested substance does not depend on the intensity of skin reaction but on the frequency of positive skin test results in the monitored sample.

Cell-mediated immunity to varicella-zoster virus as assessed by the migration inhibition test.

The migration inhibition method was used to test cell-mediated immunity to varicella-zoster (VZ) virus in 10 varicella and 11 herpes zoster patients. Control groups consisted of eight children susceptible to VZ infection on serological evidence and 49 normal persons of different age categories. Depending on the positivity criterion adopted, positive results during disease were obtained in 43% or 90% or all tests performed in varicella patients and 47% or 74% in herpes zoster patients. Irrespective of which criterion of positivity was applied, a high rate of positive results was obtained in the normal control groups; in the age range from 20--44 years it was comparable to that for patients. This finding would suggest a high activity of VZ virus among the human population. Since a positive result in migration inhibition test offers evidence of recent contact with antigen, either exogeneous contact with VZ infection or endogenous contact with latent VZ virus must have been involved.

In vitro studies of cell-mediated immunity in rabbits sensitized with Mycobacterium kansasii and Mycobacterium tuberculosis.

Rabbits, sensitized with M. kansasii, responded by a profound inhibition of migration of macrophages elicited by both antigens: the migration index for homologous antigen was 0.51 in the direct test and 0.65 in the indirect test; for heterologous antigen the indexes were 0.53 and 0.67. However, significant differences in reactivity were found in rabbits sensitized with M. tuberculosis. In the homologous system, high reactivity was maintained and the migration index reached the value of 0.53 in the direct and 0.63 in the indirect test. On the other hand, the heterologous antigen M. kansasii influenced the migration in both direct and indirect assays significantly less, the migration indexes being 0.62 and 0.72. The differences were statistically significant at 1% and 5% levels.

Autoimmunity as possible evoking factor of some so-called idiopathic bone marrow hypoplasias.

Patients with bone marrow hypoplasia or aplasia were examined using the method of lymphocyte blast transformation and the MIF production test. The experimental results demonstrated the presence of an autoimmune process in these patients. A good congruence of the methods was demonstrated. Possible role of the autoimmune process in the pathogenesis of the disease as well as the reasons for the autoimmunity development and the therapeutical implications of these findings are discussed.

In vitro production of the factors effective in the transfer of experimental autoimmune aspermatogenesis.

We studied the biological effects of the factors released into the culture medium by the lymphocytes from animals sensitized with testicular antigen. The supernatants from cultures of these lymphocytes were active in vitro in the migration inhibition tests because they transferred the sensitivity to normal spleen cells. In vivo, they transferred aspermatogenesis in the allogeneic and xenogeneic system. The results suggest a specific effect on the behaviour of macrophages rather than a direct cytotoxic effect of the active factors.