Stimulation of growth of Porphyromonas gingivalis by cell extracts from Tannerella forsythia

OBJECTIVE: In order to examine if Tannerella forsythia stimulates the growth of Porphyromonas gingivalis, an in vitro study was performed. Background: P. gingivalis and T. forsythia are often isolated simultaneously from active periodontitis sites, indicating that these bacteria somewhat interact in the periodontal environment. We reported previously that mixed infection of P. gingivalis and T. forsythia synergistically induced lesion formation in a murine abscess model, and gingipains of P. gingivalis played an important role in this synergism. One of the possible mechanisms of this synergism is growth promotion by coinfection of the two bacteria. METHODS: Cell extracts of T. forsythia were added to the nutrition-decreased medium and the promotion of growth of P. gingivalis was examined. RESULTS: Sonicated extract of T. forsythia stimulated growth of P. gingivalis in nutrition-decreased medium in a dose-dependent manner. Proteins appeared to be the nature of growth-promoting factor, and the cell extract of T. forsythia had no stimulating effect on the growth of P. gingivalis strain devoid of gingipain activities. CONCLUSION: A product or a component of T. forsythia seemed to stimulate growth of P. gingivalis under nutrition-limited conditions. Gingipains are considered to play an important role in digestion or uptake of this growth-promoting factor. The interaction between T. forsythia and P. gingivalis in growth may be in part related with the synergistic virulence in a murine model.     

不同牙周状况龈下菌斑中牙龈卟啉单胞菌和福赛斯坦纳菌的分布

目的 分析中国牙周健康者和慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌和福赛斯坦纳菌的分布情况,探讨两种细菌在中国慢性牙周炎患者中的分布及其致病机理.方法 收集65例牙周健康者、62例慢性牙周炎患者的龈下菌斑,采用16S rRNA PCR方法检测牙龈卟啉单胞菌和福赛斯坦纳菌的分布.结果 牙周健康者牙龈卟啉单胞菌和福赛斯坦纳菌的检出率分别是21.6%、12.3%,慢性牙周炎患者病变位点两菌检出率分别为74.2%、58.1%,牙周炎健康位点两菌的检出率分别是22.6%、4.8%.牙周炎病变位点两种细菌的检出率均明显高于牙周健康者和牙周炎健康位点(P＜0.001);福赛斯坦纳菌在牙周健康者中的检出率高于牙周炎健康位点(P＜0.05);牙周健康者和牙周炎健康位点牙龈卟啉单胞菌的检出率没有差异.慢性牙周炎患者两菌联合检出率为51.6%.结论 牙龈卟啉单胞菌、福赛斯坦纳菌以及两种细菌联合感染与牙周炎密切相关.     

Gingipains in the culture supernatant of Porphyromonas gingivalis cleave CD4 and CD8 on human T cells

Porphyromonas gingivalis has been shown to attack host defense systems through proteolytic cleavage of a wide variety of members of the systems. In this study, we examined the ability of P. gingivalis culture supernatant to alter the expression of human T cell surface proteins. As judged by flow cytometric analysis, detection of CD4 expression was completely eliminated by the supernatant, but CD8 was less sensitive. When the culture supernatant was added with reducing agents, proteolytic activity was enhanced, resulting in the cleavage of CD8. Mitogenic response of T cells to phytohemagglutinin or concanavalin A was decreased by the treatment of the cells with the culture supernatant of P. gingivalis. The three forms of gingipains (high molecular mass arginine-specific gingipain, arginine-specific gingipain 2 and lysine-specific gingipain) purified from the culture supernatant of P. gingivalis actively cleaved CD4 and CD8 on human T cells, indicating that proteolytic activity of the culture supernatant was due to gingipains. These results suggest that cysteine proteinases like gingipains released from P. gingivalis cleave T cell surface proteins and impede T cell function.     

