Cellular and humoral systemic and mucosal immune responses stimulated by an oral polybacterial immunomodulator in patients with chronic urinary tract infections.

An oral polybacterial immunomodulator Urostim (U), composed of killed cells and their lysates from E. coli expressing type 1 and Pili, E. coli Rc mutant, P. mirabilis, K. pneumoniae and E. faecalis was created for immunoprophylaxis and immunotherapy of urinary tract infections (UTIs). In experimental animal models, the stimulating effect of U on lymphocyte functional activity, macrophage phagocytosis and antibody producing cells, was established. In this study the immuno-modulating effects of U on the proliferating capacity and ultrastructural morphologic changes of lymphocytes, cytokine production and specific systemic humoral and mucosal immune responses in patients with UTIs have been evaluated. Patients enrolled in the study, received orally 50 mg U daily for a period of three months. On days 0, 30 and 90 a quantitative analysis was performed on lymphoproliferative responses to polyclonal mitogens, IL-2 and the specific antigen U, the production of specific serum and saliva IgA, IgM and IgG antibodies to all components of U and the concentration of pro-inflammatory cytokines. There was significant improvement of non-specific and specific lymphoproliferative responses on days 30 and 90 after the onset of treatment with U, confirmed by electronmicroscopic studies. The highest concentrations of serum proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 were registered at baseline followed by a decrease until the end of the observation period. This finding correlates with the gradual decrease of immune activation as measured by the spontaneous lymphocyte proliferation. Data from the production of specific antibacterial antibodies in serum and saliva show two types of reactions. The first type was registered in patients with low pre-treatment levels in whom the concentration of specific antibodies increased on days 30 and 90. The second type of reaction was observed in patients with high pre-treatment levels, which dropped on day 30 and were usually followed by an increase at the end of the study. These results provide evidence for the immuno-modulating effect of U. Our data show that the oral administration of the polybacterial immunomodulator Urostim stimulates adequate cellular and humoral systemic and mucosal immune responses in patients with chronic UTIs.

Immunostimulating and protective effects of an oral polybacterial immunomodulator 'Dentavax' in a rabbit experimental model.

The immunostimulating and protective effects of an oral polybacterial immunomodulator, Dentavax (D), composed of killed cells from Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Candida albicans and Lactobacillus acidophilus and their lysates, have been investigated on an experimental rabbit model. In this model, mixed suspensions of the above bacterial wild strains have been injected in six sides of oral mucosa. A long-lasting inflammation with the development of infiltrates and confluating abscesses has been observed. The influence of orally given Dentavax on the course of the model infection as well as on the dynamics of the immune response has been studied. A two-fold decrease in the duration and severity of inflammatory reaction, confirmed by the histological findings, has been registered. In immunised animals, an activation of polymorphonuclear phagocytosis, together with stimulation of humoral systemic and mucosal immunity with synthesis of specific serum (predominantly, IgG) and coproantibodies (predominantly, S-IgA) determined by ELISA, has been found. The results obtained proved the strong immunostimulating and protective effects of the preparation D, which is meant for the prophylaxis and treatment of inflammatory periodontal diseases.

Influence of RecA on in vivo virulence and Shiga toxin 2 production in Escherichia coli pathogens.

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains 933 and 86-24 as well as the uropathogenic E. coli (UPEC) strain 536 were compared with their isogenic rec A mutants and rec A trans -complemented strains in intravenous lethality and lung toxicity assays in mice. While the wild-type EHEC strains were fully virulent, the virulence of the rec A mutants was strongly reduced. Complementation of the EHEC rec A mutants with the cloned E. coli recA gene restored their virulence capacity. The stx2EHEC mutant TUV86-2 as well as its isogenic rec A mutant were completely avirulent in both assays. In contrast, RecA had no influence on the virulence of UPEC strain 536. We conclude that the lethality observed with EHEC is presumably mainly due to Shiga toxin, which is severely down-regulated in the rec A mutants as a result of lacking spontaneous phage induction. Therefore, the EHEC rec A+strains 933 and 86-24 were compared for their Shiga toxin 2 (Stx2) production with the respective rec A-counterparts. The rec A mutants of the EHEC strains were significantly reduced in toxin synthesis and were devoid of Stx2 specific phage production. Complementation of the EHEC rec A mutants with the cloned rec A gene enabled the rec A mutants to restore toxin and phage production. These results suggest that the higher level of Stx2 synthesis in the EHEC strains is the result of a higher level of spontaneous Stx2 specific phage induction, which is controlled by RecA.

