In vitro complement activation, adherence to red blood cells and induction of mononuclear cell cytokine production by four strains of Aggregatibacter actinomycetemcomitans with different fimbriation and expression of leukotoxin.

BACKGROUND AND OBJECTIVE: The periodontal pathogen Aggregatibacter actinomycetemcomitans has been proposed as pro-atherogenic, and complement-mediated adherence to red blood cells (RBCs) may facilitate its systemic spread. We investigated the ability of four strains of A. actinomycetemcomitans with differential expression of leukotoxin A (LtxA) and fimbriae to activate complement, adhere to RBCs and elicit cytokine responses by mononuclear cells (MNCs). MATERIAL AND METHODS: Aggregatibacter actinomycetemcomitans serotype b strains HK 921, HK 1651, HK 2092 and HK 2108 were fluorescence-labeled, incubated with human whole blood cells in the presence of autologous serum, and assessed for RBC adherence by flow cytometry and for capacity to induce cytokine production by cytometric bead array analysis. The levels of IgG to A. actinomycetemcomitans serotype b were quantified by ELISA, as was consumption of complement. RESULTS: The JP2 clone variants HK 1651 and, to a lesser extent, HK 2092, consumed complement efficiently, while HK 2108 (= strain Y4) consumed complement poorly. Nonetheless, the four tested strains adhered equally well to RBCs in the presence of autologous serum, without causing RBC lysis. The JP2 clone variant HK 2092, selectively lacking LtxA production, induced higher production of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and IL-10 by MNCs than did the other three strains, while the four strains induced similar production of IL-12p70. RBCs facilitated the HK 2092-induced production of TNF-α and IL-1ß, and IL-6 was enhanced by RBCs, and this facilitation could be counteracted by blockade of complement receptor 3 (CD11b/CD18). CONCLUSION: Our data suggest that the JP2 clone of A. actinomycetemcomitans, most closely resembled by the variant HK 1651, activates complement well, while strain Y4, represented by HK 2108, activates complement poorly. However, all strains of A. actinomycetemcomitans adhere to RBCs and, when capable of producing LtxA, prevent production of inflammatory cytokines by MNCs. This "immunologically silent" immune adherence may facilitate systemic spread and atherogenesis.

Microbiological and immunological characteristics of young Moroccan patients with aggressive periodontitis with and without detectable Aggregatibacter actinomycetemcomitans JP2 infection.

Cross-sectional and longitudinal studies identify the JP2 clone of Aggregatibacter actinomycetemcomitans as an aetiological agent of aggressive periodontitis (AgP) in adolescents of northwest African descent. To gain information on why a significant part of Moroccan adolescents show clinical signs of periodontal disease in the absence of this pathogen we performed comprehensive mapping of the subgingival microbiota of eight young Moroccans, four of whom were diagnosed with clinical signs of AgP. The analysis was carried out by sequencing and phylogenetic analysis of a total of 2717 cloned polymerase chain reaction amplicons of the phylogenetically informative 16S ribosomal RNA gene. The analyses revealed a total of 173 bacterial taxa of which 39% were previously undetected. The JP2 clone constituted a minor proportion of the complex subgingival microbiota in patients with active disease. Rather than identifying alternative aetiologies to AgP, the recorded infection history of the subjects combined with remarkably high concentrations of antibodies against the A. actinomycetemcomitans leukotoxin suggest that disease activity was terminated in some patients with AgP as a result of elimination of the JP2 clone. This study provides information on the microbial context of the JP2 clone activity in a JP2-susceptible population and suggests that such individuals may develop immunity to AgP.

Lamellar body counts on gastric aspirates for prediction of respiratory distress syndrome.

