Immunoglobulin class and subclass distribution of antibodies reactive with the immunodominant antigen of Actinobacillus actinomycetemcomitans serotype b

The aims of this study were to determine the immunodominant antigens of Actinobacillus actinomycetemcomitans serotype b (Aab) for the different immunoglobulin (Ig) classes and subclasses and to determine the relative levels of these different Igs in serum. Seropositive early-onset periodontitis patients were sampled, and the Ig classes IgG, IgA, and IgM and subclasses IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 were studied. Reactivity with Aab antigens was assessed by using the Western blot (immunoblot) in limiting dilution analysis and radioimmunoassay with sera from 13 early-onset periodontitis subjects. A smeared antigen in the upper portion of the immunoblots, typical of high-molecular-weight LPS, was immunodominant for IgG, IgA, IgM, IgG1, IgG2, IgG3, IgA1, and IgA2. This smeared antigen was present in every patient for all of these Igs at the endpoint. A few additional antigens were also present at the endpoint in some patients, but none were present in more than half of the subjects. The distribution of antibody titers by Ig classes reactive with the Aab immunodominant antigen was IgG > IgA > IgM. The distribution of antibody titers by IgG subclass was IgG2 > IgG1 approximately IgG3. Further quantitation by radioimmunoassay revealed that the mean concentration of IgG2 (65.7 micrograms/ml) was significantly greater than that of IgG1 (8.8 micrograms/ml). The IgA subclass distribution was IgA1 > IgA2, with IgA1 apparently being second only to IgG2. Therefore, the Aab antigen eliciting the highest antibody level in virtually all Ig classes and subclasses appeared to be lipopolysaccharide, and IgG2 was markedly elevated over all other serum Ig classes or subclasses reactive with Aab.     

Opsonization of Actinobacillus actinomycetemcomitans by immunoglobulin G antibodies to the O polysaccharide of lipopolysaccharide

Sera of localized juvenile periodontitis (LJP) patients colonized by Actinobacillus actinomycetemcomitans serotype b often contain markedly elevated levels of immunoglobulin G (IgG) antibodies to serospecific determinants in the O polysaccharide of lipopolysaccharide (LPS), as well as to outer membrane proteins of this species. IgG antibodies in LJP sera are known to opsonize A. actinomycetemcomitans for subsequent phagocytosis and killing by human neutrophils. The objective of this study was to determine whether outer membrane proteins or serospecific determinants in LPS are the primary target for opsonic IgG antibodies in LJP sera. An A. actinomycetemcomitans serotype b O-polysaccharide affinity column was constructed and subsequently used to purify LPS-specific IgG antibodies from LJP serum. The affinity-purified anti-LPS IgG antibodies were enriched in content of IgG2 (66.2%, compared with 37.0% in the total IgG fraction) and were immunospecific for A. actinomycetemcomitans serotype b LPS. In an opsonophagocytic assay using neutrophils from donors who were homozygous for the H131 allotype of Fcy receptor IIa (CD32), it was found that LPS-specific IgG antibodies exhibited substantially greater opsonic activity toward A. actinomycetemcomitans serotype b than an LJP IgG fraction that was depleted of LPS-reactive antibodies but contained antibodies against outer membrane proteins of this species. The results of this study indicate that serospecific determinants in the O polysaccharide of A. actinomycetemcomitans serotype b are a principal target for opsonic antibodies in sera of LJP subjects.     

