A monoclonal antibody against fimA type II Porphyromonas gingivalis inhibits IL-8 production in human gingival fibroblasts.

The periodontal pathogen Porphyromans gingivalis is classified into six groups (types I-V and Ib) based on the genotype of the fimbriae A (fimA) gene. Among genotypes, fimA type II strains are thought to be most strongly related to advanced periodontitis. The present study was undertaken to develop passive immunotherapy monoclonal antibodies (MAbs) against periodontitis, which are capable of inhibiting virulency and were constructed through the immunization of outer membrane vesicles (OMV) fraction of fimAII strain, TDC60, using mouse hybridoma technology. MAbs that recognized OMV by ELISA assay were identified, and 28 clones were screened by Western blot analysis. After purifying these MAbs using protein G column, the effect of the MAb on IL-8 production from human gingival fibroblasts by OMV was examined. We selected MAb TDC4-33H, which strongly inhibited the IL-8 production with a higher MAb production rate. Since the MAb showed an individual ladder-like profile against OMV by Western blotting, we further examined the reactivity against lipopolysaccharides (LPS) from TDC60, W83 (fimAIV), and ATCC33277 (fimAI). As a result, MAb TDC4-33H recognized all LPSs. Moreover, MAb TDC4-33H significantly inhibited the LPS-stimulated IL-8 production in human gingival fibroblasts. These findings suggest that MAb TDC4-33H reacts with LPS and may be useful for passive immunotherapy through neutralizing IL-8 production in gingival fibroblasts by P. gingivalis LPS.

Monoclonal antibodies produced against lipopolysaccharide from fimA Type II Porphyromonas gingivalis.

An important periodontal pathogen, Porphyromans gingivalis strains are classified into six genotypes (types I-V and Ib), based on the genotype of the fimbriae A (fimA). Among the genotypes, fimA type II strains are thought to be most strongly related to advanced periodontitis. To develop passive immunotherapy, over 300 hybridoma clones were constructed through immunization of cell extracts of fimA type II strain P. gingivalis TDC60 using hybridoma technology. Among these clones, 15 MAbs recognized TDC60 lipopolysaccharide (LPS) with an individual ladder-like structure by Western blot analysis. Further Western blotting of the 15 MAbs against LPS from TDC60, FDC381 (fimA type I), and W83 (fimA type IV) of P. gingivalis and Escherichia coli was carried out. None of these MAbs recognized E. coli LPS, and divided into at least three different Western blot patterns. To confirm the specificity to LPS, three clones were selected and competition assays were carried out using TDC60 LPS. All three MAbs reduced the reactivity against TDC60 LPS after absorption of the LPS in a dose-dependent manner. These findings suggest that MAbs recognizing different epitopes of P. gingivalis LPS were successfully constructed, and these MAbs may be useful in neutralizing P. gingivalis infection.

Functional analysis of the thioredoxin domain in Porphyromonas gingivalis HBP35.

Periodontitis is one of the most common oral diseases in humans. This caused by infection by the oral bacterium Porphyromonas gingivalis. Our strategy to prevent this infection is to establish a passive immunization system in which endogenous antibodies can be applied directly to neutralize virulent factors associated with this bacterium. We focused our attention on the P. gingivalis 35 kDa surface protein, or HBP35, since this protein is involved not only in the coaggregation with oral miroflora but also in hemin binding. In addition, nucleotide sequencing of the gene, hbp35, coding for this protein revealed the presence of a catalytic center for thioredoxin, and we further attempted to characterized the protein by amino acid substitution. A total of four Cys residues were substituted for Ser residues by combining the simple method for site-directed mutagenesis and the heterodimer system, an approach designed to construct chimeric plasmids readily. Native and mutagenized hbp35 were introduced into the Eschericha coli dsbA mutant strain, JCB 572, defective in both alkaline phosphatase and motile activities due to inefficient disulfide bond formation. Transformant harboring the native hbp35 could complement the dsbA mutation, suggesting a role of disulfide bond formation of this protein in P. gingivalis cells. Possible roles of the Cys residues in complementation are discussed.

Role of the hemin-binding protein 35 (HBP35) of Porphyromonas gingivalis in coaggregation.