Porphyromonas gingivalis invades human pocket epithelium in vitro

The present study examined the adhesive and invasive potential of Porphyromonas gingivalis interacting with human pocket epithelium in vitro . Pocket epithelial tissue, obtained during periodontal surgery of patients with advanced periodontal disease, generated a stratified epithelium in culture. P. gingivalis strains W50 and FDC 381 (laboratory strains), OMGS 712, 1439, 1738, 1739 and 1743 (clinical isolates) as well as Escherichia coli strain HB101 (non-adhering control) were tested with respect to epithelial adhesion and invasion. Adhesion was quantitated by scintillation spectrometry after incubation of radiolabeled bacteria with epithelial cells. The invasive ability of P. gingivalis was measured by means of an antibiotic protection assay. The epithelial multilayers were infected with the test and control strains and subsequently incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). The number of internalized bacteria surviving the antibiotic treatment was assessed after plating lyzed epithelial cells on culture media. All tested P. gingivalis strains adhered to and entered pocket epithelial cells. However, considerable variation in their adhesive and invasive potential was observed. E. coli strain HB101 did not adhere or invade. Transmission electron microscopy revealed that internalization of P. gingivalis was preceded by formation of microvilli and coated pits on the epithelial cell surfaces. Intracellular bacteria were most frequently surrounded by endosomal membranes; however, bacteria devoid of such membranes were also seen. Release of outer membrane vesicles (blebs) by internalized P. gingivalis was observed. These results support and extend previous work from this laboratory which demonstrated invasion of a human oral epithelial cell-line (KB) by P. gingivalis .     

Pro-inflammatory cytokine production from normal human fibroblasts is induced by Tannerella forsythia detaching factor

Background and Objective: Tannerella forsythia is a periodontal pathogen. Recently, we have reported that the cytopathic component of T. forsythia contains two distinct factors. One arrests the cell cycle at the G2 phase and the other, named forsythia detaching factor, detaches adhesion‐dependent immortalized human cells. In this study, we investigated the biological function of forsythia detaching factor using human normal fibroblasts. Material and Methods: A recombinant forsythia detaching factor, reported previously, was used. TIG‐3 cells, cultured in the absence or presence of forsythia detaching factor, were lysed and the supernatant was analyzed by western blotting with polyclonal forsythia detaching factor antibodies. The cells were subsequently fractionated to isolate the cytoplasmic, mitochondrial and remaining fractions. In order to measure the activity of mitochondria using nicotinamide adenine dinucleotide‐linked reductase, the water‐soluble tetrazolium method was used. The mitochondrial oxidative membrane potential was estimated by measuring the oxidization‐dependent fluorogenic conversion of dihydrotetramethylrosamine using flow cytometry. The concentration of interleukin‐8 in the culture supernatant was assayed using a Human IL‐8 ELISA kit. Results: Forsythia detaching factor‐treated cells detached from the substratum and aggregated from 3 to 24 h. Then, the detached cells resumed adhesion and proliferated after 48 h. The western blot analysis revealed that most forsythia detaching factor trans‐located into the mitochondrial fraction. Forsythia detaching factor suppressed the nicotinamide adenine dinucleotide‐linked reductase activity in a dose‐dependent manner and consequently increased the mitochondrial oxidative membrane potential. The production of interleukin‐8 was reinforced in forsythia detaching factor‐treated cells at 72 h through an increase of the mitochondrial oxidative membrane potential. Conclusion: The forsythia detaching factor might be involved in the virulence of T. forsythia through induction of the pro‐inflammatory cytokine interleukin‐8.     

Porphyromonas gingivalis stimulates TACE production by T cells

Introduction: Tumour necrosis factor‐α converting enzyme (TACE), also known as ADAM17, is a membrane‐bound metalloprotease and disintegrin. It is produced by a number of host cells and is known to shed and release cell‐bound cytokines, particularly members of the tumour necrosis factor family. The aim of this study was to investigate the effect of Porphyromonas gingivalis on TACE production by a human T‐cell line, to identify putative virulence factors involved in this process, and to investigate the effect of doxycycline. Methods: P. gingivalis 6‐day culture supernatants were used to challenge Jurkat T cells for 6 h. Secreted and cell‐associated TACE levels were measured by enzyme‐linked immunosorbent assay, whereas messenger RNA expression was investigated by quantitative real‐time polymerase chain reaction. To investigate the involvement of cysteine proteases or proteinaceous components in general, P. gingivalis culture supernatants were treated with the specific chemical inhibitor TLCK or heat‐inactivated, respectively. The effect of doxycycline on the regulation of TACE secretion by P. gingivalis was also investigated. Results: P. gingivalis challenge resulted in a concentration‐dependent enhancement of TACE messenger RNA expression and protein release by Jurkat cells. TLCK treatment or heat treatment of P. gingivalis culture supernatants decreased TACE release to control levels. Doxycycline inhibited TACE secretion dose dependently. Conclusion: The induction of TACE by T cells in response to P. gingivalis may in turn favour the shedding of host cell‐bound cytokines into the local microenvironment, potentially amplifying the inflammatory response. In the present experimental system, P. gingivalis cysteine proteases are involved in TACE release by T cells.     