Titer and subclass distribution of serum IgG antibody reactive with Actinobacillus actinomycetemcomitans in localized juvenile periodontitis

Most patients with localized juvenile periodontitis (LJP) manifest serum IgG antibodies specifically reactive with antigens of Actinobacillus actinomycetemcomitans serotype b (Aa-b). Whether these antibodies are protective, destructive, or irrelevant to the progress of the disease remains unclear. We report results of studies aimed at assessing the subclass IgG responses in 35 LJP patients and 35 periodontally normal control subjects using well-characterized monoclonal antibody subclass reagents in an enzyme-linked immunosorbent assay. Our data show that the mean value for total IgG reactive with antigens of Aa-b was more than sevenfold higher for patients than for normal control sera (2349.6 micrograms/ml for patients vs 332.2 micrograms/ml for controls). Individual patients and control subjects were classified as high- or low-titer, using twice the median value for total anti-Aa-b IgG in control sera as the cutoff. Of 35 patients, 26 (74%) were high-titer, and 9 (26%) were low-titer. This compares to 5 normal control subjects (14%) high-titer and 30 (86%) low-titer. IgG2 accounted for the major quantitative response in both patients and control subjects. Indeed, the mean IgG2 values for both concentration and percentage of total specific IgG were greater than the combined values for specific anti-Aa-b IgG1, IgG3, and IgG4. Of the 26 high-titer sera, IgG2 predominated in 24, with IgG1 and IgG3 predominating in 1 each; IgG2 predominated in only 2 of the low-titer sera.     

Binding of the capsule-like serotype-specific polysaccharide antigen and the lipopolysaccharide from Actinobacillus actinomycetemcomitans to human complement-derived opsonins

We investigated the molecular mechanism of resistance of Actinobacillus actinomycetemcomitans to complement-dependent chemiluminescence response by human polymorphonuclear leukocytes. Whole cells of serotype b-specific polysaccharide antigen-defective mutants ST2 and ST5 were constructed by inserting transposon Tn916 into A. actinomycetemcomitans strain Y4. These strains induced strong chemiluminescence response by human polymorphonuclear leukocytes and markedly bound to human complement-derived opsonins. In contrast, strain Y4 induced weak chemiluminescence response and weakly bound to complement-derived opsonins. The biosensor analysis revealed that lipopolysaccharide from strain Y4 strongly bound to human C3b, but serotype b-specific polysaccharide antigen did not. The serotype b-specific polysaccharide antigen molecule might sterically hinder the interaction between complement-derived opsonins and lipopolysaccharide to reduce complement-dependent chemiluminescence response by human polymorphonuclear leukocytes.     

Characterization of serologically nontypeable Actinobacillus actinomycetemcomitans isolates

Our previous studies have shown that Actinobacillus actinomycetemcomitans isolates of a given arbitrarily primed PCR (AP-PCR) genotype belong to the same serotype (of serotypes a through e). In the present study we investigated whether the AP-PCR genotypes of nonserotypeable A. actinomycetemcomitans isolates match those of the serotypeable isolates. The isolates were additionally characterized by restriction analysis of the apaH PCR amplification products. The material included 75 nonserotypeable and 18 serotypeable A. actinomycetemcomitans isolates from 34 epidemiologically unrelated subjects. The serotypeable isolates were obtained from subjects who also harbored nonserotypeable isolates. Eight AP-PCR genotypes were distinguished among the isolates; six genotypes matched those detected in our previous studies, whereas two genotypes were new. Intraindividually, the A. actinomycetemcomitans isolates produced identical AP-PCR banding patterns, regardless of whether they were serotypeable or nonserotypeable, in 22 of 23 subjects participating with multiple isolates. AP-PCR genotype 3, corresponding to serotype c, was by far the most common among the nonserotypeable isolates (62% of subjects). Results obtained with the apaH restriction analysis confirmed the results obtained with AP-PCR for 31 of the 34 subjects. The results suggest that nonserotypeable A. actinomycetemcomitans isolates originate from serotypeable isolates, especially from serotype c isolates, and the likelihood of the existence of additional serotypes is small.     

