Search PubMed⌕ Search

Biomedical subjects

P Novotny

Publications and source records attributed to P Novotny.

At least 37 records · Page 2Linked to original sources
PubMed1991

A novel bivalent acellular pertussis vaccine based on the 69 kDa protein and FHA.

A combination of the 69 kDa outer membrane protein and filamentous hemagglutinin (FHA), both isolated from pertussis toxin (ptx) minus mutants of B. pertussis, is protective in the mouse intracerebral challenge potency test ("Kendrick test"). A combination of the same 69 kDa protein and lymphocytosis promoting factor (LPF, pertussis toxin, PTX) is approximately 15 times less effective. The data show that the 69 kDa protein in tandem with FHA is the most relevant combination for mouse protection, and may be a more suitable acellular pertussis vaccine candidate than the LPF/FHA combination which has never been satisfactorily protective in the mouse test. However, the preparation of the 69 kDa protein of acceptable quality is still difficult. Attempts were made at screening the most suitable batches of the preparation by exploiting some recently discovered properties of the 69 kDa protein, i.e. its chromatofocusing pattern and lymphocyte affinity. Although both these properties may differentiate 69 kDa preparations, none of them was able to replace the mouse intracerebral challenge potency test for the final quality assessment.

Adhesins, Bacterial↗
PubMed1991

Analysis of antibody profiles in children with whooping cough.

Development of antibody titres in non-vaccinated children with whooping cough of different duration (all confirmed by positive culture) were investigated by ELISA using lymphocytosis promoting factor (LPF, pertussis toxin), filamentous haemagglutinin (FHA), 69 kDa protein and lipopolysaccharide (LPS) as antigens. The antibody responses occur in three different patterns: Firstly, the LPF antibody response develops very quickly starting with the first day of clinical cough with all three classes, IgG, IgM and IgA appearing simultaneously; LPF antibody appears to be a dominant feature. Secondly, FHA and 69 kDa antibodies appear, starting as IgM with the shift to IgG and IgA later. The third pattern is represented by LPS antibody, the IgA appearing early, but with IgM predominant. Higher titres of IgG reacting with LPS were observed in vaccinated children. Transplacental transfer of antibody was also studied. All antibody titres determined in maternal blood and cord blood were proportional except for anti-LPS antibody which was retarded. Most IgG antibody was IgG1 subclass; surprisingly the 69 kDa antibody consisted of a mixture of approx. 90% IgG1 and 10% IgG4.

Antibodies, Bacterial↗
PubMed1990

[Asymptomatic myocardial ischemia].

Silent (asymptomatic) myocardial ischemia (SMI) is defined as a transient alteration in myocardial perfusion in the absence of chest pain or the usual anginal equivalents. Patients may be classified as having one of the three types of SMI: type A--totally asymptomatic patients with no history of angina or myocardial infarction; type B--asymptomatic patients with previous myocardial infarction; type C--patients with angina and asymptomatic ischemic episodes. SMI has been found in 2.5% of all healthy males aged 40-59 and in 20% of all postinfarction patients. In type C-patients, 80% has been found to have asymptomatic ischemic episodes in addition to typical angina pectoris. The frequency of SMI may be up to three or four times that of anginal attacks. SMI patients have generally reduced sensitivity to pain an differences in severity an duration of ischemic episodes. Diagnosis is based on screening by means of exercise testing in patients working in specific professions (like pilots, busdrivers etc.), in postinfarction patients and in patients after unstable angina pectoris and after coronary bypass surgery or coronary angioplasty. Prognosis is the same as in asymptomatic ischemia. SMI is an indicator of instability in certain groups of patients (post infarction, after unstable angina pectoris). SMI persisting after medical therapy of unstable angina is associated with adverse short-term-prognosis, therefore coronary surgery or angioplasty is indicated.

Adult↗
PubMed1990

Identification of a 68-kilodalton outer membrane protein as the major protective antigen of Bordetella bronchiseptica by using specific-pathogen-free piglets.

