Antibacterial activity of Wratizolin.

Wratizolin in the concentration of 1-10 micrograms/ml was found to inhibit the growth of Gram-positive bacteria such as: Staphylococcus, Streptococcus, Sarcina, Corynebacterium, and some representatives of aerobic bacilli. Gram-negative bacteria of Enterobacteriaceae and Pseudomonas were not sensitive to Wratizolin even in the concentration of 100 micrograms/ml.

Serologic relationship of fimbriae among Enterobacteriaceae.

The existence of serological relationship of fimbriae among serotypes of the same genus and among genera of Enterobacteriaceae was determined by agglutination and absorption procedures. It was found that fimbriae of different serotypes of the same genus and among the genera a) Escherichia, Shigella and Klebsiella, and b) Salmonella, Arizona and Citrobacter were more or less antigentically related. No relationship was found among fimbriae of the Edwardsiella, Enterobacter, Hafnia, Serratia, Proteus and Providencia. It was also found that in Klebsiella, MS and MR fimbriae differed antigenically. On the basis of the results obtained nine antigentically distinct types of fimbriae were distinguished.

Studies on virulence of Shigella flexneri.

The antigenic structure and some biological properties were compared in virulent S. flexneri strains of differences in: 1) antigenic composition; 2) biochemical activity; 3) sensitivity to a set of Shigella phages and 4) susceptibility to phagocytosis. The only difference found concerned the LD50 for mice; it was 10 to 100 times larger for avirulent than for virulent strains of S. flexneri. The toxic products of the strains were also compared. The lipopolisaccharide and free endotoxin purified from virulent and avirulent variants behaved similarly when tested for: LD50, pyrogenicity and the local Shwartzman reaction. A striking difference was demonstrated in the ultrastructure of lipopolisaccharide and free endotoxin isolated from virulent and avirulent variant of S. flexneri 3a.

Effect of outer membrane proteins (OMP) of Shigella on interleukin 2 (IL-2) production by spleen cells of mice.

The kinetics of interleukin (IL-2) release from mouse spleen cells incubated with different doses of outer membrane proteins (OMP) from Shigella was investigated. OMP induced very low activity of IL-2 after 2 and 4 h, and only a slightly higher level of the cytokine was detected after 6 h. However, IL-2 activity increased markedly after 20 and 24 h of incubation, and doses of 5 and 10 micrograms of OMP were found to be the most effective. Spleen cells cultured with OMP for 48 h contained reduced concentration of IL-2.

Humoral response in mice immunized with outer membrane proteins of Shigella flexneri.

Intraperitoneal immunization of mice with outer membrane proteins (OMP) of Sh. flexneri induced in the animals a synthesis of specific antibodies. Their level determined by ELISA test was found to be relatively low in the sera of animals immunized with a single dose (10 micrograms) of OMP; it was markedly higher in mice immunized with two doses of OMP, and very high after three fold immunization. The specific antibodies maintained in the animals for 8-16 weeks after immunization. Anti-OMP sera given to normal mice by intraperitoneal route protected them not only against challenge with homologous Shigella but also against Proteus and Escherichia.

Characterization of outer membrane proteins of virulent phase I Shigella sonnei strains and their avirulent phase I derivatives.

Comparison of polyacrylamide gel electrophoretic protein profiles of four isogenic sets of virulent phase I Sh. sonnei strains and their avirulent phase I cells revealed no differences in outer membrane protein composition between virulent and avirulent derivatives. However, significant qualitative and quantitative differences were found in composition of major outer membrane proteins between strains of different origin. All four strains tested contained one major protein of 33K. This protein was susceptible to proteolytic enzymes and was found to be heat modifiable. Other major proteins of 35K and 37K present in three strains and 36K present in one strain were identified as peptidoglycan associated proteins.

Humoral response in mice immunized with outer membrane proteins of Hafnia alvei. Protective activity of anti-OMP-antibodies.

Intraperitoneal immunization of mice with outer membrane proteins (OMP) of Hafnia alvei induced in the animals a synthesis of specific antibodies. The antibody levels, determined by ELISA test, were found to be relatively low in the sera of mice immunized with a single dose (5 micrograms) of OMP and after a second immunization. However, they were higher in mice immunized with three doses of OMP. The antibodies were present in circulation for a relatively short time after immunization. Serum containing anti-OMP antibodies given intraperitoneally to normal mice protected them only against challenge with a homologous Hafnia strain.

