M. Mára

Effect ofBacillus firmus and other sporulating aerobic microorganisms onin vitro stimulation of human lymphocytes. A comparative study

B. firmus activates human peripheral blood lymphocytesin vitro. Bacteria inactivated by heat or by formaldehyde were about equally effective, stimulating the blastic transformation of lymphocytes at doses of 10–200 mg/L and Ig formation in the culture at 10–500 mg/L. The action of formaldehyde treatedB. firmus was compared with that of analogously inactivatedB. subtilis, B. polymyxa, B. coagulans, B. megaterium, B. pumilus, B. cereus andB. lentus at a concentration of 100 mg/L. All these bacilli midly stimulated blastic transformation and most of them substantially stimulated Ig formation, butB. firmus was the most efficient in stimulating the formation of Ig of all classes, in particular IgM and IgA. Its effect on Ig formation was comparable with that of PWM and was unusually high as compared with that of other bacteria.B. firmus is apparently a strong polyclonal activator of B lymphocytes. Its cells or their components could be potentially used for modulating immune reactions.

Resistance to infection and activation of the monocyto-macrophage system caused by Bacillus firmus and its fractions

Crude lipids isolated fromBacillus firmus, but not from other bacilli, were previously found to induce significant resistance againstListeria monocytogenes infection in mice. In this study, formaldehyde-and heat-killed bacterins of eightBacillus species and some cellular fractions ofB. firmus were prepared and tested for further immunomodulatory activities. Crude lipids, their aqueous extract, LTA, Protodyne and Pex-residue preparations exhibited a strong anti-infection activity, whereas Pextract, P40 and all bacterins tested had no effect. Formaldehyde-killed bacterins, live bacteria and the P40 preparation of bothB. firmus strains, as well as bacterins of bothB. subtilis strains, induced pronounced splenomegaly in mice. Peptidoglycan and Pex-residue induced significant depression of cytochrome P-450 in mouse liver microsomes after application of 0.1 mg per mouse. Optimal conditions for obtaining a bacterial suspension exhibiting these immunomodulatory properties were elaborated.

Prolonged survival of AVN wistar rats with transplanted yoshida sarcoma and increase of granular lymphocytes after administration ofBacillus firmus and their crude lipids

Bacillus firmus is a Gram-positive, aerobic, sporulating, nonpathogenic air contaminant which, according to earlier findings, is a strong polyclonal activator of B lymphocytes. The crude lipids of this microbe induced significant resistance of mice against listerial infection. The administration of bacterin, like that of crude lipids obtained by the extraction of cell suspension with chloroform—methanol to rats, strain AVN Wistar, transplanted later with Yoshida sarcoma, significantly prolonged the survival of the animals in comparison with the control group. At the same time the number of granular lymphocytes was increased. The destruction of tumor cells in the peritoneal exudate of immunostimulated rats was also determined.

Phenol—water extracts of gram-positivelisteria monocy togenes and gram-negativesalmonella typhimurium. comparison of biological activities

Using phenol—water extraction, a lipopeptidopolysacoharide complex (LPPS) was isolated fromListeria monocytogenes. Some biological and immunological properties of LPPS were compared with lipopolysaccharide isolated by the same procedure fromSalmonella typhimurium. LPPS possesses low pyrogenicity, but the immunological activity is comparable with LPS: slightly lower adjuvant and polyolonal stimulating effect, almost equal mitogenic effect on mouse spleen cells and higher mitogenic effect oh human peripheral blood cells, The results are discussed in connection with the chemical structure of both substances.

Immunomodulatory effects of Bacillus firmus

Abstract3-day-old miniature piglets were stimulatedin vivo withBacillus firmus by the intraperitoneal or intragastric route for 1 d. Cells containing IgA and IgG2 were detected in the ileum in all stimulated but not in control animals. The frequency of blood CD3+ cells increased after intraperitoneal administration ofB. firmus, the ratio of polymorphonuclears to lymphocytes increased in all stimulated piglets.B. firmus induced antitumor immunity in rats with transplanted Yoshida sarcoma cells. Granular lymphocytes and dead tumor cells were found in peritoneal exudate of stimulated animals.B. firmus induced IFN-γ synthesis in human blood lymphocytes stimulatedin vitro for 1 d. The amount of TNF-α produced by these stimulated human peripheral blood mononuclears (PBMC) was lower than that of PBMC stimulated with some other bacterial immunomodulators. Cells containing TGF-β or IL-8 were not found in human PBMC stimulated withB. firmus.