Maternal antibody to an outer membrane 68-kilodalton (kDa) protein of Bordetella bronchiseptica was shown to be protective in experiments on specific-pathogen-free piglets. After challenge with B. bronchiseptica, 100% (n = 19) control piglets from nonimmunized sows developed pneumonia, coughing, and sneezing, and 74% of the animals developed severe atrophic rhinitis. In 12 piglets from a sow immunized with 68-kDa protein, pneumonia occurred only in 34% of offspring, coughing was reduced, the duration of coughing bouts was shortened, and severe atrophic rhinitis occurred in one animal only (8%). The difference in the occurrence of atrophic rhinitis and of pneumonia in immunized and nonimmunized offspring was statistically significant (P less than 0.05). Sera of protected piglets had high titers (enzyme-linked immunosorbent assay) of antibodies that showed a high specificity for the 68-kDa protein isolated from B. bronchiseptica, whereas their reactivity with an analogous 69-kDa protein isolated from Bordetella pertussis was low or absent. The 68-kDa protein of B. bronchiseptica appeared to be the major protective antigen in B. bronchiseptica infection; however, isolated protein alone did not induce such a solid protection, as observed in a previous study after the application of an effective whole cell vaccine.

Animals↗
PubMed1990

Construction and characterization in vivo of Bordetella pertussis aroA mutants.

A DNA fragment encoding a kanamycin resistance determinant was used to insertionally inactivate the cloned aroA gene of Bordetella pertussis in Escherichia coli K-12, and a conjugative shuttle vector system based on the suicide vector pRTP1 was used to deliver the mutations from E. coli back into B. pertussis CN2992FS and BP1. The aroA mutation was introduced by allelic exchange into the chromosome of B. pertussis, resulting in otherwise isogenic parental and aroA mutant pairs. The B. pertussis aroA mutants grew well on laboratory medium supplemented with aromatic compounds but failed to grow on unsupplemented medium. The B. pertussis aroA mutants expressed the normal B. pertussis extracellular, virulence-associated proteins; inactivated, whole-cell vaccines prepared from the mutants protected mice as efficiently as vaccines made from the parent strains against intracerebral challenge with the virulent B. pertussis 18323. Live B. pertussis aroA bacteria inefficiently colonized the lungs of NIH/S mice after they were challenged with aerosol, unlike the wild-type B. pertussis organism. Mice exposed to three separate aerosols of live B. pertussis aroA bacteria were protected against lung colonization after being exposed to an aerosol containing the virulent parental B. pertussis strain. High-level antibodies against B. pertussis rapidly appeared in the sera of mice immunized by aerosol with the B. pertussis aroA strains and challenged with the virulent parent.

Aerosols↗
PubMed1989

Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis.

Protein P.69 is localized on the outer membrane of Bordetella pertussis and is one of the virulence factors believed to contribute to the disease state of whooping cough. We demonstrate that protein synthesis of P.69 is under genetic control of the vir locus. Using oligonucleotide probes derived from the protein sequence of a cyanogen bromide fragment, we have cloned the gene for P.69 from B. pertussis CN2992. Analysis of the DNA sequence reveals a G + C-rich gene capable of encoding a protein of 910 amino acids with a Mr of 93,478, suggesting that P.69 is a processed form of a larger precursor. In common with some of the genes in the pertussis toxin operon, the sequence CCTGG was found 5' to the ATG initiation codon. At the 3' end, 29 bases after the TAA stop codon, the sequence GTTTTTCCT was found and may have some function in transcription termination. A full-length clone of the gene for P.69 carried by the cosmid pBPI69 was unable to direct the expression of P.69 protein in an Escherichia coli host. The generation of P.69-fusion products allowed the detection of P.69-specific protein products synthesized in E. coli.

Amino Acid Sequence↗
PubMed1989

[Therapeutic coronary effectiveness of retard gallopamil following repeated administration in comparison with placebo].