Studies on immunity in mice immunized with outer membrane proteins of Hafnia.

Nonspecific protection induced in mice after administration outer membrane proteins of Hafnia alvei against infection with homologous and heterologous bacteria was transferred into other mice with lymphocytes isolated from spleens of mice immunized with outer membrane proteins. It was also found that mice sensitized with outer membrane proteins derived from H. alvei or with living bacteria induced in animals delayed hypersensitivity (DTH) in homologous and heterologous systems. The observed type of hypersensitivity was transferable to normal mice by lymphocytes obtained from donor animals which were previously sensitized with OMP. The experiments revealed that immunity induced with outer membrane proteins of Hafnia alvei is cell-mediated.

Effect of outer membrane proteins from Shigella on humoral immunity induced in mice by SRBC.

Shigella flexneri outer membrane proteins (OMP) which had been earlier found to exert immunomodulatory effect on cell mediated immune response were also found to act as immunomodulator of the humoral immune response. Effects of OMP were investigated in the experiments in vitro and in vivo, where the level of humoral immune response, measured as the number of plaque-forming cells (PFC) to SRBC in the spleen was evaluated. We demonstrate that small doses of OMP (1-5 micrograms) stimulate, whereas higher doses (10-50 micrograms) suppress the humoral immunity.

Some biological properties of outer membrane proteins of Hafnia.

Outer membrane proteins (OMP) isolated from four antigenically different strains of Hafnia alvei were tested for the toxicity, pyrogenicity, ability to induce Shwartzman reaction as well as for their influence on the leukocyte system. LD50 doses for the studied preparations determined on inbred mice were 18, 20, 28 and 34 mg/kg. These differences in the toxicity of the preparations were reflected in manifestation of Shwartzman reaction; more toxic preparations induced strongest necrohemorrhagic changes at the site of injection. The OMP preparations injected intravenously to rabbits caused moderate increase of body temperature. They induced changes in the number of leukocytes in the animals comparable with those of other preparates of bacterial origin.

Fraction of spleen cells responsible for suppression of cellular immune response to SRBC in mice treated with outer membrane proteins of Shigella.

Effect of splenocytes isolated from mice immunized with suppressive dose of OMP from Shigella on delayed hypersensitivity, induced in mice with sheep red blood cells was investigated. Only the population of T lymphocytes was found to suppress the delayed hypersensitivity, as measured by the footpad reaction. The results suggest that OMP of Shigella are able to induce in the spleens of animals active T cells which are responsible for the suppression of cellular response induced by SRBC.

Studies on specificity of protection induced by immunization with outer membrane proteins of Shigella.

Immunization of C3H/HeJ mice with outer membrane proteins (OMP) and with peptidoglycan associated proteins (PGP) isolated from Shigella flexneri 3a and from Shigella sonnei phase I protected the animals against lethal dose of homologous and heterologous bacteria and against various serotypes of Shigella flexneri. Neither of the protein preparations protected the animals against challenge with Escherichia, Klebsiella, Citrobacter, Salmonella, Serratia, Proteus and Pseudomonas. OMP preparations however, isolated from these species protected the animals not only against challenge with homologous bacteria but also against Shigella flexneri 3a.

Transfer of immunity by means of spleen cells from mice immunized with outer membrane proteins of Shigella flexneri.

Intraperitoneal immunization of mice with a single dose (5 micrograms) of outer membrane proteins (OMP) of Shigella flexneri was found to evoke in the spleen the appearance of cells by means of which immunity to lethal dose of Shigella could be transferred into other mice. Active cells capable of transferring immunity appeared in the spleen of the animals as early as on day 3, reached the strongest protective activity on day 4 and disappeared on day 8 after immunization. Active cells from animals immunized with two doses of OMP maintained in the spleens for 19 days. The experiments revealed that immunity to Shigella could be transferred only with lymphocytes; macrophages were found to be inactive.

Antifungal activity of Wratizolin.

Wratizolin was found to inhibit completely or delay markedly the growth of fungi of Microsporum, Trichophyton and Epidermophyton genus. Mould fungi, yeasts, and yeast-like organisms, with a few exceptions, were not sensitive to Wratizolin.

Some biological properties of outer membrane proteins of Shigella.