AIM: To develop a rapid method for diagnosing lung maturity at birth with the purpose of administering surfactant early to infants with immature lungs and to spare infants with mature lungs from this treatment. METHODS: Lamellar body counts (LBC) on gastric aspirates from 191 newborns were counted in the platelet window in automatic blood cell counters. A preliminary study was performed on 108 aspirates from 2000 in infants with <32 weeks' gestation. Furthermore, 83 aspirates from 2004 to 2005 in infants with <30 weeks' gestation were analysed. RESULTS: Lamellar bodies in gastric aspirate were identified by electron microscopy. Seventy of the aspirates from 2004 to 2005 were analysed with a Sysmex XE-2100 (Sysmex, Holbaek, Naestved, Odense and Rigshospitalet, Denmark) counter. Twenty-four of these infants developed moderate to severe respiratory distress syndrome (RDS). The best cut-off value was 8000/µL with a sensitivity of 75% and a specificity of 72%. Forty-four of the 70 aspirates from 2004 to 2005 were analysed by Sysmex, Advia 120 and Cell-Dyn 4000. Thirteen other aspirates from 2004 to 05 were analysed by Sysmex and Coulter Counter LH755. Using Advia and Coulter the results were similar to Sysmex, but LBC obtained with Cell-Dyn were not correlated with the development of RDS. CONCLUSION: Lamellar body counts on gastric aspirate is a promising tool for prediction of development of RDS in infants of <30 weeks` gestation.

Allergen-reactive antibodies are found in nasal fluids from patients with birch pollen-induced intermittent allergic rhinitis, but not in healthy controls.

BACKGROUND: Increased levels of allergen-reactive immunoglobulins (Igs) have been reported in nasal fluids from patients with intermittent allergic rhinitis (IAR) sensitive to ragweed and grass. The aims of this study were to make a detailed characterization of nasal fluid Igs in birch pollen-induced IAR. METHODS: Nasal fluids were obtained from 23 patients with birch pollen-induced IAR during and after the birch pollen season, and from 20 healthy controls. Nasal fluid total and Bet v 1-reactive (IgA), IgE and IgG as well as albumin were analyzed by immunoassays. The integrity of IgA and IgG, and the molecular form of IgA were assessed by Western blotting and column fractionation, respectively. RESULTS: Nasal fluid total IgE and IgG, but not IgA, were higher in patients compared with controls. Western blotting indicated no significant degradation of IgA (including S-IgA) and IgG. Most of the IgA, including Bet v 1-reactive antibodies, was of the secretory form and of the IgA1 subclass. Bet v 1-reactive IgA and IgG were present in all patients, but was mostly nondetectable in controls. No significant differences in the levels of Bet v 1-reactive IgA and IgG were found in patients during the birch pollen season compared with off season. Both Bet v 1 and Bet v 2-reactive IgE were nondetectable in most samples. CONCLUSIONS: Nasal fluid Bet v 1-reactive IgA and IgG were found in all patients with birch pollen-induced IAR, but not in controls. However, no significant differences were found between patients during and after the birch pollen season.

DNA microarrays to study gene expression in allergic airways.

BACKGROUND: Allergic rhinitis results from interactions between a large number of cells and mediators in different compartments of the body. DNA microarrays allow simultaneous measurement of expression of thousands of genes in the same tissue sample. OBJECTIVE: To study gene expression in nasal mucosal biopsies from patients with allergic rhinitis using DNA micro-arrays. METHODS: Nasal biopsies were obtained from 14 patients with symptomatic birch pollen-induced allergic rhinitis and five healthy controls. RNA was extracted from the biopsies and pooled into one patient pool and one control pool. These were analysed in duplicate with DNA micro-arrays containing more than 12 000 known genes. RESULTS: Approximately half of the genes were expressed in the patient and control samples. Guided by the current literature we chose 32 genes of possible relevance to allergic airway inflammation and investigated their relative expression. Among these, transcripts encoding immunoglobulins and their receptors were most abundant. The expression of cytokines and growth factors was low, whereas their corresponding receptors and cell surface markers displayed higher expression levels. IgA had the highest expression of all 12 626 genes. RT-PCR showed that IgA1 was the predominant subclass. This was confirmed by the protein level in nasal fluids. Allergen-specific IgA was significantly higher in patients than in controls and correlated significantly with eosinophil granulae proteins. CONCLUSION: DNA micro-array analysis can be used to identify genes of possible relevance to allergic airway inflammation. In this study, the expression profile in the nasal mucosa was quantitatively dominated by immunoglobulins, particularly IgA. Protein analyses in nasal fluids indicated a role for allergen-specific IgA in eosinophil degranulation.