Transmission of Porphyromonas gingivalis between spouses

Abstract Porphyromonas gingivalis has been associated with severe forms of periodontitis. The question can be raised about the origin of this suspected periodontal pathogen. The purpose of the present investigation was to study the possibility of transmission of P. gingivalis between spouses. 18 patients were selected with severe periodontitis and colonized with P. gingivalis. 10 of their spouses appeared to be colonized with P. gingivalis too. 8 of the patients and their spouses were investigated further clinically and microbiologically. Microbiological evaluation revealed mostly high %s of P. gingivalis in the pockets, not only of the patients (5–48% of the cultivable flora), but also in 7 of the 8 spouses (0.2–61%). Furthermore, this species was isolated often from the saliva, the tongue, the buccal mucosa and the tonsillar area from both patients and spouses. For typing purposes, bacterial DNA was isolated, treated with the restriction endonucleases Bam HI or Pst 1, after which the DNA fragments were separated by agarose gelelectrophoresis. With one exception, each individual was colonized with only one clonal type of P. gingivalis. The DNA patterns of all P. gingivalis isolates from unrelated individuals were found to be distinct. In contrast, in 6 of the 8 couples, the DNA patterns of isolates from husband and wife were indistinguishable. From these data, it can be concluded that it is most likely, that P. gingivalis can be transmitted between spouses. It remains to be investigated whether transmission of P. gingivalis is a risk factor for developing periodontal destruction.     

Prevalence and serotyping of Porphyromonas gingivalis in an Indonesian population

In this study, the prevalence and serotype distribution of Porphyromonas gingivalis in an Indonesian population (n=158) is described. The relationship between subgingival P. gingivalis and periodontal attachment loss was investigated. The serotype distribution and periodontal parameters were studied. Serotyping was also used to study person-to-person transmission between siblings and between spouses. Approximately 50% of the subjects had periodontal attachment loss > or =3 mm at 1 or more recorded sites. The population was divided into 2 subgroups based on the presence or absence of P. gingivalis. No differences in plaque index, pocket depth, clinical attachment loss, bleeding upon probing, % of sites with > or =3 mm clinical attachment loss and % of sites with probing pocket depth > or =5 mm, were observed between both sub-populations. All known 6 capsular serotypes were found in the study population, with the exception of the K1 type. Detection of 1 of the known P. gingivalis serotypes was not related with the amount of clinical attachment loss. In 3 out of 29 sibships with more than one member positive for P. gingivalis, an identical P. gingivalis serotype was found. None of the 15 couples in the study shared an identical P. gingivalis serotype, indicating that transmission is probably not a common phenomenon in this population.     

Oral bacterial DNA differ in their ability to induce inflammatory responses in human monocytic cell lines

Background: Deoxyribonucleic acids (DNA) of periodontal pathogens, Porphyromonas gingivalis (Pg) and Tannerella forsythia, stimulate cytokine production in human monocytic cells (THP‐1) through Toll‐like receptor 9 (TLR‐9) and nuclear factor‐κB signaling. Fusobacterium nucleatum (Fn) is one of the most frequently isolated bacteria in periodontally diseased tissues and is reported to synergize with Pg, enhancing the pathogenicity. We investigate inflammatory mediator production in THP‐1 cells challenged with Fn and Streptococcus sanguinis (Ss) DNA, a non‐pathogenic oral bacteria, and further assess whether cytokines triggered by whole pathogens or Pg lipopolysaccharide (LPS) are affected by TLR‐9 signaling inhibitors (chloroquine). Methods: THP‐1 cells were stimulated with Pg‐DNA (100 ng/μL), Fn‐DNA (100 ng/μL), Ss‐DNA (100 ng/μL), Pg‐LPS (10 ng/μL), and heat‐killed whole bacteria (multiplicity of infection, 1:100) for 16 hours with or without chloroquine pretreatment (10 μg/mL). Interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor‐α levels were determined using enzyme‐linked immunosorbent assay. Statistical analyses included analysis of variance with multiple comparisons using Dunnett or Tukey methods and paired t test. A value of P <0.05 was significant. Results: Inflammatory mediator levels were increased in response to all the stimuli with the exception of Ss‐DNA (P <0.05). Chloroquine pretreatment significantly decreased cytokine production from THP‐1 cells with the exception of IL‐6 production triggered by whole Fn and Ss (P <0.05). Conclusions: Differences exist among oral bacterial DNA in inducing immune responses. By altering the conditions in cytosolic compartments, we can interfere with cellular responses triggered by extracellular receptor activation. Thus, alternative treatment approaches targeted to intracellular receptors might be of benefit in controlling periodontal inflammation.     