Stimulating effect of an oral polybacterial immunomodulator on the proliferative activity of guinea pig lymphocytes.

A preparation for the prophylaxis and treatment of inflammations of oral mucosa and parodont Dentavax (D) was investigated in guinea pigs. Animals were given orally D for 5 consecutive days and a month later the procedure was repeated. On day 3, 10, 21, and 28 after immunization and reimmunization lymphoproliferative responses to PHA, rIL-2, LPS and D were measured by the radiometric blast transformation assay in peripheral blood, spleen, mesenteric lymph nodes (MLN) and Peyer's patches (PP). The percentage of cells entering S and G2/M-phases of cell cycle was assessed by the flow cytometric DNA analysis. A correlation in proliferative activity of cells after in vitro stimulation with PHA and LPS has been established by both methods. Peak values of lymphocyte stimulation were found on day 10, especially after the second administration of D in all organs tested, mainly in MLNs and spleen. Electron-microscopic studies demonstrated an extensive development of the endoplasmatic reticulum in plasmatic cells from spleen, PPs, mesenteric, bronchial and inguinal lymph nodes. The results obtained may be considered a proof of the immunostimulating effect of Dentavax.

Experimental investigations on the immunomodulating activity of polybacterial preparation for peroral immunotherapy and immunoprophylaxis of uroinfections.

The immunomodulating effect of a polybacterial immunostimulator Urostim in experimental animal models was investigated. The preparation was created for immunotherapy and immunoprophylaxis of uroinfections. It consists of killed bacterial cells and their lysates of four microbial species: Escherichia coli expressing type 1 pili, Rc mutant of E. coli, Klebsiella pneumoniae, Proteus mirabilis and Enterococcus faecalis, BALB/c mice and guinea-pigs were treated orally with Urostim for 5 or 10 days, respectively. A stimulatory effect of Urostim on phagocytosis (increased phagocytic index and phagocytic number) of mice peritoneal exudate cells was observed. An increased number of plaque-forming cells and elevated titres of haemagglutinating antibodies in mice demonstrated activation of humoral immunity. Lymphoproliferative studies showed a significant response to phytohaemagglutinin (PHA) and Urostim, especially after the second application of the preparation. At the same time no changes in the absolute number of peripheral blood lymphocytes were found. An active protection of mice against a systemic infection and of guinea-pigs against uroinfection caused by Gram-negative bacteria was obtained. In conclusion, the results obtained characterize Urostim as an effective immunostimulator. Its peroral application leads to the activation of innate resistance, cell-mediated, humoral and local immunity.

Technological aspect of the production of live dysentery vaccines for oral administration.

An experimental technology for the production of live freeze-dried vaccines prepared from attenuated Shigella flexneri 2a and Shigella sonnei I strains was developed. It is based on the cultivation of bacterial strains in a fermentor under conditions which ensure high yields. The strains grow in S-form, their antigenic structure is preserved and they remain non-virulent. The attenuating markers are stable. The freeze-dried vaccines retain good immunogenicity when applied intra-intestinally to rats.

[Stability of Shigella flexneri attenuated strain 2A 77 ADE- RIF-r after cultivation in a fermenter and lyophilization]. / Issledovaniia stabil'nosti attenuirovannogo shtamma Shigella flexneri 2A 77 ADE- RIF-r posle kul'tivirovaniia v reaktore i liofilizatsii.

The method for the cultivation of S. flexneri attenuated strain 2a 77 ade- rifr in a fermenter with the use of casein fermentative broth containing 200-250 mg% of amino nitrogen has been developed. No changes in the properties of the initial strain have been found after its cultivation in a fermenter or after the lyophilization of the cultures grown in a fermenter. After cultivation in casein fermentative broth with 100 mg% of amino nitrogen the presence of S-R dissociation has been established, the S colonies having the properties of the initial strain. The reversion of the R colonies to prototrophism in adenine and differences in the level of their resistance to rifampicin are observed. The study of the plasmid profile of the initial strain and its ade+ revertants has revealed the disappearance of plasmids with a molecular weight of 140 MD in these revertants. All clones under study (ade+ and ade-) show negative results in the keratoconjunctival test. The conditions of cultivation and the causes of reversion are discussed.

Technological investigations with attenuated strains of Shigella for production of live vaccines. 1. Cultivation in a fermenter of an attenuated strain S. flexneri 2a with two markers.

