Immune responses to porphyromonas gingivalis infection suppress systemic inflammatory response in experimental murine model.

Periodontitis is a localized infectious disease caused by periodontopathic bacteria such as Porphyromonas gingivalis (P. gingivalis), and the severity correlates to significance of immune responses. Recently, it has been reported that periodontitis is associated with the development of systemic disease such as diabetes and atherosclerosis because of increasing invasion of oral pathogens to the circulation. However, the association between local and systemic infectious responses is still unclear. In the present study, we examined the differences of biological responses in animals with or without bacterial infection. After Balb/c mice were infected subcutaneously with live P. gingivalis W83, serum, skin and liver were collected according to experimental protocol. The skin and liver tissues were observed pathologically by haematoxylin-eosin staining, and serum IL-6 levels were measured using ELISA method. Throughout the experimental period, conditions of the mice were observed continuously. As expected, severe infiltration of leukocytes were observed at inflamed skin corresponding to the number of bacterial challenges. Although no inflammatory appearance of skin was observed, serum IL-6 levels were increased dramatically (P <0.01, Student's t-test) and liver tissues were injured in the mice without bacterial challenge. Interestingly, although severe inflammatory appearance of the skin was observed, serum IL-6 levels were not increased and no inflammatory responses were observed in the liver of the 3-times bacterially challenged group. Importantly, immunoglobulin G against P. gingivalis W83 was detected in the blood of mice with 3-times bacterial challenge corresponding to improvement of weight loss and survival. In conclusion, although multiple infections develop severe localized inflammation, the immune system should be sufficient to protect the systemic inflammatory responses.

Nucleotide sequencing and transcriptional analysis of two tandem genes encoding glucosyltransferase (water-soluble-glucan synthetase) in Streptococcus cricetus HS-6.

Two tandem genes encoding glucosyltransferase synthesizing water-soluble glucan (GTF-S) were cloned from the lambda gene library of Streptococcus cricetus HS-6 (serotype a) using anti-GTF-S antibody, and the nucleotide sequences were analyzed. The two genes (ORF1 and ORF2) were identified as streptococcal glucosyltransferases based on the following evidence: [1] the deduced amino acid sequences of their products have an active site for catalytic action and C-terminal repeated units for dextran binding, and [2] a homology search revealed that the ORF1 and ORF2 products are homologous to the GtfS protein (77.4%) of S. downei Mfe28 and GtfT protein (83.8%) of S. sobrinus OMZ176, respectively, which are both known to have GTF-S activity. Therefore, ORF1 and ORF2 might be designated gtfS and gtfT of S. cricetus, respectively. A Northern blotting and RNase protection assay suggested that the gtfS and gtfT of S. cricetus are transcribed as a single bicistronic mRNA as well as separate monocistronic mRNAs. Primer extension analysis indicated multiple transcriptional start points for each gene.

Heterogeneous post-translational modification of Actinobacillus actinomycetemcomitans fimbrillin.

Fresh isolates of Actinobacillus actinomycetemcomitans produce bundle-forming fimbriae. The exact molecular mass of A. actinomycetemcomitans fimbrillin, a structural subunit of fimbriae, was determined by liquid chromatography-electrospray ionization mass spectrometry. Three major molecular species with 6,226.0, 6,366.0, and 6,513.0 Da were detected in a purified fimbrial fraction from the strain 310-a. These molecular masses were significantly higher than the molecular weight (5,118 Da) calculated from nucleotide sequence data of the fimbrillin gene, flp, suggesting that the fimbrial peptides were post-translationally modified. Modification of the fimbrial peptides was also suggested by an N-terminal amino acid sequence analysis of fimbrillin peptic fragments, with the modified amino acids being due to seven serine or asparagine residues located in the C-terminal region. A periodate oxidation/biotin-hydrazide labeling assay of fimbrillin suggested that it might be glycosylated.

[A case of metastatic extradural seminoma suspected intradural invasion by the measurements of HCG beta concentration in CSF].

