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Biomedical subjects

M Kilian

Publications and source records attributed to M Kilian.

At least 73 records · Page 4Linked to original sources
PubMed1996

High rate of nasopharyngeal carriage of potential pathogens among children in Greenland: results of a clinical survey of middle-ear disease.

Nasopharyngeal and middle-ear colonization with bacteria and viruses, including Mycoplasma pneumoniae and chlamydiae, was investigated in a survey of 54 children with acute otitis media (AOM) and 201 control children without AOM in Greenland. In total, 98% with AOM and 91% without AOM carried potentially pathogenic bacteria in the nasopharynx. Two or more potentially pathogenic species were carried by 78% with AOM and 57% without AOM. Haemophilus influenzae was found in 92% and 77%, respectively, but only Streptococcus pneumoniae was found significantly more often in the nasopharynx of children with AOM than in age-matched controls (P < .03). The two species were found in 22 of 24 ear-discharge specimens. Nine children (three with AOM) had chlamydiae in the nasopharynx, and seven of them reported rhinitis. Enteroviruses or rhinoviruses were detected in 23 nasopharyngeal specimens from 39 children with AOM, in 13 such specimens from 39 children without AOM (P = .040), and in 4 of 14 ear-discharge specimens. The potentially pathogenic load in the nasopharynx was massive, suggesting an association with the high prevalence of otitis media among children in Greenland.

Bacterial Infections↗
PubMed1996

Actinobacillus minor sp. nov., Actinobacillus porcinus sp. nov., and Actinobacillus indolicus sp. nov., three new V factor-dependent species from the respiratory tract of pigs.

The results of DNA-DNA relatedness experiments and comparisons of sequences of genes coding for 16S rRNA were used to determine the genetic relationships of selected V factor-dependent species belonging to the family Pasteurellaceae and obtained from the porcine respiratory tract. These results showed that the Minor group and taxa C, D plus E, and F are distinct phylogenetic groups that are separate from each other and from other members of the family Pasteurellaceae. On the basis of these results, three new species, corresponding to the Minor group, taxa D plus E, and taxon F, are proposed; the names of these new species are Actinobacillus minor (type strain, NM305), Actinobacillus porcinus (type strain, NM319), and Actinobacillus indolicus (type strain, 46KC2), respectively.

Actinobacillus↗
PubMed1996

Biological significance of IgA1 proteases in bacterial colonization and pathogenesis: critical evaluation of experimental evidence.

IgA1 protease activity, which allows bacteria to cleave human IgA1 in the hinge region, represents a striking example of convergent evolution of a specific property in bacteria. Although it has been known since 1979 that IgA1 protease is produced by the three leading causes of bacterial meningitis in addition to important urogenital pathogens and some members of the oropharyngeal flora, the exact role of this enzyme in bacterial pathogenesis is still incompletely understood owing to lack of a satisfactory animal model. Cleavage of IgA1 by these post-proline endopeptidases efficiently separates the monomeric antigen-binding fragments from the secondary effector functions of the IgA1 antibody molecule. Several in vivo and in vitro observations indicate that the enzymes are important for the ability of bacteria to colonize mucosal membranes in the presence of S-IgA antibodies. Furthermore, the extensive cleavage of IgA sometimes observed in vivo, suggests that IgA1 protease activity results in a local functional IgA deficiency that may facilitate colonization of other microorganisms and the penetration of potential allergens. It has been hypothesized that IgA1 protease activity of Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae, under special immunological circumstances, allows these bacteria to take advantage of specific IgA1 antibodies in a strategy to evade other immune factors of the human body. The decisive factor is the balance between IgA antibodies against surface antigens of the respective bacteria and their IgA1 protease. Recent studies have shown that serine-type IgA1 proteases of H. influenzae, meningococci, and gonococci belong to a family of proteins used by a diverse group of Gram-negative bacteria for colonization and invasion.

Amino Acid Sequence↗
PubMed1996

Characterization of the Streptococcus pneumoniae immunoglobulin A1 protease gene (iga) and its translation product.