The aim of this randomized double-blind crossover trial was to evaluate the therapeutic efficacy of 100 mg slow-release gallopamil compared to placebo. The results of multistage exercise tests with computer-assisted ECG were analyzed in 16 patients (14 men and two women) aged 39 to 66 years who fulfilled the angiographical evidence of coronary heart disease with stable angina pectoris. All cardioactive medication was withdrawn for the duration of the study except for the trial medication and sublingual nitroglycerin as required. Patients began the therapy with a 5-day placebo run-in period. After this regimen, they were randomized to placebo or slow-release gallopamil (200 mg/day) in two divided doses for 7 days, at the end of which time each patient crossed over to the alternate regimen. Exercise testing was repeated at the end of the run-in period and after 1 week of placebo or active drug therapy. At highest comparable load the ST-segment depression was reduced about 70% before the morning administration (i.e., 12 h after the evening administration) and 6 h after the morning administration. The work load (W x min) increased in 54% or 61% at the maximal possible load. The number of angina pectoris attacks under bicycle ergometer exercise decreased in about 50%. PR-interval was prolonged from 0.14 to 0.15 s. QRS- and OTc-intervals were not influenced. No clinically relevant changes in heart rate and blood pressure were observed. The results indicate great antianginal efficacy and a good toleration of slow-released gallopamil.

Administration, Oral↗
PubMed1988

Identification of a 69-kilodalton nonfimbrial protein as an agglutinogen of Bordetella pertussis.

Cells of Bordetella pertussis BP353, a nonfimbriated Eldering serotype 1.3 strain, were used as an immunogen to produce three monoclonal antibodies, BPE3, BPD8, and BPE8, that agglutinated the immunizing cells, as well as certain other nonfimbriated and fimbriated serotype 3-containing B. pertussis strains. The antibodies did not agglutinate serotype 1 or nontypable B. pertussis cells. These monoclonal antibodies specifically detected a 69-kilodalton (kDa) band on Western blots (immunoblots) containing whole B. pertussis cell lysates of Eldering agglutinogen serotypes 1.3, 1.3.6, 1.2.3.4, and 1.2.3.4.6. This 69-kDa antigen was released from the bacteria by cell incubation for 60 min at 60 degrees C, and it was purified by affinity chromatography with a BPE3-agarose affinity matrix. Purified material was used to produce a polyclonal antiserum that agglutinated all nonfimbriated and fimbriated B. pertussis cells containing serotype 3 agglutinogen. Immunogold electron microscopy and indirect immunofluorescence studies demonstrated that it is an outer membrane constituent but nonfimbrial in appearance. BPE3 did not detect purified fimbriae on Western blots, and antibodies to these fimbriae did not bind to the 69-kDa component. Although B. bronchiseptica and B. parapertussis cells were not agglutinated by the monoclonal antibodies, antigenically similar proteins were detected in extracts of the bacteria. These results identify the 69-kDa protein as a nonfimbrial agglutinogen present on all virulent strains of B. pertussis. The monoclonal antibodies described here should be useful for further studies on the structure and function of this protein.

Agglutinins↗
PubMed1988

Molecular cloning and analysis of P. 69, a vir-controlled protein from Bordetella pertussis.

The cloning sequencing and analysis of an important antigenic component of Bordetella pertussis is described. The gene for P.69, in common with a variety of other so called "virulence" genes, (e.g., adenylate cyclase (AC), pertussis toxin (PT) and filamentous haemagglutinin (FHA)), is under control of the vir locus. The protein P.69 is externally localised on cells and protein preparations are protective as judged by the mouse intra-cerebral challenge test. The gene encoding the P.69 antigen was isolated by hybridization of mixed oligonucleotide probes against B. pertussis genomic DNA. These oligonucleotides were designed from the protein sequence data obtained from a cyanogen bromide digest of the P.69 protein. DNA sequence analysis reveals a G:C rich gene capable of encoding a protein of 910 amino acids and Mr of 93478, whose likely promoter and ribosome binding sites show little homology to their E.coli counterpart. In common with some of the genes in the PT operon the sequence 5'-CCTGG-3' was found 5' to the ATG initiation codon. At the 3', end 29 bases after the TAA stop codon, the sequence 5'GTTTTTCCT-3' was found in an equivalent position to the same sequence in the PT operon. Examination of the protein sequence reveals two regions with directly repeated elements (GGAVP)3(GGFGP)2 and (PQP)5.

Amino Acid Sequence↗
PubMed1986

Correlation of fetal heart rate monitoring and acid-base status with periventricular/intraventricular hemorrhage in the low birthweight neonate.