Population dynamics of Streptococcus mitis in its natural habitat.

The purpose of this study was to examine the genetic structure of the typical commensal Streptococcus mitis biovar 1 in its natural habitat in the human oral cavity and pharynx and to investigate the role that selected microbial properties and host, spatial, and temporal factors play in determining the structure of the bacterial population. Consecutive samples were collected from buccal and pharyngeal mucosal surfaces of two infants, their four parents, and two elderly individuals over a period of approximately 1 year. A total of 751 isolates identified as S. mitis biovar 1 were typed by restriction endonuclease analysis (REA) and representative clones were typed by multilocus enzyme electrophoresis (MLEE). The genetic diversity of the S. mitis biovar 1 isolates collected from single infant hosts over a period of 9 to 10 months was found to be between 0.69 and 0.76, which is considerably higher than that previously observed for intestinal populations of Escherichia coli. The study provides evidence of the existence of both transient and persistent clones in adult individuals. In the two infants, however, none of 42 demonstrated clones were detected on more than a single occasion. Statistical calculations showed that the ability to persist was not distributed at random in the S. mitis biovar 1 population. However, neither immunoglobulin A1 protease activity nor the ability to bind alpha-amylase from saliva was a preferential characteristic of persistent genotypes. In contrast to current concepts of climax ecosystems, the species niche in the habitat appears to be maintained predominantly by a succession of clones rather than by stable strains. Several lines of evidence suggest that the major origin of "new" clones is the many other habitats in the respiratory tract that are occupied by this species.

Immunoglobulins in nasal secretions of healthy humans: structural integrity of secretory immunoglobulin A1 (IgA1) and occurrence of neutralizing antibodies to IgA1 proteases of nasal bacteria.

Certain bacteria, including overt pathogens as well as commensals, produce immunoglobulin A1 (IgA1) proteases. By cleaving IgA1, including secretory IgA1, in the hinge region, these enzymes may interfere with the barrier functions of mucosal IgA antibodies, as indicated by experiments in vitro. Previous studies have suggested that cleavage of IgA1 in nasal secretions may be associated with the development and perpetuation of atopic disease. To clarify the potential effect of IgA1 protease-producing bacteria in the nasal cavity, we have analyzed immunoglobulin isotypes in nasal secretions of 11 healthy humans, with a focus on IgA, and at the same time have characterized and quantified IgA1 protease-producing bacteria in the nasal flora of the subjects. Samples in the form of nasal wash were collected by using a washing liquid that contained lithium as an internal reference. Dilution factors and, subsequently, concentrations in undiluted secretions could thereby be calculated. IgA, mainly in the secretory form, was found by enzyme-linked immunosorbent assay to be the dominant isotype in all subjects, and the vast majority of IgA (median, 91%) was of the A1 subclass, corroborating results of previous analyses at the level of immunoglobulin-producing cells. Levels of serum-type immunoglobulins were low, except for four subjects in whom levels of IgG corresponded to 20 to 66% of total IgA. Cumulative levels of IgA, IgG, and IgM in undiluted secretions ranged from 260 to 2,494 (median, 777) microg ml(-1). IgA1 protease-producing bacteria (Haemophilus influenzae, Streptococcus pneumoniae, or Streptococcus mitis biovar 1) were isolated from the nasal cavities of seven subjects at 2.1 x 10(3) to 7.2 x 10(6) CFU per ml of undiluted secretion, corresponding to 0.2 to 99.6% of the flora. Nevertheless, alpha-chain fragments characteristic of IgA1 protease activity were not detected in secretions from any subject by immunoblotting. Neutralizing antibodies to IgA1 proteases of autologous isolates were detected in secretions from five of the seven subjects but not in those from two subjects harboring IgA1 protease-producing S. mitis biovar 1. alpha-chain fragments different from Fc(alpha) and Fd(alpha) were detected in some samples, possibly reflecting nonspecific proteolytic activity of microbial or host origin. These results add to previous evidence for a role of secretory immunity in the defense of the nasal mucosa but do not help identify conditions under which bacterial IgA1 proteases may interfere with this defense.