Bacterial immunoglobulin A1 (IgA1) proteases constitute a very heterogenous group of extracellular endopeptidases which specifically cleave human IgA1 in the hinge region. Here we report that the IgA1 protease gene, iga, of Streptococcus pneumoniae is homologous to that of Streptococcus sanguis. By using the S. sanguis iga gene as hybridization probe, the corresponding gene from a clinical isolate of S. pneumoniae was isolated in an Escherichia coli lambda phage library. A lysate of E. coli infected with hybridization-positive recombinant phages possessed IgA1-cleaving activity. The complete sequence of the S. pneumoniae iga gene was determined. An open reading frame with a strongly biased codon usage and having the potential of encoding a protein of 1,927 amino acids with a molecular mass of 215,023 Da was preceded by a potential -10 promoter sequence and a putative Shine-Dalgarno sequence. A putative signal peptide was found in the N-terminal end of the protein. The amino acid sequence similarity to the S. sanguis IgA1 protease indicated that the pneumococcal IgA1 protease is a Zn-metalloproteinase. The primary structures of the two streptococcal IgA1 proteases were quite different in the N-terminal parts, and both proteins contained repeat structures in this region. Using a novel assay for IgA1 protease activity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we demonstrated that the secreted IgA1 protease was present in several different molecular forms ranging in size from approximately 135 to 220 kDa. In addition, interstrain differences in the sizes of the pneumococcal IgA1 proteases were detected. Southern blot analyses suggested that the S. pneumoniae iga gene is highly heterogenous within the species.

Amino Acid Sequence↗
PubMed1996

Population structure of Streptococcus agalactiae reveals an association between specific evolutionary lineages and putative virulence factors but not disease.

To evaluate the genetic diversity and relationships in a collection of 85 Danish strains of Streptococcus agalactiae (group B streptococcus) we have performed restriction fragment length polymorphism analysis on EcoRI- and MspI-digested whole-cell DNA using as probes rRNA, DNA fragments representing the genes encoding hyaluronidase, C5a-peptidase, alpha-antigen, and beta-antigen as well as two randomly selected genomic DNA fragments for which the coding potential is unknown. In addition, we have assayed for expression of hyaluronidase activity and beta-antigen. Combined analyses of our data and those previously obtained by multilocus enzyme electrophoresis and serotyping revealed a population separating into six major lineages that correlate with individual serotypes. The significant linkage disequilibrium of alleles indicates that the S. agalactiae population examined is predominantly clonal. Notably, strains expressing the serotype III capsule divide into two distant evolutionary lineages, of which one lacks expression of hyaluronidase activity. Six North American isolates of serotype III clustered together with multiple Danish serotype III strains, showing that the combinations of characters on which the phylogenetic tree was based are conserved worldwide. Occurrence of beta-antigen correlated with a specific version of the alpha-antigen gene and was exclusively associated with a single major phylogenetic lineage. Comparisons with the clinical history of the strains revealed no evidence of differences in pathogenic potential among the six major genetic divisions.

Adhesins, Bacterial↗
PubMed1996

Highly toxic clone of Actinobacillus actinomycetemcomitans in geographically widespread cases of juvenile periodontitis in adolescents of African origin.

The bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis as the etiologic agent on the basis of several lines of circumstantial evidence. A matter of extensive debate is whether A. actinomycetemcomitans is an exogenous contagious pathogen or an opportunistic pathogen that resides in the normal oral microflora. Here we show evidence of a single clone of A. actinomycetemcomitans isolated from multiple patients with juvenile periodontitis in members of families of African origin living in geographically widespread areas. The clone is characterized by a 530-bp deletion in the leukotoxin gene operon, resulting in a significantly increased production of leukotoxin.

Adolescent↗
PubMed1995

Neisseria gonorrhoeae IgA1 proteases share epitopes recognized by neutralizing antibodies.