Structural and segregational stability of various replicons in Actinobacillus actinomycetemcomitans

To understand the role of specific fats on carcinogenesis, we have studied the effects of lipids derived from the ascites fluids of ovarian cancer patients on oncogenic components, associated with the regulation of proliferation. The treatment of tumor cells with patient-derived fats produced increased cell proliferation, as indicated by an increase in the number of S-phase cells. A similar enhancement in cell proliferation was not observed in normal fibroblasts, following lipid treatment. The effects of patient-derived lipids on the expression of c-jun, c-fos, and c-erbB2 gene products were examined. The cellular expression of the proto-oncogene product, c-fos, was increased in all three ovarian tumor cell lines, following lipid treatment. Expression of c-jun gene product was not detected in SKOV-3 or OVCAR-3 and was not induced by fat treatment. UL-1 cells did not express detectable levels of c-jun prior to fat treatment and treatment with patient-derived fat induced significant levels of c-jun product. All three ovarian tumor cell lines expressed the c-erbB2 gene product and it was generally enhanced by treatment with patient-derived lipids. When specific fatty acids were tested, 14:0, 16:1, and 18:1 were principally responsible for the observed enhancement of c-erbB2 levels, while the fatty acids, 18:0 and 20:4, produced the greatest increase in c-fos expression. Many alterations caused by fats are consistent with the loss of normal growth regulation and may account for the epidemiologic link between certain fats and the risk for ovarian cancer.     

Serum antibodies to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in juvenile periodontitis and adult periodontitis (part I)

Recent microbiological studies support the concept that specific gram negative bacteria play a major role in the etiology and pathogenesis of human chronic inflammatory periodontal disease. Actinobacillus actinomycetemcomitans has been isolated frequently from juvenile periodontitis and Porphyromonas gingivalis has been shown to be a prominent species in adult periodontitis in humans. The purpose of this study was to determine levels of the specific antibodies to A.actinomycetemcomitans and P.gingivalis in 17 patients with juvenile and 15 patients with adult periodontitis and 24 healthy subjects. IgG and IgM antibody titers against these antigens were determined by enzyme-linked immunosorbent assay (ELISA). The levels of antibodies against A.actinomycetemcomitans were significantly higher in the juvenile periodontitis compared to the adult periodontitis patients and controls. Anti-P.gingivalis antibodies were elevated in adult periodontitis compared to juvenile periodontitis patients and controls.     

A statistical approach to the ecology of Actinobacillus actinomycetemcomitans in subgingival plaque

Experimental renal disease models establish glomerular hypertension as a crucial determinant in glomerulosclerosis progression and demonstrate that glomerular capillary pressure reduction delays sclerosis development. An oscillating pressure (OP) chamber was constructed as an in vitro model to study human mesangial cells. Cell cultures were grown under atmospheric pressure (AP) and a controlled OP corresponding to intraglomerular capillary pressure. We show that OP significantly decreases mesangial cell proliferation within 24 hours and attenuates DNA synthesis throughout a 7-day period. To explore the effects of OP on cell metabolism, cell-associated and medium-secreted extracellular (CA and EC, respectively) collagen synthesis were measured by [3H]proline incorporation. In subconfluent cultures, total CA and EC collagen synthesis was unaffected by OP, while in confluent cultures total EC collagen [3H]proline incorporation was increased. To determine whether OP influenced mesangial cell growth induction, the effects of increasing glucose in the cell culture media were investigated. Our data show that the high glucose growth stimulatory effect on cell number and DNA synthesis was suppressed by OP. Under high glucose conditions, total CA collagen synthesis was increased in confluent cultures, whereas the EC collagen fraction remained unchanged. In these cultures, OP caused an additional increase in CA collagen synthesis. This study shows that mesangial cell growth and collagen synthesis are influenced by hyperbaric OP, supporting the hypothesis that glomerular capillary pressure plays a role in progressive glomerulosclerosis development.     