We investigated the correlation of fetal heart rate patterns and fetal acid-base findings with the occurrence of early periventricular/intraventricular hemorrhage and variants. Ninety-five consecutive singleton neonates with birthweights less than or equal to 2000 gm were investigated. One-third of them showed early periventricular/intraventricular hemorrhage or variants. Fetal heart rate scores and umbilical arterial/venous acid-base status showed no correlation with results of scanning. The implications of these findings are discussed.

Acid-Base Equilibrium↗
PubMed1985

Identification of a 68-kilodalton protective protein antigen from Bordetella bronchiseptica.

A 68-kilodalton (kd) outer membrane protein antigen of Bordetella bronchiseptica has been identified by using monoclonal antibodies that recognized two nonoverlapping determinants. Antibody BB05 also reacted with homologous proteins from Bordetella pertussis and Bordetella parapertussis but not with another 12 organisms from various bacterial genera. Passive injection of BB05 antibody protected mice from aerosol infection with B. bronchiseptica as shown by reduced mortality and reduced pathology of turbinate bones. The 68-kd B. bronchiseptica antigen was purified by BB05-based affinity chromatography and evaluated for its potency to immunize mice actively against either intraperitoneal or aerosol challenge with B. bronchiseptica. Immunization with the 68-kd antigen in incomplete Freund adjuvant significantly reduced the levels of mortality in intraperitoneally challenged mice. In the aerosol infection model, injection of the 68-kd antigen with complete or incomplete Freund adjuvant or saponin reduced the bacterial counts in the lungs of infected mice. These results suggest that the 68-kd protein may represent a potential "protective" antigen of B. bronchiseptica.

Animals↗
PubMed1985

Evaluation of Bordetella bronchiseptica vaccines in specific-pathogen-free piglets with bacterial cell surface antigens in enzyme-linked immunosorbent assay.

The progenies of specific-pathogen-free sows which had been immunized with Bordetella bronchiseptica vaccines of various origin before parturition were challenged intranasally with B. bronchiseptica within 5 days of birth. Sera of piglets were taken weekly and investigated by enzyme-linked immunosorbent assay against a mixture of B. bronchiseptica cell surface antigens containing curled fibers and fimbriae, lipopolysaccharide, and a mixture of proteins mostly derived from the outer membrane. The serological response to this antigenic mixture was paradoxical; the highest titers were obtained with the least effective vaccines. Antibodies which did relate to protection were oriented against the outer-membrane-derived proteins, one of which, of 68,000 molecular weight, appeared to be particularly important for two reasons. First, its concentration within the antigenic mixture was dependent upon cultural conditions; of all the proteins present in virulent strains, it was the first to disappear upon modulation. Second, it was absent from a strain which was unable to induce atrophic rhinitis in specific-pathogen-free piglets. Although all vaccines tested had some beneficial effect on the various clinical manifestations of the disease, only two vaccines were effective (P less than 0.001) in the prevention of nasal pathological changes. These two vaccines also stimulated the highest titers against the 68,000-molecular-weight protein. A mouse protection test utilizing a lethal intraperitoneal challenge failed to monitor the efficacy of vaccines for protection against atrophic rhinitis.

Animals↗
PubMed1985

Adenylate cyclase activity of a 68,000-molecular-weight protein isolated from the outer membrane of Bordetella bronchiseptica.

A method was developed which is suitable for the isolation of substantial quantities of outer membrane proteins of Bordetella species in a water-soluble form. The extracted material may then be further fractionated in the absence of detergents by ion-exchange chromatography and preparative flat-bed isoelectrofocusing. These procedures facilitated the isolation of one of the proteins, of molecular weight 68,000, for which the antibody titer correlated with the degree of protection against nasal changes induced in specific-pathogen-free piglets by Bordetella bronchiseptica infection (P. Novotny, M. Kobisch, K. Cownley, A. P. Chubb, and J. A. Montaraz, Infect. Immun. 50:190-198). This protein, which banded between 7.0 and 7.6 pH in preparative isoelectrofocusing, may be further purified with a monoclonal immunosorbent. Immunopurified protein showed adenylate cyclase activity. The enzymatic activity was found to be unstable during processing; i.e., although the crude extract showed up to 150 nmol of cyclic AMP per mg/min, the immunopurified protein showed a maximum of only 200 nmol of cyclic AMP per mg/min. Two strains of B. bronchiseptica, isolated from herds of healthy pigs showing no signs of atrophic rhinitis, did not produce the 68,000-molecular-weight protein and were negative for adenylate cyclase. However, it is not known whether the 68,000-molecular-weight protein is a component of adenylate cyclase or whether it is an unrelated protein associated with this enzyme in some unknown way. Adenylate cyclase activity from culture supernatants of B. bronchiseptica, B. pertussis, and B. parapertussis can be absorbed equally to the same monoclonal immunosorbent.