Hemin-binding protein 35 (HBP35) in Porphyromonas gingivalis is one of the outer membrane proteins and has been reported to be a non-fimbrial coaggregation factor. In this study, a P. gingivalis HBP35-deficient mutant (MD774) was constructed from wild-type strain FDC381 by insertion mutagenesis in order to provide a better understanding of this protein's role in coaggregation. The intact cells and vesicles in FDC381 were found to have strong aggregation activities with Gram-positive bacteria. But neither the vesicles nor the intact cells showed aggregation activity in MD774. In addition, MD774 reduced autoaggregation activity. Immunoblot analysis of MD774 showed the presence of a non-maturated 45-kDa fimbrillin protein. Electron microscopy showed that the MD774 had no long fimbriae on the cell surface. Arg- and Lys-gingipain activity in MD774 was significantly decreased, compared with FDC381. Real-time RT-PCR demonstrated a significant reduction in the expression of gingipain-associated genes rgpA, rgpB, and kgp. In conclusion, we suggest that the reduction in coaggregation was caused by the combined reduction of a variety of molecules, including HBP35, gingipains, and fimbriae. Our results suggest that the HBP35 protein directly influences not only coaggregation as an adhesion molecule but also indirectly influences the expression of other coaggregation factors.

Antibody responses to Porphyromonas gingivalis hemagglutinin A and outer membrane protein in chronic periodontitis.

BACKGROUND: Hemagglutinin and outer membrane protein (OMP) are major virulence factors associated with colonization of Porphyromonas gingivalis in the gingival crevice. The genes for the 200-kDa antigenic protein (200-kDa AP) and 40-kDa OMP of P. gingivalis have been successfully cloned. Additionally, the 200-kDa AP gene has been shown to constitute the hemagglutinin A (hagA) gene of P. gingivalis. Therefore, this study was constructed to evaluate the distributions and serum levels of immnoglobulin G (IgG) antibodies specific for 200-kDa AP and 40-kDa OMP in periodontitis patients. METHODS: Fifty patients with chronic periodontitis and 59 controls without periodontal destruction were enrolled in this study. We cloned the genes for 200-kDa AP and 40-kDa OMP from P. gingivalis and constructed the purified recombinant proteins. Serum levels of IgG subclass antibodies specific for both recombinant 200-kDa and 40-kDa OMP were determined in patients and controls by an enzyme-linked immunosorbent assay (ELISA). RESULTS: The serum IgG subclass distribution for patients and controls was IgG1>IgG4>IgG2>IgG3 in the anti-200-kDa AP response, which was almost identical to that in the anti-40-kDa OMP response. The patient group showed significantly higher serum IgG responses to the 40-kDa OMP than the control group (P<0.01). In contrast, IgG subclass responses to the 200-kDa AP were not different between the patients and controls. Serum levels of antibodies reactive with both 200-kDa and 40-kDa proteins did not have a significant association with mean probing depth. CONCLUSION: These results suggested that serum IgG responses against P. gingivalis OMP rather than the hagA may be more active in chronic periodontitis.

The trans-chromosomic mouse-derived human monoclonal antibody promotes phagocytosis of Porphyromonas gingivalis by neutrophils.

BACKGROUND: As a safe immunotherapeutic approach, human monoclonal antibody (hMAb) may be effective in clearing periodontopathic bacteria. The trans-chromosomic (TC) technology has recently been applied to construction of the TC mouse, which enables us to incorporate entire human chromosome fragments containing immunoglobulin (Ig) gene cluster. The aim of this study is to establish TC mouse-derived hMAb, and to test the in vitro opsonophagocytic activity. METHODS: Human Ig-producing TC mouse was immunized by recombinant 40-kDa outer membrane protein (r40-kDa OMP) of Porphyromonas gingivalis 381, and the spleen cells were fused with the mouse myeloma cell line. The specificity of antir40- kDa OMP hMAb was evaluated with the enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance assays. Flow cytometric analyses were performed to assess the opsonophagocytic activity. RESULTS: We successfully constructed 99 IgG isotype clones (IgG1: 84; IgG2: 11; IgG4: four clones), which were specifically reactive with r40-kDa OMP. The anti-r40-kDa OMP IgG1 hMAbs promoted phagocytosis of P. gingivalis by neutrophils. Futhermore, an increased opsonophagocytic activitity of anti-r40-kDa OMP IgG1 hMAbs was observed not only in P. gingivalis 381, but also in the W50, W83, and Su63 strains. CONCLUSION: Our results document the TC mouse-derived hMAb to promote neutrophil phagocytosis of P. gingivalis, suggesting an immunotherapeutic option for clearance of P. gingivalis.

Construction of novel human monoclonal antibodies neutralizing Porphyromonas gingivalis hemagglutination activity using transgenic mice expressing human Ig loci.

Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. One potential virulence factor, hemagglutinin, may mediate bacteria attachment onto and penetration into host cells, as well as agglutinate and lyses erythrocytes to intake heme, an absolute requirement for growth. Toward the development of passive immunotherapy, the construction of a human type monoclonal antibody, which is capable of inhibiting the hemagglutinating ability, will be significant and important. The human mAbs, both exhibiting a high degree of specificity and affinity against the recombinant 130 kDa hemagglutinin domain protein have been prepared using XenoMouse technology. The constructed Xeno-mAbs, IgG2 subclass, significantly inhibited hemagglutination of P. gingivalis and its vesicles. The newly constructed Xeno-mAbs may prove to be useful for the development of passive immunization against periodontal diseases caused by P. gingivalis infection, pending the results of fertility study in disease mode.

Transcutaneous immunization with a 40-kDa outer membrane protein of Porphyromonas gingivalis induces specific antibodies which inhibit coaggregation by P. gingivalis.

This study seeks to assess the potential of a 40-kDa outer membrane protein of Porphyromonas gingivalis (40k-OMP) as a transcutaneous vaccine against chronic periodontitis. Transcutaneous immunization (TCI) of mice with 40k-OMP alone elicited 40k-OMP-specific IgG antibody (Ab) responses in both serum and saliva. When administered with cholera toxin (CT) as adjuvant, TCI with 40k-OMP not only elevated IgG Abs as noted above, but also induced IgA responses in serum but not in saliva. Salivary IgG from mice given 40k-OMP alone or 40k-OMP plus CT showed higher binding levels to the 40k-OMP than did that of non-immunized mice. Ab-forming cell (AFC) analysis revealed high numbers of 40k-OMP-specific IgG AFCs in the spleen but low numbers in the salivary glands of mice given 40k-OMP alone or 40k-OMP plus CT. Since 40k-OMP-specific IgG inhibited the coaggregation of P. gingivalis vesicles and S. gordonii, TCI with 40k-OMP may be a useful tool in the quest to prevent P. gingivalis infection.

Expression of the gtfI gene from Streptococcus sobrinus in Streptococcus anginosus using integration-mediated transformation system.

We have constructed a Streptococcus anginosus transformant expressing the gtfI gene from Streptococcus sobrinus, using a previously developed integration-mediated transformation system to introduce foreign genes onto the oral streptococcal chromosome, and attempted to evaluate the gene expression. In this system, one cloning plasmid and three pACYC184 derivatives, anchor, heterodimer, and integration plasmids were used for the construction of a series of integrants via homologous recombination. A portion of S. sobrinus gtfI gene devoid of approximately 1 kb of the 5'-region derived from pMD39 was cloned into the integration plasmid and introduced onto the S. anginosus chromosome. Next, the polymerase chain reaction product corresponding to 2.0 kb of the 5'-region of the gtfI gene from S. sobrinus chromosome was further cloned into the cloning plasmid, and the intact gtfI gene was reconstructed following integration. The final S. anginosus integrant successfully secreted the enzymatically active gtfI gene products and extracellular enzyme was characterized. This enzyme produced water-insoluble glucans and glucan-forming activity was stimulated by the addition of dextranT10. When this integrant was grown in Todd-Hewitt broth supplemented with sucrose, the integrant adhered to the glass surface in vitro and this integrant exhibited the different colony morphology on Mitis-Salivarius agar plates compared to S. sobrinus and S. anginosus. These observations strongly suggest that the construction of S. anginosus integrant expressing S. sobrinus gtfI gene using this transformation system may be an effective means of analysis of cariogenic biofilm formation.

Targeting of Porphyromonas gingivalis with a bispecific antibody directed to FcalphaRI (CD89) improves in vitro clearance by gingival crevicular neutrophils.

Phagocytosis and killing of pathogens by polymorphonuclear neutrophils (PMN) from gingival crevicular fluid (GCF) is diminished in chronic periodontitis patients. As an approach to improve targeting of PMN toward a periodontopathogen, Porphyromonas gingivalis, the efficacy of a bispecific antibody (BsAb) directed against both recombinant 130 kDa hemagglutinin domain (r130k-HMGD) of P. gingivalis, and PMN Fc receptor (FcR) was evaluated. GCF PMN exhibited higher IgA FcR (FcalphaRI) levels, and lower IgG FcR (FcgammaRIIa and FcgammaRIIIb) levels than PB PMN. Functional studies revealed that GCF PMN exhibited a higher capacity to phagocytose and kill P. gingivalis opsonized with a BsAb targeting P. gingivalis r130k-HMGD to FcalphaRI as compared to an anti-r130k-HMGD antibody. However, phagocytosis and killing activity of PB PMN that were incubated with the two antibodies proved comparable. These data support targeting of pathogens toward FcalphaRI as an option to improve antibacterial immunity in chronic periodontitis patients.