A 34-year-old man underwent left orchidectomy for his left testicular seminoma. One month later, he developed paraplegia, hypesthesia under Th10 level and vesicorectal disturbance. He was diagnosed as having compressive myelopathy secondary to metastatic neoplasm at thoracic vertebra 10 and its extradural space which were revealed on magnetic resonance imaging. After administration of combination chemotherapy with cisplatin, etoposide and bleomycin, the extradural lesions diminished and the neurological symptoms gradually improved. In this case, intradural invasion of tumor cells was suspected because the level of human chorionic gonadotrophin beta subunit (HCG beta) concentration in cerebrospinal fluid (CSF) was higher than that in plasma, while radiographic scanning demonstrated regional tumor located at extradural space of Th10 level. It is important to evaluate the spread of tumor cells for the choice of therapy and the monitoring of HCG beta (plasma:CSF ratio) was considered to be one of the useful methods to detect the presence of central nerve system metastases from HCG-producing tumor.

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

Fimbriae preparation from Actinobacillus actinomycetemcomitans was found to contain an abundant low-molecular-weight protein (termed Flp) with an apparent molecular mass of approximately 6.5 kDa, in addition to a small amount of 54-kDa protein. Immunogold electron microscopy localized the Flp protein at the bacterial fimbriae but not at the cell surface. The DNA fragment including the flp gene was cloned from A. actinomycetemcomitans 304-a and its nucleotide sequence was determined. An open reading frame of the flp gene was composed of 225 bp encoding a protein of 75 amino acids. Comparison of the translated amino acid sequence with the sequence of native Flp determined by Edman degradation indicated that the N-terminal part of 26 amino acids is leader peptide. The N-terminal sequence of mature Flp exhibited some similarity to type-IV pilin. Furthermore, the processing site of premature Flp is also similar to that of type-IV prepilin, and a gene encoding a protein homologous to type-IV prepilin-like protein leader peptidase was found downstream of the flp gene. These findings indicate that Flp is the major component protein of A. actinomycetemcomitans fimbriae.

Isolation and characterization of a 53 kDa major cell envelope protein antigen from Treponema denticola ATCC 35405.

A major cell envelope protein was purified from the cell envelope fraction of Treponema denticola ATCC 35405 by ion exchange chromatography after extraction with Zwittergent 3-14. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a relative molecular mass of 53 kDa for this protein with a pI of 6.3-6.8. Amino acid analysis revealed that this protein contained high proportions of hydrophobic amino acids (40.4%), and no cysteine could be detected. The N-terminus of the protein was blocked to Edman degradation. Rabbit antiserum raised against the purified 53 kDa protein reacted with the outer envelope of the T. denticola cell surface as confirmed by immunoelectron microscopy. This rabbit antiserum reacted with 4 of the 11 strains of treponemes tested in this study. Sera from 9 to 18 periodontitis patients reacted strongly with this 53 kDa cell envelope protein of T. denticola as determined by immunoblotting analysis.

Humoral immune response to an antigen from Porphyromonas gingivalis 381 in periodontal disease.

The humoral immune responses of patients with periodontitis were evaluated to characterize the host response to Porphyromonas gingivalis. A sonic extract of P. gingivalis 381 from whole cells was fractionated by gel chromatography and ion-exchange chromatography. The fractionated extracts were evaluated by Western blot (immunoblot) analyses with patient sera. A dominant antigen was identified from the sonic extract with an apparent molecular mass of 53 kDa. The 53-kDa protein antigen (Ag53) was purified by affinity chromatography by using a monoclonal antibody. Ag53 was detected on the vesicle surface of P. gingivalis 381 by immunoelectron microscopy by using the monoclonal antibody and was detected as a major protein in the outer membrane and in vesicles by Western blot analysis. Monoclonal antibody cross-reactivity to Ag53 in the sonic extracts of P. gingivalis ATCC 33277, P. gingivalis 1021, and Porphyromonas endodontalis ATCC 35406 was revealed. Seventy-seven patients with periodontitis were examined for their responses to Ag53. Serum immunoglobulin G (IgG) from 54 patients reacted strongly to Ag53; however, serum IgG from the remaining 23 patients did not exhibit detectable reactivity at all to Ag53, even though the patients had high serum IgG titers to the sonic extract. Ag53 is a new marker that represents an interesting aspect of the humoral immune response to P. gingivalis in patients with periodontitis.