Immunogenicity of a Glycoconjugate from Hydrazine-Detoxified Lipopolysaccharide of Vibrio cholerae O139.

A glycoconjugate (dLPS-CBSA) from hydrazine-detoxified lipopolysaccharide (LPS) of Vibrio cholerae O139 and BSA was used for immunization of BALB/c mice. The immunogenicity (concentration of induced IgG antibodies) of the conjugate was determined by enzyme-linked immunosorbent assay. The highest level of LPS-specific IgGs was found in sera collected 2 weeks after the third immunization. Both IgM and IgG antibodies present in the anti-V. cholerae O139-specific sera showed long-lasting vibriocidal activity. The glycoconjugate also stimulated production of IL-4, IL-6, IFNγ, TNFα and IL-10. Immunization with the conjugate induced significant production of both Th1 and Th2 cytokine sets, indicating balanced Th1/Th2 immune response polarization. The quantitative analysis of total and LPS-specific IgM and IgG antibodies production by B lymphocytes (from both the spleen and the bone marrow) was performed by ELISPOT assay. From the analysis of antibodies produced directly by B lymphocytes in the bone marrow (of dLPS-CBSA immunized mice), we were able to identify the IgM-IgG switch in the antibodies produced.

Bacteremia due to Pseudomonas aeruginosa: results from a 3-year national study in the Slovak Republic.

Risk factors, mortality and antimicrobial susceptibility of Pseudomonas aeruginosa bacteremias isolated from 148 patients from all University Hospitals in Slovakia were analyzed. Only 1.2% of 169 strains of P. aeruginosa were resistant to meropenem, 4.1% to piperacillin/tazobactam, 7.7% to ceftazidime as well as cefepime and 12% to amikacin. More than 30% of P. aeruginosa were resistant to ciprofloxacin. Our analysis of risk factors for antimicrobial resistance to the particular antimicrobials, indicated no difference in risk factors and outcome in cases infected with P. aeruginosa bacteremias resistant to amikacin, piperacillin/tazobactam or ceftazidime in comparison to episodes caused by P. aeruginosa due to susceptible isolates. When comparing risk factors for P. aeruginosa bacteremia in children vs. adults, cancer vs. non-cancer patients, several differences in risk factors were observed. Neither antimicrobial resistance to amikacin, ceftazidime or piperacillin/tazobactam, nor appropriateness of therapy according to two separate analyses were associated with better outcome.

Prospective national survey of viridans streptococcal bacteraemia risk factors, antibacterial susceptibility and outcome of 120 episodes.

The aim of this study was to prospectively investigate 120 cases of viridans streptococcal bacteraemia (VSB) in 117 patients in major university hospitals in Slovakia in 2000-2002 (3 y) for antibacterial susceptibility, risk factors and outcome. From 127 episodes, 16 (13%) of VSB were caused by PEN-R strains and 13 (10%) by ERY-R strains. 32 cases had cancer as underlying disease (20 haematological), 41 had endocarditis and 35 were elderly (>65 y of age) patients. Concerning mortality, 29 of 127 patients died (24%). There were several risk factors associated with mortality. Solid tumour as underlying disease (p<0.02), stroke (p<0.002), concomitant lung infection (p<0.01), endoscopic procedure (p<0.036), intubation (p<0.0008), ventilatory support (p<0.002), and coma (p<0.009) were associated with more deaths. A comparison of 115 bacteraemias to 13 bacteraemias caused by erythromycin-resistant strains of Streptococcus viridans was performed. There were no significant differences in underlying disease, risk factors and mortality. Erythromycin resistance in bacteraemias caused by S. viridans did not have significant impact on outcome of the patients, nor did it show specific relation to analysed risk factors in our study. 14.5% of VSB were cause by PEN-resistant viridans streptococci. Risk factors for penicillin resistance were ventilatory support (p<0.01), intubation (p<0.001) and resistance to other antibiotics: 8 of 16 (50%) of PEN-R VSB were resistant also to erythromycin or cotrimoxazole or tetracycline compared with 9% of PEN-R VSB (p<0.005). Endoscopic procedures in the upper respiratory system were at risk for development of PEN-R VSB. There was also difference in outcome; 71% vs 22.5% (p<0.0002) of cases infected with PEN-R VSB died compared to PEN-S VSB. PEN-R is therefore clinically significant in VSB.

Monoclonal antibodies against hepatitis B e antigen: production, characterization, and use for diagnosis.

Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SP2/0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples.

Detection of antibodies against hepatitis B core antigen using the avidin-biotin system.

An enzyme avidin-biotin assay for the detection of anti-HBcAg antibody in human sera was developed. The assay uses genetically engineered HBcAg. HBcAg is immobilized on the surface of the wells of microtitre plates and the test serum sample, biotin-labelled HBcAg and streptavidin-labelled horseradish peroxidase are added. The assay was found to be specific and was compared with a commercial radioimmunoassay kit for sensitivity by testing 96 human clinical sera for anti-HBcAg antibody. Both assays gave identical results.

Expression of hepatitis B virus large envelope protein in Escherichia coli and Saccharomyces cerevisiae.

The gene coding for hepatitis B large envelope protein was cloned under the lac promoter in bacterial vector pUC-8 and under the ADH1 promoter in yeast expression shuttle vector pVT103-U, and expression of HBsAg in bacteria and yeast was determined. The strongest expression of large envelope protein was obtained after transformation of the protease-deficient yeast strain BJ1991. The recombinant large envelope protein did not form complex 22-nm particles and was not secreted into medium.

A suppressive mechanism counteracts the production of anti-idiotype antibody.

BALB/c mice were repeatedly, at two-week intervals, immunized with monoclonal antibody against hepatitis B surface antigen and their sera were titrated for anti-idiotype antibody. The highest titres appeared after 1-4 immunizations. Further immunizations led to fast disappearance of the anti-idiotype antibody. These data suggest that anti-idiotype antibody production is a rather complicated process in which some suppressive mechanism is involved.