Subcloning of the 200-kDa Porphyromonas gingivalis antigen gene and inhibition of hemagglutination by an antibody against the recombinant protein.

Porphyromonas gingivalis is a major etiologic agent of periodontitis and exhibits hemagglutinating and adherence activities. We previously succeeded in molecular cloning the 200-kDa cell-surface antigenic protein (200-k AP), designated pMD101, that is recognized in sera from periodontitis patients, and identified the 200-k AP as a hemagglutinin A (HagA) derivative. HagA is one of the hemagglutinins known to be a useful vaccine against periodontitis. HagA has four large, contiguous, direct repeats and the repeat unit is believed to contain the hemagglutinin domain. Because production of 200-k AP was low in the Escherichia coli host, it was difficult to obtain large amounts of recombinant protein. In this study, we attempt to subclone the gene encoding the useful antigen from pMD101 in an effort to obtain large quantities. A subclone, designated pMD160, encoding a fusion protein of 80-kDa HagA and maltose-binding protein was successfully constructed, and the novel clone produced relatively large amounts of recombinant protein. DNA nucleotide sequences of the pMD160 insert demonstrated that the 80-kDa protein contained a short hemagglutinin motif and a direct repeat unit region. The recombinant protein was purified to homogeneity and rabbit antiserum was raised. The antibody was capable of inhibiting the hemagglutinating activity of P. gingivalis. These findings suggest that novel 80-kDa HagA derivative proteins can be produced efficiently from E. coli hosts and these may be useful in developing immunotherapy against periodontitis infected by P. gingivalis.

Production of functional ScFv inhibiting Streptococcus mutans glucosyltransferase activity from a hybridoma P126.

Streptococcus mutans has been considered the principal etiologic agent of dental caries in humans. The glucosyltransferase-I (GTF-I), which synthesized adhesive water-insoluble glucans from sucrose, has been demonstrated to be an important cariogenic property. Water-insoluble glucans (WIG) synthesized by S. mutans GTF-I can mediate sucrose-enhanced colonization on tooth surfaces and form dental plaque. It has been suggested that inhibition of WIG synthesis decreases bacterial colonization and cariogenicity. Indeed, the use of GTF enzymes as a vaccine antigen resulted in protection from experimental dental caries in rodents. However, it is preferable to eliminate unwanted immune response during active immunization of humans. To prevent this incidence, we attempted to produce the single-chain variable fragment (ScFv) antibody against GTF-I to develop passive immunization for dental caries. Hybridomas producing monoclonal antibody (MAb) that inhibited WIG synthesis by GTF-I have been constructed. Using mRNA from an IgG1 hybridoma P126, cDNAs encoding the variable fragments of the L and H chains of IgG1 from the hybridoma P126 were cloned by RT-PCR-based techniques and then transformed into an Escherichia coli expression system. The purified ScFv antibody recognized the recombinant (r) GTF-I proteins and was capable of inhibiting the WIG synthesis of rGTF-I.

Characterization of the gene encoding 200-kDa Porphyromonas gingivalis protein that reacts to sera from periodontitis patients.

Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, is considered to be one of the major etiologic agents of adult periodontitis. We previously succeeded in molecular cloning of a 200-kDa antigenic protein (200-k AP) from P. gingivalis 381 by immunoscreening using sera from severe periodontitis patients and designated it as pMD101. We also identified amino acid sequences of the short peptide from a lysyl endopeptidase digested recombinant (r), 200-k AP, and found that the short peptide had exactly the same amino acid sequence as the hemagglutinin A (hagA) of P. gingivalis, which is thought to have potential use in a vaccine against periodontitis. In this study, we attempted to confirm whether 200-k AP was a molecule identical to hagA. DNA sequences of pMD157, a subclone encoding the 25-kDa antigenic region of pMD101, were identical to the same part of the hagA gene. The r200-k AP was purified for homogeneity and rabbits were immunized with it. The antibody against r200-k AP previously showed a similar Western-blot pattern as P. gingivalis lysate by monoclonal antibodies against hagA by literature and reacted to r130-kDa hemagglutinin. These findings suggest that 200-k AP is identical to hagA and that r200-k AP may be useful as an immunotherapy agent against periodontitis caused by P. gingivalis infection.