The antigenic diversity among IgA1 proteases of 61 Neisseria gonorrhoeae strains isolated during a period of 23 years and on four continents was examined in enzyme neutralization assays employing rabbit antisera raised against selected IgA1 proteases. The antigenic analyses were compared with results of iga gene-region RFLP patterns and enzyme cleavage specificity for substrate IgA1. Type 1 IgA1 proteases were antigenically uniform while six different antigenic types were detected among type 2 enzymes. Extensive cross-reactions of antibodies against the different antigenic types suggested only minor differences in relevant epitopes. Epitopes previously found to be common to all Neisseria meningitidis IgA1 proteases were also shared by all N. gonorrhoeae IgA1 proteases in the collection. Human sera from patients with gonorrhoea showed broadly cross-reactive neutralizing activity at titers comparable to those of sera from immunized rabbits. In conclusion, N. gonorrhoeae IgA1 proteases show a remarkable lack of diversity of epitopes recognized by enzyme-neutralizing antibodies. If future studies confirm that cleavage of IgA1 is an important step in gonococcal infections, Neisseria IgA1 proteases may be attractive vaccine candidates.

Animals↗
PubMed1995

Confirmation of the species Prevotella intermedia and Prevotella nigrescens.

The elevation of the two genotypes of Prevotella intermedia to species rank as P. intermedia and Prevotella nigrescens has increased the need for reliable differentiation between the two taxa. In this study, 53 strains, including strains whose species affiliations were known as well as fresh dental plaque isolates, were subjected to a multilocus enzyme electrophoretic analysis, DNA analyses in which we used whole genomic DNA, rRNA sequences, and an oligonucleotide specific for the former P. intermedia genotype II (P. nigrescens) as probes, and a sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of soluble cellular proteins. All of these tests consistently separated the strains into the same two distinct groups corresponding to P. intermedia and P. nigrescens, confirming that the two species constitute two distinct populations of bacteria. Each of the tests used independently provided reliable identification to the species level. A previously reported heterogeneity in the pattern of human immunoglobulin A1 (IgA1) degradation was not confirmed. No differences between species were observed. All of the strains induced total degradation of IgA1 within 48 h, a property that may be a virulence factor in periodontal disease development. The enzymes responsible for IgA1 degradation were not inactivated by the physiological proteinase inhibitors alpha 2-macroglobulin and alpha 1-proteinase inhibitor.

Alleles↗
PubMed1995

Comparative characterization of the iga gene encoding IgA1 protease in Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae.

Cloning and sequencing of the IgA1 protease gene (iga) from Neisseria meningitidis strain HF13 showed an overall structure equivalent to iga genes from Neisseria gonorrhoeae and Haemophilus influenzae, although no region corresponding to the gonococcal alpha-peptide was evident. An additional 18 N. meningitidis and 3 H. influenzae iga genes were amplified by the polymerase chain reaction technique and sequenced corresponding approximately to the N-terminal half of the mature enzyme. Comparative analyses of a total of 29 iga genes showed that pathogenic Neisseria have iga genes with a significantly lower degree of heterogeneity than H. influenzae iga genes. Recombinational events indicated by mosaic-like structures corresponding to those found among N. gonorrhoeae protease genes were detected among N. meningitidis iga genes. One region showed characteristic differences in sequence and length which correlated with each of the different cleavage specificities. Meningococci were extremely conserved in this region with no evidence of recombination between isolates of different cleavage specificities. Sequences further downstream showed no obvious relationship with enzyme cleavage type. This region consisted of conserved areas interspersed with highly variable areas. Amino acid sequence homologies in the variable regions of meningococci reflected the antigenic types defined by using polyclonal neutralizing antibodies.

Amino Acid Sequence↗
PubMed1995

Clonal diversity of the Streptococcus mitis biovar 1 population in the human oral cavity and pharynx.