Resident aerobic microbiota of the adult human nasal cavity.

Recent evidence strongly suggests that the microbiota of the nasal cavity plays a crucial role in determining the reaction patterns of the mucosal and systemic immune system. However, little is known about the normal microbiota of the nasal cavity. The purpose of this study was to determine the microbiota in different parts of the nasal cavity and to develop and evaluate methods for this purpose. Samples were collected from 10 healthy adults by nasal washes and by swabbing of the mucosa through a sterile introduction device. Both methods gave results that were quantitatively and qualitatively reproducible, and revealed significant differences in the density of the nasal microbiota between individuals. The study revealed absence of gram-negative bacteria that are regular members of the commensal microbiota of the pharynx. Likewise, viridans type streptococci were sparsely represented. The nasal microbiota was dominated by species of the genera Corynebacterium, Aureobacterium, Rhodococcus, and Staphylococcus, including S. epidermis, S. capitis, S. hominis, S. haemolyticus, S. lugdunensis and S. warneri. These studies show that the microbiota of the nasal cavity of adults is strikingly different from that of the pharynx, and that the nasal cavity is a primary habitat for several species of diphtheroids recognized as opportunistic pathogens. Under special circumstances, single species, including IgA1 protease-producing bacteria, may become predominant in a restricted area of the nasal mucosa.

Nasal continuous positive airway pressure and early surfactant therapy for respiratory distress syndrome in newborns of less than 30 weeks' gestation.

OBJECTIVE: To determine whether early versus late treatment with porcine surfactant (Curosurf) reduces the requirement of mechanical ventilation in very preterm infants primarily supported by nasal continuous positive airway pressure (nasal CPAP). DESIGN: Multicenter randomized, controlled trial. PATIENTS: The study population comprised 60 infants <30 weeks' gestation with respiratory distress syndrome (RDS) who had an arterial to alveolar oxygen tension ratio (a/APO2) of 0.35 to 0.22. The cohort from which the study population was generated comprised 397 infants. RESULTS: The need for mechanical ventilation or death within 7 days of age was reduced from 63% in the late-treated infants to 21% in early-treated infants. Increasing numbers of antenatal steroid doses also improved the outcome, especially in the early-treated infants. Six hours after randomization mean a/APO2 rose to 0.48 in the early-treated infants compared with 0.36 in the late-treated. The need of mechanical ventilation before discharge was reduced from 68% in the late-treated to 25% in the early-treated infants. CONCLUSIONS: Nasal CPAP in combination with early treatment with Curosurf significantly improves oxygenation and reduces the subsequent need for mechanical ventilation in infants <30 weeks' gestational age with RDS.

A comprehensive genetic study of streptococcal immunoglobulin A1 proteases: evidence for recombination within and between species.