Specific antibodies induced by nasally administered 40-kDa outer membrane protein of Porphyromonas gingivalis inhibits coaggregation activity of P. gingivalis.

In this study, we have assessed the efficacy of the 40-kDa outer membrane protein (40k-OMP) of Porphyromonas gingivalis as a nasal vaccine for the prevention of adult periodontitis. Mice nasally immunized with 40k-OMP and cholera toxin as mucosal adjuvant displayed significant levels of 40k-OMP-specific serum IgG1, IgG2b and IgA as well as mucosal IgA antibodies (Abs) in saliva and nasal secretions. Ab-forming cell (AFC) analysis confirmed the antibody titers by detecting high numbers of 40k-OMP-specific AFCs in spleen, salivary glands and nasal passages. Because 40k-OMP-specific IgG inhibited coaggregation of P. gingivalis vesicles and S. gordonii, it may be an important tool for the prevention of adult periodontitis.

Production of antibody against a synthetic peptide of Porphyromonas gingivalis 40-kDa outer membrane protein.

Porphyromonas gingivalis has been implicated as a major pathogen in periodontal diseases. We previously cloned a 40-kDa outer membrane protein (OMP) gene from P. gingivalis 381 and succeeded in producing sufficient quantities of the recombinant protein for experimental use. Since antibodies against the recombinant (r) 40-kDa OMP have potent ability to kill P. gingivalis cells by complement activation and opsonization, r40-kDa OMP has been the subject of considerable interest as a possible vaccine candidate. In this study, in order to develop a component vaccine, the immunodominant domain in 40-kDa OMP was identified. Peptides corresponding to portions of the N-terminal regions of 40-kDa OMP were synthesized chemically and their immunoreactivities with antibody against r40-kDa OMP were tested. The 16-mer peptide, LDDEYKERVFQTFVHY, was found to react strongly with the antibody. Furthermore, a rabbit antibody was prepared by immunization with the 16-mer peptide, cross-linked with dehydrofolate reductase, and its immunoreactivity was then examined. In a BIAcore experiment the antibody clearly reacted with r40-kDa OMP as well as P. gingivalis strains. These findings suggest that the 16-mer synthetic peptide may be useful for development of a component vaccine against P. gingivalis.

Production of a single-chain variable fraction capable of inhibiting the Streptococcus mutans glucosyltransferase in Bacillus brevis: construction of a chimeric shuttle plasmid secreting its gene product.

Periodontitis and dental caries are common oral diseases, in these days, and the passive immunization is one of the most effective approaches for prevention. For this purpose, we have constructed mouse and human monoclonal antibodies to inhibit the Porphyromonas gingivalis-associated hemagglutination and coaggregation. In addition, an artificial antibody, single-chain variable fraction, or scFv, which also inhibited the hemagglutination, was constructed. Specifically for dental caries, mouse and human monoclonal antibodies that inhibited the glucosyltransferase (GTF) activity, responsible for biofilm formation, were also constructed. The advantage of scFv over the native antibody is that the former molecule does not induce possible side-effects due to Fc, such as autoimmune disease, because it consists only of variable regions originating from both heavy and light chains. To increase the abilities of the antibody preparations, we attempted to construct an additional scFv using Bacillus brevis, a secretion-proficient gram-positive bacterium, as a host cell. An scFv protein possessing the same biological activity as that of the parental antibody was successfully secreted from a B. brevis transformant following the construction of a chimeric shuttle plasmid, which was accomplished by employing a new heterodimer system.

Human monoclonal antibody inhibits Porphyromonas gingivalis hemagglutinin activity.