Some biological, biochemical and morphological changes and their correlations in selected BCG vaccine strains

Changes in the biological, biochemical and morphological properties of three BCG strains, Copenhagen BCG 1331 and two of its derivatives, Praha BCG 725 SH and Praha BCG 725 SAS, were studied and correlated. These changes were caused by chemical (culture media) and physical factors (freeze-drying). The highest immunizing potency and the highest lipid content were displayed, both prior to and after freeze drying, by the Danish strain. Long-term cultivation of the Danish strain on different media in Prague resulted in a decrease of its immunizing potency and lipid content. Both of the Czechoslovak strains derived in this manner, BCG 725 SH and BCG 725 SAS, differed mutually by their immunizing potency, lipid content and percentage of intact cells. The immunizing potency and lipid content of these two strains were in inverse proportion; so were the immunizing potency and percentage of intact mycobacteria. The experiments suggested that substantial changes develop after physical action (freeze-drying) only in strains being maintained on rich culture media. Freeze-drying changed the relationship between immunizing potency and lipid content into a direct one in both of the Czechoslovak strains. The relationship between immunizing potency and percentage of intact mycobacteria remained inverse. In BCG 725 SH, total lipids and immunizing potency decreased and the percentage of intact cells increased after freeze drying. In BCG 725 SAS on the other hand the immunizing potency increased, lipids remained on a stable level and the percentage of intact cells decreased. The substantial changes in these parameters induced by freeze-drying subsequently returned to the original values irrespective of whether the strains had undergone a number of freeze-drying procedures or had been passaged directly after a single freeze-drying run. An essential effect was thus only produced by the first freeze-drying, and the changes appear not to be stable.

Class IgG, IgM and IgA Antibodies against Staphylococcus aureus Antigens in Human Serum and Saliva

Using the ELISA method antibodies against the sonicate, teichoic acid (TA) and exoproducts ofStaphylococcus aureus were determined in sera and saliva of healthy individuals. Main serum antibodies against all the antigens used were shown to be class IgG antibodies. However, antigens of the sonicate stimulated significantly even the systemic IgA response. In the saliva class IgA antibodies predominated, but IgG antibody levels against TA and exoproducts approached the level of IgA antibodies. Levels of IgM antibodies against all antigens tested were low in both the serum and saliva which corresponds with the anamnestic type of response. On the basis of these results one may assume that not only IgG, but also IgA antibodies are important in the systemic immunity against staphylococcal infection and in the immunity of mucous membranes; besides IgA, even class IgG antibodies play an important role.

Bacterial oxidation of cholic acid byArthrobacter sp

Some bacteria possess the ability to convert cholic acid (CA) to pharmacologically important chenodeoxycholic acid (CDCA) or to its precursor 3α, 7α-dihydroxy-12-oxo-5β-cholan-24-ic acid (12-oxo-CDCA). The ability of several bacterial strains to convert CA to 12-oxo-CDCA was tested in our work. The highest conversion was reached by one strain ofArthrobacter sp. which converted up to 61% CA to 12-oxo-CDCA in a complex medium containing 4% (W/V) CA under aerobic conditions. 12-Oxo-CDCA was further oxidized to 3,12-dioxo-7ì-hydroxy-5β-cholan-24-ic acid (3,12-dioxo-7-OHCA) that represented up to 10% of total bile acids in the cultivation medium after a 1-d cultivation. The concentration of 12-oxo-CDCA increased rapidly during the first hours of cultivation, maximal yields were achieved between 20 and 24 h of cultivation and they decreased after that due to further bacterial oxidation.

Resistance toMycobacterium tuberculosis H37RV infection induced byListeria and mycobacterial lipids

The avirulent strainListeria monocytogenes (Welshimer) induced upon a single injection 4 weeks prior to challenge withMycobacterium tuberculosis H37RV resistance in guinea-pigs, which was manifested by a significant decrease of spleen weight and Feldman’s index in immunized animals. The degree of resistance was dependent on the immunizing dose and time of administration. Repeated high doses ofListeria yielded only a low or no effect. Further increase of resistance was obtained using the BCG lipids; however, stimulation of resistance with listeria lipids was not successful and, finally, Freund’s incomplete adjuvant was significantly effective in the Feldman index only. The BCG lipids, extracted with ethanol, which contained nitrogen and water-soluble substances, induced a significant resistance againstM. tuberculosis infection. The chloroform-methanol BCG lipids were also effective, however, significantly less than the ethanol-extracted material. The listeria factor Ei itself or together with Freund’s incomplete adjuvant possessed only low effectiveness against mycobacterial infection. However, if injected together with ethanol-extracted BCG lipids, it produced a significant degree of resistance which was higher than that induced by lipids only. The degree of resistance was comparable with the effect of living BCG strain which served as a source of isolated lipids.

Accumulation of activated lymphocytes in liver of Listeria factor Ei treated rabbits

Factor Ei isolated fromListeria monocytogenes caused in rabbits 1 d after intravenous administration activation of peripheral blood lymphocytes in terms of rRNA biosynthesis. Increase of the number of these active lymphocytes was observed not only in peripheral blood, but also in spleen, lung, kidney and liver. When the number of lymphocytes was related to the amount of the organ tissue cells, the increment appeared significant only in the liver where the number of active lymphocytes exceeded the control value by one order of magnitude. Based upon this observation we concluded that the accumulation ofListeria factor Ei activated lymphocytes occurred in liver. This characteristic is considered an additional immunomodulative property ofListeria factor Ei similar to theEscherichia coli lipopolysaccharide.