Subcellular localization and cytotoxic activity of the GroEL-like protein isolated from Actinobacillus actinomycetemcomitans

The subcellular locations, ultrastructure, and cytotoxic activity of the GroEL-like protein from Actinobacillus actinomycetemcomitans were investigated. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) clearly indicated that synthesis of the GroEL-like protein is substantially increased after a thermal shock. Analysis of the purified native GroEL-like protein by transmission electron microscopy revealed the typical 14-mer cylindrical molecule, which had a diameter of about 12 nm. A. actinomycetemcomitans cells grown at 35 degreesC and heat shocked at 43 degreesC were fractionated, and fractions were separated by SDS-PAGE and analyzed by Western immunoblotting using antibodies to GroEL- and DnaK-like proteins. The GroEL-like protein was found in both the soluble and membrane fractions, whereas the DnaK-like protein was mostly found in the cytoplasm. An increase in specific proteins, including the GroEL- and DnaK-like proteins, was found in heat-shocked cells. The subcellular localization of the GroEL-like protein was examined by immunoelectron microscopy of whole cells. More GroEL-like protein was detected in stressed cells than in unstressed cells, and most of it was found not directly associated with outer membranes but rather in extracellular material. The native GroEL-like protein was assessed for cytotoxic activities. The GroEL-like protein increased the proliferation of periodontal ligament epithelial cells at concentrations between 0.4 and 1.0 microgram/ml. The number of cells in the culture decreased significantly at higher concentrations. A cell viability assay using HaCaT epithelial cells indicated that the GroEL-like protein was strongly toxic for the cells. These studies suggest the extracellular nature of the GroEL-like protein and its putative role in disease initiation.     

Functional analysis of pVT745, a plasmid from Actinobacillus actinomycetemcomitans

The expansion of myeloma cells is regulated by cytokines, among which IL-6 is a major growth factor. It has been recently suggested that serum transforming growth factor beta 1 (TGF beta 1), a cytokine found in large amounts in alpha-granules of platelets, might play a role in multiple myeloma (MM). It was the purpose of this study to determine serum TGF beta 1 levels in MM patients and to seek a correlation with disease parameters. Measurements were done by ELISA. We studied 35 MM patients (19 stage II, 16 stage III, 20 IgG, 8 IgA and 6 BJ, 1 IgD) in different phases of the disease, 27 healthy individuals and 17 thrombocytopenic patients with other haematological diseases (three MDS, three congenital thrombocytopenia, 11 ITP). Overall samples from MM patients were included: 10 at diagnosis, 18 in remission and 32 in relapse. In normal controls TGF beta 1 serum levels ranged from 1 to 33 ng/ml (median 16.5 ng/ml). In both thrombocytopenic controls with other diseases and thrombocytopenic MM patients (seven samples), TGF beta 1 serum levels were very low (median 3.2 and 4.5 ng/ml respectively). In MM patients with PLT > 100 x 10(9)/L (53 samples), TGF beta 1 serum levels were in the normal range in patients without immunoparesis (1 to 27 ng/ml, median 16.6 ng/ml), whereas they were higher in patients with immunoparesis (polyclonal immunoglobulins (Igs) below lower normal reference values) ranging from 10.2 to 45 ng/ml (median 26.8 ng/ml) (P < 0.01). Serum TGF beta 1 levels fluctuated in the same patient at different times but not according to relapse or remission. Correlation was found only between serum TGF beta 1 levels and immunoparesis and not between serum TGF beta 1 levels and disease stage or Ig subtype nor with prognostic factors for MM (serum CRP, beta 2M or IL-6). This finding suggests that the remaining normal plasma cells are sensitive to the inhibitory action of TGF beta 1 on Ig production. In conclusion TGF beta 1 serum levels are very low in thrombocytopenic patients confirming that platelets are the major source of this cytokine. Furthermore, a strong correlation was found between TGF beta 1 serum levels and immunoparesis in MM patients.     