Adenylyl Cyclases↗
PubMed1985

Bordetella adenylate cyclase: a genus specific protective antigen and virulence factor.

Most of the adenylate cyclase (AC) present in Bordetella species is localized in the outer membrane, partly exposed to the cell surface. An isolation procedure to obtain the cell-bound enzyme was applied to Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis. Passive transfer of B. bronchiseptica anti-AC antibody, either in the form of monoclonal antibody or antibody transferred to offspring from female mice previously immunized with immunopurified B. bronchiseptica AC, prevented both death and the development of "atrophic rhinitis" in mice infected by an aerosol of virulent B. bronchiseptica. Immunopurified AC from both B. bronchiseptica and B. pertussis protected mice against intracerebral challenge with strain 18323, although five to six times more protein of B. bronchiseptica was necessary to produce the same level of protection as with B. pertussis-derived protein. The AC of Bordetella species differ in molecular weight and, despite sharing at least two common non-overlapping epitopes (BB05, BB07), antigenic differences are likely: sera from piglets artificially infected with virulent B. bronchiseptica developed high ELISA titers against AC from B. bronchiseptica but low titers against B. pertussis-derived antigen. Strains of B. bronchiseptica which did not induce "atrophic rhinitis" in pigs were found to be AC-less variants. When tested in our laboratory model, such a strain was also unable to induce "atrophic rhinitis" in mice. We conclude that AC is a mandatory component of any future subcellular vaccine against Bordetella species.

Adenylyl Cyclases↗
PubMed1983

Modulation of Bordetella pertussis by nicotinic acid.

Growth of Bordetella pertussis in a high concentration of nicotinic acid (NA) had a modulating effect on several properties and activities of the bacteria. Compared with normally grown cells, those grown in a high concentration of NA had reduced capacity for taking up both NA and nicotinamide (ND); they had reduced adenylate cyclase activity and showed loss of agglutinogen factors 2 and 3, but an increase in factor 1. By contrast, cells grown in a high concentration of ND showed only a slightly decreased capacity for uptake of ND and none of the other changes. Modulation of B. pertussis by NA varied with the strain and culture conditions and appeared to be distinct from the antigenic modulation induced by high Mg2+ in the culture medium. Evidence is presented for the association of a small proportion of the extracytoplasmic adenylate cyclase with the outer membrane of B. pertussis.

Adenylyl Cyclases↗
PubMed1978

Strain related infectivity of Neisseria gonorrhoeae for the guinea-pig subcutaneous chamber and the variability of the immune resistance in different breeds of guinea-pig.

The ability of gonococci to infect the subcutaneous chamber in guinea-pigs is strictly strain related. This ability was usually present in prototrophic strains and auxotrophs requiring proline only which were obtained from patients with uncomplicated gonorrhoea, but it was invariably absent in Neisseria gonorrhoeae auxotypes requiring arginine, uracil, and hypoxanthine, or arginine only for growth. All the strains obtained from disseminated gonococcal infections (all dependent upon arginine, uracil, and hypoxanthine) were unable to infect the guinea-pig chamber. Hence, the high invasiveness of N. gonorrhoeae for man and its infectivity for guinea-pig chambers appear to be unrelated properties. Although guinea-pigs of the same origin (Dunkin-Hartley) were used throughout, the degree of immune resistance was found to differ between the lines supplied by various breeders--that is, after a standard immunisation schedule using whole cell gonococcal vaccines, the homologous immune resistance to challenge varied from weak or non-existent in some lines, to highly resistant in others.

Animals↗