A total of 250 isolates of oral streptococci were recovered from swabs of oropharyngeal surfaces of 3 members of one family. All isolates were examined by biochemical and serological means, and 106 isolates were identified as Streptococcus mitis biovar 1. These were typed by restriction endonuclease analysis using the enzymes EcoRI and HaeIII and further characterized by their whole-cell polypeptide profile patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, rabbit antisera raised against 8 reference strains of oral streptococci were used to characterize representative isolates both by their carbohydrate and protein antigens by Ouchterlony and Western blot analyses. Very limited biochemical diversity was observed among the 106 S. mitis biovar 1 isolates. In contrast, 24 different genotypes defined by restriction endonuclease analysis were detected, and each individual carried 6-13 types. Limited sharing of genotypes was observed between the 3 members of the same family and between the pharyngeal and buccal mucosa of single individuals. The antigenic analyses showed remarkable antigenic diversity between the 24 genotypes. The results provide a basis for studying the population dynamics of an oral commensal species and its interaction with the salivary immune system.

Adult↗
PubMed1995

In vivo cleavage of immunoglobulin A1 by immunoglobulin A1 proteases from Prevotella and Capnocytophaga species.

Immunoglobulin A1 (IgA1) proteases secreted by oral Prevotella and Capnocytophaga species specifically cleave IgA1 at the same peptide bond in the hinge region, leaving intact monomeric Fab and Fc fragments. Assuming that Prevotella- and Capnocytophaga-induced Fab fragments of IgA1 expose a specific immunogenic neoepitope at the cleavage site, we established an enzyme-linked immunosorbent assay to measure human serum antibodies to this neoepitope as indirect evidence of in vivo activity of Prevotella and Capnocytophaga IgA1 proteases. The assay used a monoclonal antibody with specificity for the neoepitope, and the ability to block binding of the monoclonal antibody to the neoepitope was investigated. Absorption of sera with Prevotella melaninogenica-induced Fab fragments of IgA1 resulted in removal of antibodies blocking binding of the monoclonal antibody, whereas absorption with Fab fragments induced by bacterial IgA1 proteases of other cleavage specificities did not remove blocking antibodies. Consequently, we assume that the antibodies detected had been induced by a neoepitope an the Fab fragment of IgA1 exposed exclusively after cleavage with IgA1 proteases from Prevotella and Capnocytophaga, indicating in vivo activity of these IgA1 proteases. Evidence, though indirect, of in vivo activity of Prevotella and Capnocytophaga IgA1 proteases was present in 42 of 92 sera examined and in a significantly higher proportion of sera from adults with periodontal disease compared with control individuals. No correlation with disease was observed for the juvenile periodontitis groups.

Adolescent↗
PubMed1995

Distinct antigenic and genetic properties of the immunoglobulin A1 protease produced by Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever in Brazil.

All examined Haemophilus influenzae biogroup aegyptius isolates of the clone associated with Brazilian purpuric fever (the BPF clone) produced type 2 immunoglobulin A1 (IgA1) proteases encoded by identical iga genes that were distinct from the iga genes of other Brazilian H. influenzae biogroup aegyptius isolates. A partial nucleotide sequence analysis revealed close similarities to the iga genes of H. influenzae serotype c and one noncapsular H. influenzae biotype III strain isolated from a case of conjunctivitis in Tunisia, suggesting an evolutionary relationship. Epitopes recognized by neutralizing antibodies differed for the IgA1 proteases of the BPF clone and of other H. influenzae strains, including Brazilian H. influenzae biogroup aegyptius isolates from patients with noninvasive conjunctivitis. The low probability of developing cross-reacting neutralizing antibodies to the IgA1 protease of the BPF clone may contribute to the pathogenic potential of this virulent phenotype in Brazil.

Amino Acid Sequence↗
PubMed1995

Evidence for absence in northern Europe of especially virulent clonal types of Actinobacillus actinomycetemcomitans.

Genetic analysis of an Actinobacillus actinomycetemcomitans population consisting of 88 clinically well characterized Finnish isolates performed by multilocus enzyme electrophoresis confirmed that the five serotypes divide into two phylogenetic lineages, one comprising serotypes b and c and one comprising serotypes a, d, and e. There was no association between any subpopulation and the periodontal health status of the subject from whom the isolates originated, suggesting that the role of A. actinomycetemcomitans in periodontitis is largely opportunistic in the population examined. Southern blot analyses of genomic DNA digested with each of the restriction endonucleases MspI, RsaI, and TaqI revealed extremely limited genetic polymorphism of the structural leukotoxin gene, ltxA, and its associated promoter. All isolates hybridized to a 530-bp DNA fragment derived from the promoter region of the leukotoxin gene operon of a minimally leukotoxic A. actinomycetemcomitans strain. Deletion of the 530-bp sequence has been associated with significantly increased toxin production detected among isolates from patients with juvenile periodontitis in North America but was detected neither among the 88 isolates in the present collection analyzed nor among more than 60 strains in another population of northern European A. actinomycetemcomitans isolates analyzed previously.