Cloning of the gene encoding the Actinobacillus actinomycetemcomitans serotype b OmpA-like outer membrane protein

The gene encoding an outer membrane protein A (OmpA)-like, heat-modifiable Omp of Actinobacillus actinomycetemcomitans ATCC 43718 (strain Y4, serotype b) was cloned by a PCR cloning procedure. DNA sequence analysis revealed that the gene encodes a protein of 346 amino acid residues with a molecular mass of 36.9 kDa. The protein expressed by the cloned gene reacted with a monoclonal antibody to the previously described 29-kDa Omp (Omp29) of strain Y4. This monoclonal antibody reacted specifically with Omp29 of A. actinomycetemcomitans (serotype b), but not with any Omp of Escherichia coli, including OmpA. This protein exhibited characteristic heat modifiability on sodium dodecyl sulfate-polyacrylamide gels, showing an apparent molecular mass of 29 kDa when unheated and a mass of 34 kDa when heated. The N-terminal amino acid sequence of the protein expressed in E. coli perfectly matched those deduced from the purified Omp29 of strain Y4. The deduced amino acid sequence of the gene coding for Omp29 from serotype b matched completely (except for valine at position 321) that of a recently reported omp34 gene described for A. actinomycetemcomitans serotype c (NCTC 9710). Because of the conserved nature of the gene within these serotypes, we designated the gene described herein from serotype b as omp34.     

放线共生放线杆菌菌毛重组蛋白的提取及纯化

目的:对大肠杆菌表达的放线共生放线杆菌菌毛重组蛋白LTB-FAP进行提取和纯化,为其用于牙周病的治疗提供理论依据.方法:将重组质粒pET28a/LTB和pET28a/LTB-FAP分别转化到大肠杆菌中进行表达,通过超声裂解法获得以包涵体形式表达的重组外源蛋白LTB-FAP;用2,4,6和8 mol·L-1尿素缓冲液对重组蛋白进行洗涤、溶解;然后用阴离子交换和凝胶过滤柱层析对重组蛋白进行纯化,采用薄层扫描仪对蛋白纯度进行测定.结果:目的包涵体蛋白经过3次4 mol·L-1尿素洗涤,6 mol·L-1尿素溶解后,经薄层扫描可获得纯度为60%的目的蛋白;经离子交换层析纯化,蛋白纯度可达约75%;Sepharose 6B行凝胶过滤层析获得了纯度可达90%的重组蛋白LTB-FAP.结论:阴离子交换和凝胶过滤柱层析的方法可提取、纯化重组蛋白LTB-FAP,获得的LTB-FAP可以用于免疫机体.     

Subacute bacterial endocarditis due to Actinobacillus actinomycetemcomitans. Report of a case with a review of the literature

A case of subacute bacterial endocarditis due to Actinobacillus actinomycetemcomitans is reported. The patient was successfully treated first by a combination of gentamicin and ampicillin and then, because of severe allergic reactions, ampicillin was replaced by co-trimoxazole; symptoms did not recur and blood cultures remained sterile. A synoptic table is presented of 19 reported cases of infection caused by A. actinomycetemcomitans not connected with actinomycosis, with particular regard to their clinical features, treatment, and outcome.     

An antiserum to a synthetic fimbrial peptide of Actinobacillus actinomycetemcomitans blocked adhesion of the microorganism

The purpose of this study is to certify the importance of the fimbriae as an attachment factor of Actinobacillus actinomycetemcomitans, a human periodontopathic bacterium, and the significance of anti-fimbrial antibody function as an attachment inhibitor. Fimbrial antigen was prepared from the A. actinomycetemcomitans 310-a strain. Oligopeptides were synthesized according to the amino acid sequence of the fimbrial protein. The peptide antigen was conjugated with branched lysine polymer resin beads. The peptide antigen was suspended in PBS emulsified with incomplete Freund's adjuvant and used to immunize rabbits. A rabbit antiserum reacted with an approximately 54 kDa protein of the fimbriae protein from A. actinomycetemcomitans 310-a and with those of other fimbriated strains. This antiserum strongly inhibited the attachment of fimbriated A. actinomycetemcomitans strains to saliva-coated hydroxyapatite beads, buccal epithelial cells, and a fibroblast cell line, Gin-1. Such a synthetic fimbrial peptide antigen may be effective in inducing antibodies which inhibit adhesion and subsequent colonization by A. actinomycetemcomitans.     