An analysis of 13 immunoglobulin A1 (IgA1) protease genes (iga) of strains of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguis was carried out to obtain information on the structure, polymorphism, and phylogeny of this specific protease, which enables bacteria to evade functions of the predominant Ig isotype on mucosal surfaces. The analysis included cloning and sequencing of iga genes from S. oralis and S. mitis biovar 1, sequencing of an additional seven iga genes from S. sanguis biovars 1 through 4, and restriction fragment length polymorphism (RFLP) analyses of iga genes of another 10 strains of S. mitis biovar 1 and 6 strains of S. oralis. All 13 genes sequenced had the potential of encoding proteins with molecular masses of approximately 200 kDa containing the sequence motif HEMTH and an E residue 20 amino acids downstream, which are characteristic of Zn metalloproteinases. In addition, all had a typical gram-positive cell wall anchor motif, LPNTG, which, in contrast to such motifs in other known streptococcal and staphylococcal proteins, was located in their N-terminal parts. Repeat structures showing variation in number and sequence were present in all strains and may be of relevance to the immunogenicities of the enzymes. Protease activities in cultures of the streptococcal strains were associated with species of different molecular masses ranging from 130 to 200 kDa, suggesting posttranslational processing possibly as a result of autoproteolysis at post-proline peptide bonds in the N-terminal parts of the molecules. Comparison of deduced amino acid sequences revealed a 94% similarity between S. oralis and S. mitis IgA1 proteases and a 75 to 79% similarity between IgA1 proteases of these species and those of S. pneumoniae and S. sanguis, respectively. Combined with the results of RFLP analyses using different iga gene fragments as probes, the results of nucleotide sequence comparisons provide evidence of horizontal transfer of iga gene sequences among individual strains of S. sanguis as well as among S. mitis and the two species S. pneumoniae and S. oralis. While iga genes of S. sanguis and S. oralis were highly homogeneous, the genes of S. pneumoniae and S. mitis showed extensive polymorphism reflected in different degrees of antigenic diversity.

Comparative analysis of immunoglobulin A1 protease activity among bacteria representing different genera, species, and strains.

Immunoglobulin A1 (IgA1) proteases cleaving human IgA1 in the hinge region are produced constitutively by a number of pathogens, including Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, and Streptococcus pneumoniae, as well as by some members of the resident oropharyngeal flora. Whereas IgA1 proteases have been shown to interfere with the functions of IgA antibodies in vitro, the exact role of these enzymes in the relationship of bacteria to a human host capable of responding with enzyme-neutralizing antibodies is not clear. Conceivably, the role of IgA1 proteases may depend on the quantity of IgA1 protease generated as well as on the balance between secreted and cell-associated forms of the enzyme. Therefore, we have compared levels of IgA1 protease activity in cultures of 38 bacterial strains representing different genera and species as well as strains of different pathogenic potential. Wide variation in activity generation rate was found overall and within some species. High activity was not an exclusive property of bacteria with documented pathogenicity. Almost all activity of H. influenzae, N. meningitidis, and N. gonorrhoeae strains was present in the supernatant. In contrast, large proportions of the activity in Streptococcus, Prevotella, and Capnocytophaga species was cell associated at early stationary phase, suggesting that the enzyme may play the role of a surface antigen. Partial release of cell-associated activity occurred during stationary phase. Within some taxa, the degree of activity variation correlated with degree of antigenic diversity of the enzyme as determined previously. This finding may indicate that the variation observed is of biological significance.

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.

The effect of microwave heating on vitamins B1 and E, and linoleic and linolenic acids, and immunoglobulins in human milk.

Breast milk was treated with (1) conventional heating (in water bath) vs microwave heating; (2) microwave heating at two power levels (30% and 100%); (3) increasing final temperatures; and (4) microwave thawing vs refrigerator thawing and examined for changes in specific immunoglobulins to a pool of E. coli and poliovirus type 1 antigens, vitamins E and B1, and the polyunsaturated fatty acids linoleic and linolenic acid. Immunoglobulin activities were stable until final milk temperatures of around 60-65 degrees C were reached, and total inactivation occurred at 77 degrees C. Heating even to high final temperatures did not change contents of vitamins and polyunsaturated fatty acids. No differences in immunoglobulins and nutrients were demonstrated between microwave heating and conventional heating, and between power levels or thawing methods. The study shows that microwave heating of human milk can be performed without significant losses of examined immunoglobulins and nutrients, provided that final temperatures are below 60 degrees C.

A method for titration of inhibiting antibodies to bacterial immunoglobulin A1 proteases in human serum and secretions.