A method was developed for cultivation of the strain Shigella flexneri 2a 77 with two attenuated markers. It ensures the preservation of the initial properties of the strain. The regimen for control of dissolved oxygen level was optimized. It was established that yeast extract is a necessary component of the nutritive medium used. The role of the phase and quantity of the inoculum for growth of the bacterial population has been clarified. Fresh fermenter culture, applied in rats, shows a high immunogenic activity. After lyophilization, the cultures retain 50-70% of the immunogenic activity of the non-lyophilized cultures. The method makes preconditions for the production of a live attenuated dysentery vaccine for oral administration.

[2-cycle culture of Vibrio cholerae in a fermenter]. / Dvukhtsiklicheskoe kul'tivirovanie kholernykh vibrionov v fermentere.

The method for the two-cycle cultivation of V. cholerae, reference strains Inaba and Ogawa, in a fermenter has been developed. In the first and second cultivation cycles no difference in the yield of the biomass and in the ultrastructure of the vibrios has been established. In the second cycle a decrease in the minimum time of generation has been observed. The vaccines obtained in the first and second cultivation cycles have not been found to differ in their antigenic and immunogenic activity. The vaccines meet the WHO requirements.

[Standardization of cholera vaccine and preparation of a national reference standard]. / Standartizatsiia kholernoi vaktsiny, izgotovlenie natsional'nogo referens-preparata.

The antigenic potency of the proposed national reference preparations in comparison with that of the corresponding international reference preparations was studied by means of the active protection test in mice. The antigenic potency of the proposed national reference preparations for Inaba and Ogawa was found to be the same or even greater than the antigenic potency of the international reference preparations for cholera vaccine. A high level of antigenic activity was observed during comparison of a production lot of cholera divaccine with the international reference preparation and the national reference preparation in parallel tests. The proposed national reference preparations for Inaba and Ogawa may be used for evaluating the antigenic potency of the lot of cholera vaccine produced in Bulgaria as the standard preparation.

[Effect of certain factors on Vibrio cholerae multiplication in fermenter]. / Vliianie nekotorykh faktorov na razmnzhenie kholernykh vibrionov v fermentere.

The influence of the level of dissolved oxygen, as well as the phase of growth and the amount of inoculum introduced, on the growth of bacterial population in the process of the cultivation of cholera vibrios in the fermenter "Novopalias" has been studied. The results of this study indicate that the cultivation of cholera vibrios with the level of dissolved oxygen constituting 50% of the maximum air saturation of the broth ensures a better yield of the biomass than cultivation with the level of dissolved oxygen equal to 10% and 30%. The introduction of inoculum in an amount of 1 : 20 (inoculum/medium) does not ensure a better yield of the biomass than an amount of 1 : 100 introduced at the phase of stationary growth. Cultivation at 50% pO2 and the introduction of inoculum in an amount of 1 : 100 at the phase of stationary growth have demonstrated some technical advantages in comparison with the introduction of inoculum at the phase of exponential growth, which allows one to use this technique for the production of cholera vaccine.

[Cholera vibrio culture in a fermenter with automatic control of a number of parameters]. / Kultivirovanie kholernykh vibrionov v fermentere s avtomaticheskim kontrolem riada parametrov.

The cultivation of V. cholerae was carried out in casein broth in a fermenter. 2 kinds of cultivation conditions were compared. The results thus obtained indicate that the cultivation of V. cholerae under the conditions of the controlled level of dissolved oxygen (pO2--50%) ensures the optimum conditions for proliferation and the high yield of biomass; it also allows obtaining vaccine whose immunogenic properties are not inferior to those of the WHO reference vaccine.

[Local Immune response in rabbits following enteral immunization with live attenuated bacterial Enterobacteriaceae vaccines]. / Lokale Immunantwort nach enteraler Impfung von Kaninchen mit attenuierten bakteriellen Enterobacteriaceae-Lebendimpfstoffen.

Streptomycin-dependent and inactivated Shigella flexneri 2a and Shigella sonnei strains were intra-intestinally applied to rabbits for immunisation. Rosette and plaque tests and well as indirect haemagglutination gave short-time secretion of low titres of specific copro-antibody, following monovaccines and bivaccines. High titres of secretory antibody were induced, depending on doses, by re-immunisation. No antigen competition was established. The localised immune response caused by Shigella live vaccines was found to be much stronger than that induced by inactivated vaccines