A single-chain Fv reactive with the Goodpasture antigen

Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expressed product. HBR-3 was shown by ELISA, immunoblotting, and immunohistologic techniques, to have the same specificity for alpha 3(IV)NC1 as P1 and autoantibodies from patients with Goodpasture's disease. HBR-3 and P1 were shown to have similar affinity for their mutual ligand. On sections of normal human kidney, the scFv bound only to glomerular basement membrane and distal tubular basement membrane. It did not bind to the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigenic form. We have, therefore, generated a scFv which reproduces the specific binding properties of the parent monoclonal antibody, P1. The potential of HBR-3 as a diagnostic reagent in Alport's syndrome has been demonstrated. The development of this recombinant molecule should permit new approaches to the investigation of Goodpasture's disease.     

Molecular characterization of an outer membrane protein of Actinobacillus actinomycetemcomitans belonging to the OmpA family

The major outer membrane protein (OMP) of Actinobacillus actinomycetemcomitans is an OmpA homolog that demonstrates electrophoretic heat modifiability. The gene encoding this protein was isolated from a genomic library of A. actinomycetemcomitans NCTC 9710 by immunoscreening with serum from a patient with localized juvenile periodontitis. Expression of the cloned gene in Escherichia coli and subsequent Western blot analysis revealed a protein with an approximate molecular mass of 34 kDa. The amino acid sequence predicted from the cloned gene demonstrated that the mature protein had a molecular mass of 34,911 Da and significant identity to members of the OmpA family of proteins. We have named the major OMP of A. actinomycetemcomitans Omp34, and its corresponding gene has been named omp34.     

Molecular characterization of low-molecular-weight component protein, Flp, in Actinobacillus actinomycetemcomitans fimbriae

OBJECTIVE: To demonstrate the use of aggregated, locally collected birth notification data to examine trends in birth-weight specific survival for singleton and multiple births. DESIGN: Retrospective analysis of 171,527 notified births and subsequent infant survival data derived from computerised community child health records. Validation of data completeness and quality was undertaken by comparison with birth and death registration records for the same period. SETTING: Notifications of births in 1989-1991 to residents of the North Thames (East) Region (formerly North East Thames Regional Health Authority). OUTCOME MEASURES: Birthweight specific stillbirth, neonatal, and postneonatal death rates. RESULTS: There was close correspondence between the notification and registration data. For 96% of the registered deaths a birth notification record was identified and for the majority of these the death was already known to the Community Child Health Computer. Completeness of birth-weight data, particularly at the lower end of the range, was substantially better in birth notification data. Comparison with the most recent published national data relating to birthweight specific survival of very low birthweight singleton and multiple births suggests that the downward trend of mortality is continuing, at least in this Region. CONCLUSIONS: The use of routinely collected aggregated birth notification data provides a valuable adjunct to existing sources of information about perinatal and infant survival, as well as other information regarding process and outcome of maternity services. Such data are required for comparative audit and may be more complete than that obtained from registration or hospital generated data.     

Cloning and sequencing of part of the S10 operon from Actinobacillus actinomycetemcomitans FDC Y4

Linear dye penetration was evaluated in teeth with open apices in which calcium phosphate cement was used as an apical barrier to facilitate obturation. The apical foramens of 42 extracted single-rooted human teeth were opened to a size 90 file. Half the teeth received apical barriers consisting of calcium phosphate cement (CPC) followed by obturation using a customized gutta-percha cone/ lateral condensation technique. The other half were obturated without benefit of apical barriers. Linear dye penetration was measured after 48 h exposure to India ink. The teeth receiving apical CPC barriers before obturation had significantly less dye penetration than teeth without apical barriers. Based on its proven biocompatibility and osteconductive potential, calcium phosphate cement may serve well as a replacement for calcium hydroxide in a single-visit immediate apical barrier apexification technique.