Bacterial IgA1 proteases specifically cleave IgA1, including S-IgA1, molecules into Fab alpha and Fc alpha fragments. Hereby these enzymes interfere with the protective functions of antibodies belonging to this isotype. Antibodies inhibiting IgA1 proteases have been detected in humans, but the titration of such antibodies is a matter of methodological concern. Because human serum and secretions contain IgA1 substrate, it is impossible to provide uniform substrate conditions for samples of IgA1 protease incubated with inhibitors differing in their origin and state of dilution. This study demonstrates that such variations in substrate are not prohibitive for a reliable titration of inhibiting antibodies. This was evident from experiments demonstrating that the variations do not interfere with the quantification of residual IgA1 protease activity provided the activity is measured in terms of the proportion of IgA1 substrate cleaved during incubation. Proportions of cleaved IgA1 were measured by exploiting the differential reactivity of cleaved and intact IgA1 molecules in an ELISA using anti-Fc alpha and enzyme-conjugated anti-light chain antibodies for catching and development, respectively. A protocol for the titration of IgA1 protease-inhibiting antibodies based on this ELISA is described. By application of the protocol to chromatographic fractions of saliva, IgA1 protease-inhibiting activity was found to co-purify with salivary S-IgA.

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.

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.

Neonatal Immune Thrombocytopenia Due to Allo-or Autoantibodies: Clinical and Immunological Analysis of 83 Cases.

Serological and clinical data were collected in 59 cases of suspected neonatal alloimmune thrombocytopenia (NAIT) and in 24 thrombocytopenic newborn of mothers with presumed autoimmune thrombocytopenic purpura (AITP). In the NAIT group, the anti-HPA-la (anti-Zw(a), anti-Pl(AI)) and the anti-HPA-5b (anti-Br(a)) account for about 68% and 11% respectively of the serologically proven cases. The findings of a high frequency (45%) of maternal anti-HLA antibodies in the HPA-la positive group, suggests an association of NAIT and maternal HLA alloimmunisation. In the AITP group, 83% of the women had increased amounts of platelet-associated IgG (PAIgG) with a predominance of IgGl and IgG3. In one third of the cases, maternal circulating autoantibodies, mainly against GPIIb/IIIa and GPIb/IX, were found. The finding of circulating platelet autoantibodies in 16% of the HPA-la positive non-thrombocytopenic mothers makes it possible that these women are suffering from compensated AITP. In the AITP group, neither maternal platelet count, maternal increased amounts of PAIgG, the pattern of PAIgG subclasses, circulating autoantibodies, the specificity of the autoantibodies nor maternal splenectomy could be used to predict the severity of the neonatal thrombocytopenia. In the NAIT group, intracerebral hemorrhage occured in 10% and in the AITP group in 4%.

Similar proportions of immunoglobulin A1 (IgA1) protease-producing streptococci in initial dental plaque of selectively IgA-deficient and normal individuals.

By comparing the initial colonization of cleaned teeth in immunoglobulin A (IgA)-deficient, IgM-compensating individuals with that in normal individuals, no significant difference in the proportion of IgA1 protease-producing streptococci was found. Thus, as one of several bacterial means of immune evasion, the ability to cleave secretory IgA1 does not appear essential to the successful adherence of oral streptococci.

A comparative genetic study of serologically distinct Haemophilus influenzae type 1 immunoglobulin A1 proteases.

The bacterial immunoglobulin A1 (IgA1) proteases are putative virulence factors secreted by a number of human pathogens capable of penetrating the mucosal barrier. Among Haemophilus influenzae strains, the IgA1 protease is found in several allelic forms with different serological neutralizing properties. A comparison of the primary structures of four serologically distinct H. influenzae IgA1 proteases suggests that this variation is caused by epitopes of the discontinuous conformational type. Analysis of the homologies among the four iga genes indicates that the variation results from transformation and subsequent homologous recombination in the iga gene region among H. influenzae strains. We find evidence for gene rearrangements, including transpositions in the iga gene region encoding the secretory part of the IgA1 preprotease. The amino acid sequence of the C terminus of the preprotease (the beta-core), which is assumed to be involved in secretion of the protease by forming a pore in the outer membrane, is highly conserved. In contrast to conserved areas in the protease domain, the nucleotide sequence encoding the beta-core showed a striking paucity of synonymous site variation.