Actinobacillus Infections↗
PubMed1995

Increased proportions of bacteria capable of cleaving IgA1 in the pharynx of infants with atopic disease.

Based on the observation that children with a history of atopic disease show significantly increased levels of cleaved secretory IgA in nasopharyngeal secretions, we have previously formulated the hypothesis that bacteria-induced local deficiencies of the immune barrier of the upper respiratory tract may be a contributing factor in the development and perpetuation of atopic diseases. To evaluate this hypothesis, 25 infants were subjected to clinical, bacteriologic, and immunologic examination at the age of 18 mo, 30 mo, and 5 y. The 11 infants, who showed clinical and immunologic evidence of atopic disease at the age of 18 mo, harbored significantly higher proportions of IgA1 protease-producing bacteria (median, 36%; range, 14-64%) than the 14 healthy infants (median, 5%; range, 0.4-14%). No statistically significant differences were observed at the two subsequent examinations, but healthy children showed a statistically significant increase in proportions of IgA1 protease-producing bacteria in the pharynx with increasing age. IgA1 protease-producing bacteria detected included Streptococcus mitis biovar 1, Haemophilus influenzae, Haemophilus parahaemolyticus, Streptococcus pneumoniae, and Neisseria meningitidis, of which the first mentioned species was mainly responsible for the difference observed at the 18-mo examination. Percentage proportions of IgA1 protease-producing bacteria were significantly related to passive smoking which may stimulate the premature and more pronounced pharyngeal colonization of the atopic infants with the IgA1 protease-producing variant of S. mitis biovar 1. The results of the study support the hypothesis that IgA1 protease-producing bacteria colonizing the upper respiratory tract jeopardize the local immune barrier and, thereby, may facilitate the penetration of potential allergens resulting in atopic disease.

Bacterial Infections↗
PubMed1995

Antisperm antibodies in infertile women: subclass distribution of immunoglobulin (Ig) A antibodies and removal of IgA sperm-bound antibodies with a specific IgA1 protease.

OBJECTIVE: To determine the immunoglobulin (Ig) A subclass distribution of antibodies in the serum and cervical mucus (CM) of infertile women and to evaluate the effect of an IgA1 protease on the removal of sperm-bound antibodies. METHODS: Twenty infertile women with antisperm antibodies in serum (n = 10) or in CM (n = 10) were recruited for this study. Monoclonal antibodies to human IgA1 and IgA2 were conjugated to immunobeads and the IgA subclass distribution of antisperm antibodies was determined for positive serum and CM samples. The effect of an IgA1 protease (isolated from Neisseria meningitidis strain HF13) on sperm-bound antibodies was evaluated by immunobead binding. RESULTS: In serum, IgA1 subclass antisperm antibodies predominated (89%) when compared to IgA2 (11%). In CM IgA1 accounted for 62% and IgA2 accounted for 38% of the total IgA antisperm antibodies. Enzyme treatment was able to reduce dramatically the amount of serum IgA antibodies bound to sperm from 88% to 10%. Similarly, a significant reduction in CM antisperm antibodies was observed after enzymatic treatment with no loss in sperm motility. CONCLUSION: Cervical mucus antisperm antibodies have a higher proportion of IgA2 subclass suggesting a local production of IgA. Specific IgA1 protease treatment is capable of reducing the amount of immunobead-detectable IgA on sperm. Hamster sperm penetration assays are ongoing to determine if this treatment might improve sperm penetration rates with antibody positive sperm.

Antibodies↗