BACKGROUND: Porphyromonas gingivalis has been implicated as an important pathogen in the development of chronic periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. One potential virulence factor, hemagglutinin, may mediate bacteria attachment onto and penetration into host cells, as well as agglutinate and lyse erythrocytes to intake heme, an absolute requirement for growth. We previously cloned the gene encoding the 130 kDa hemagglutinin domain (130k HMGD) and identified its functional domain. The construction of a human monoclonal antibody that is capable of inhibiting the hemagglutinating ability is significant and important toward the development of passive immunotherapy. METHODS: Human lymphocytes isolated from a donor, who had high antibody titer against the recombinant 130k HMGD (r130k HMGD), were immortalized by Epstein-Barr virus, and specific antibody-producing B cells were established by panning using the r130k HMGD. RESULTS: The constructed HuMAb-HMGD1, IgG subclass, recognized the r130k HMGD as well as the 43 and 49 kDa major bands in P. gingivalis cells and vesicles. The HuMAb-HMGD1 significantly inhibited hemagglutinating activity of P. gingivalis vesicles in a dose-dependent manner. Furthermore, the HuMAb-HMGD1 recognized the synthetic peptide, EGSNEFAPVQNLTGSSVG, which contains the functional domain of 130k HMGD. CONCLUSION: The newly constructed HuMAb-HMGD1 may prove to be useful for the development of passive immunization against periodontal diseases caused by P. gingivalis infection, pending the results of fertility study in disease mode.

Further development of an electroosmotic medium pump system for preparative disk gel electrophoresis.

A simple and practical 6.8-cm-diameter (36.30-cm(2) cross-sectional-area) preparative disk gel electrophoresis device, based on the design of M. Hayakawa et al. (Anal. Biochem. 288 (2001) 168), in which the elution buffer is driven by an electroosmotic buffer flow through the membrane into the elution chamber from the anode chamber was constructed. We have found that the dialysis membranes employed provide suitable flow rates for the elution buffer, similar to those of an earlier 3.6-cm-diameter device, resulting in the prevention of excess eluate dilution. The efficiency of this device was demonstrated by the fractionation of a bovine serum albumin (BSA) Cohn V fraction into monomer, dimer, and oligomer components using nondenaturing polyacrylamide gel electrophoresis (native-PAGE). The maximum protein concentration of the eluate achieved was 133 mg/ml of BSA monomer, which required a dilution of the eluate for subsequent analytical PAGE performance. As a practical example, the two-dimensional fractionation of soluble dipeptidyl peptidase IV (sDPP IV) from 50 ml fetal bovine serum (3.20 g protein) per gel is presented. The sDPP IV enzyme protein was recovered in a relatively short time, utilizing a 6.5% T native-PAGE and subsequential sodium dodecyl sulfate-PAGE system. This device enhances the possibility of continuous electrophoretic fractionation of complex protein mixtures on a preparative scale.

A 35-kDa co-aggregation factor is a hemin binding protein in Porphyromonas gingivalis.

It has been known that Porphyromonas gingivalis has an obligate requirement for hemin or selected heme- or Fe-containing compounds for its growth. In addition, the influence of hemin on the expression of several putative virulence factors produced by this bacterium has also been recently documented; however, the mechanisms involved in hemin uptake are poorly defined. We succeeded in cloning the gene coding for the 35-kDa protein, which was specifically expressed in P. gingivalis and seemed to confer colonizing activities. Recently, we have constructed the P. gingivalis 381 mutant defective in the 35-kDa protein by insertion mutagenesis. The beige mutant exhibited little co-aggregation and the virulence was also decreased. Based on these results and homology search analysis, we focused on assessing the hemin bindings and found the heme regulatory motif (HRM) as a hemin direct binding site. The 35-kDa protein did possess the binding ability of selected protoporphyrins involving the hemin. These results demonstrated that 35-kDa protein is one of the hemin binding proteins in P. gingivalis and suggested that hemin binding ability of 35-kDa protein is important for the expression of virulence in P. gingivalis.

Characterization of rat monoclonal antibodies against human beta-defensin-2.

Defensins are a family of cationic antimicrobial peptides that participate in host defense. Human beta-defensin (hBD)-2 has a potent bactericidal activity against a wide spectrum of microorganisms. Because human gingival epithelium is constantly exposed to a variety of microbial challenges, it is considered that hBD-2 has an important role in the protective mechanisms against oral bacterial infection. However, little is known about the production of hBD-2 in tissues of the oral cavity. Six rat monoclonal antibodies (MAbs) raised against chemically synthesized hBD-2 have been characterized. Rat MAbs were specific for the conformational epitopes on hBD-2, but not to hBD-1. To identify the epitope on hBD-2, a series of six overlapping peptides covering the hBD-2 whole sequence were synthesized and the immunoreactivities of six MAbs were examined. The FCPRRYK domain in hBD-2 was recognized by all six MAbs and suggested to be an epitope region. By immunocytochemistry, hBD-2 was localized focally in the epidermis of the human gingival tissue using the MAbs. The MAbs specifically recognized against hBD-2 will be a useful tool to study the functional role of antimicrobial agents and an important asset in the imaging of oral infection processes.