Outer membrane proteins (OMP) derived from two antigenically different representatives of Shigella (Sh. flexneri 3a and Sh. sonnei phase I) were tested for the toxicity, pyrogenicity, ability to induce Shwartzman reaction as well as for their influence on the leukocyte system. LD50 dose determined on mice was 28 mg/kg for OMP of Sh. flexneri and 23 mg/kg for OMP of Sh. sonnei. Both preparations injected intravenously to rabbits caused moderate increase of body temperature, expressed by the value 1.8 degrees C. Intravenous administration of protein preparations to rabbits, induced at first leukopenia and then transient leukocytosis. When injected subcutaneously in the dose of 500 micrograms and after 24 h intravenously in the dose 100 micrograms, they produced slight hemorrhagic changes at the site of administration.

Effect of outer membrane proteins of Shigella on delayed hypersensitivity in mice to sheep red blood cells.

Small doses of outer membrane proteins (OMP) of Shigella flexneri injected intraperitoneally into mice 1 to 3 days before or 3 days after sensitization of animals with sheep erythrocytes were found to increase delayed hypersensitivity as measured by the footpad reaction. In contrast, administration of higher doses of OMP resulted in suppression of hypersensitivity response. Cell transfer experiments showed that the spleen cells from sensitized and OMP treated mice transferred stimulating and suppressing activity to normal recipients. Suppression of hypersensitivity was also observed when recipients were injected with OMP 24 h before they were infused with spleen cells obtained from donor mice sensitized with sheep erythrocytes.

Phagocytic and bactericidal properties of macrophages of mice immunized with outer membrane proteins (OMP) of Shigella.

Macrophages obtained from peritoneal exudates of mice immunized with the single doses (1 and 5 micrograms) of OMP were shown to have stronger phagocytic as well as bactericidal properties in relation to Shigella flexneri bacilli than nonactivated macrophages. Macrophages from the animals immunized with 10, 20 and 30 micrograms OMP doses showed phagocytic and bactericidal properties similar to those of nonactivated macrophages while the immunization with the dose 50 micrograms resulted in their suppression. Likewise, activity of macrophages from mice immunized twice or three times with various doses of OMP did not differ much from that obtained after immunization with single OMP doses. On the other hand, immunization of mice with a sublethal dose of live Shigella flexneri did not activate either phagocytic or bactericidal properties of macrophages. Besides, phagocytic and bactericidal activity of macrophages of mice immunized with OMP of Shigella was determined in relation to Salmonella typhimurium. The doses 1 and 5 micrograms of OMP resulted in slight activation of macrophages which manifested itself by a little increase in their phagocytic and bactericidal ability. When used in the dose 10 micrograms, OMP remained without any effect on the above activity of macrophages. Only the dose 50 micrograms, slightly suppressed their phagocytic properties.

Outer membrane protein composition of Hafnia alvei.

The outer membrane proteins (OMP) obtained from 33 strains of Hafnia of different origin, were analysed by SDS-polyacrylamide gel electrophoresis. About 21 protein bands could be distinguished in OMP of each strain. The major proteins of the outer membrane were of apparent molecular weight 34.000 (34K), 37.000 (37K), 39.000 (39K) and 41.000 (41K). On the basis of the electrophoregrams obtained, four groups of strains possessing different composition of major OMP were distinguished: (1) 34K and 37K, (2) 34K, 37K and 39K, (3) 34K, 37K and 41K, (4) 34K, 39K and 41K. The major OMP of 34K present in all strains examined was susceptible to proteolytic enzymes and was found to be heat modifiable. The remaining major OMP of 37K, 39K and 41K were identified as peptidoglycan associated proteins.

Studies on nonspecific protection induced with OMP of Shigella flexneri and other genera of Enterobacteriaceae.

Outer membrane proteins (OMP) isolated from Shigella flexneri, Escherichia coli, Proteus vulgaris and Salmonella typhimurium were tested for their protective activity. Each OMP preparate given to mice intraperitoneally in a single injection (5 micrograms/per mouse) protected the animals not only in homologous but also in varying intensity in heterologous systems. Evidence was obtained that this nonspecific protection is cell mediated.

Immunogenic and protective properties of outer membrane proteins of Hafnia alvei.

Outer membrane proteins (OMP) extracted from antigenically distinct or related strains of Hafnia alvei containing defined composition of major proteins proved to be immunogenic. Intraperitoneal immunization of mice with a single dose of such preparations protected the animals against homologous and heterologous Hafnia strains. The OMP preparations were also found to induce protection with varying intensity against Escherichia, Proteus, Shigella and Salmonella.