Porphyromonas gingivalis invades oral epithelial cells in vitro

The aim of the present study was to analyze the adhesive and invasive potential of a number of P. gingivalis strains, in an in vitro system utilizing cultures of human oral epithelial cells (KB cell line, ATCC CCL 17). P. gingivalis strains W50 and FDC 381 (laboratory strains) and OMGS 1738, 1743 and 1439 (clinical isolates) as well as E. coli strain HB 101 (non-adhering, non-invasive control) were used. Adherence was assessed by means of scintillation counting and light microscopy, after incubation of radiolabelled bacteria with epithelial cells. In the invasion assay, monolayers were infected with the P. gingivalis and E. coli strains and further incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). Invasion was evaluated by (i) assessing presence of bacteria surviving the antibiotic treatment, and (ii) electron microscopy. All P. gingivalis strains adhered to and entered into the oral epithelial cells. After 3 hours of incubation, bacteria were frequently identified intracellularly by means of electron microscopy. The cellular membranes, encapsulating the microorganisms in early stages of the invasive process, appeared later to disintegrate. The presence of coated pits on the epithelial cell surfaces suggested that internalization of P. gingivalis was associated with receptor-mediated endocytosis (RME). Formation of outer membrane vesicles (blebs) by intracellular bacteria indicated that internalized P. gingivalis was able to retain its viability. E. coli strain HB 101 neither adhered to nor invaded epithelial cells.     

Clonal stability of Porphyromonas gingivalis in untreated periodontitis

Objectives: The objective of the present investigation was to study the clonal stability of Porphyromonas gingivalis in a population of Indonesian subjects, deprived of dental care and with varying degrees of periodontitis over a period of 8 years.
Material and Methods: In 1994, 105 subjects and in 2002, 103 subjects were P. gingivalis culture positive. Multiple isolates from each of these subjects were used for amplified fragment length polymorphism (AFLP) typing.
Results: Sixty-six individuals were P. gingivalis culture positive at both time points. In 31 subjects (47%) an exact identical P. gingivalis genotype distribution was found in 1994 and in 2002. In 26 of these subjects one genotype, in eight subjects two identical genotypes were found at both time points. In 70% of the subjects at least one P. gingivalis genotype was found in 1994 and 2002, whereas other genotypes were either newly detected or were no longer detectable. Identical genotypes were found in 26% of the sibships. Clonal stability in siblings was 39%. Horizontal transmission of P. gingivalis was only found in 2002 and was low (11%). In total, 56 P. gingivalis genotypes were identified in 1994 and 61 in 2002. Twenty-four appeared unique, whereas other genotypes were found in multiple subjects within as well as without families. One genotype occurred in 11 different subjects.
Conclusions: The clonal stability of P. gingivalis under natural conditions is high. Complete different genotype distribution was found in only 27% of the subjects. Transmission of P. gingivalis occurred frequently among siblings but not among spouses.     

Porphyromonas gingivalis induction of mediator and cytokine secretion by human gingival fibroblasts

We hypothesized that bacterial viability and strain characteristics of Porphyromonas gingivalis could affect the induction of pro-inflammatory mediator secretion by human gingival fibroblast cultures. Both killed and viable P. gingivalis elicited production of prostaglandin E2, interleukin-1 beta (IL-1 beta), IL-6 and IL-8, although killed P. gingivalis induced generally higher levels, particularly IL-6 and IL-8, compared with the viable bacteria. P. gingivalis strains, which exhibited wild-type levels of trypsin-like protease activity, stimulated human gingival fibroblasts to secrete increased levels of prostaglandin E2 and IL-1 beta, although minimal levels of IL-6 and IL-8 were noted in supernatants from the gingival fibroblast cells. P. gingivalis strains BEI and NG4B19, which have either decreased or undetectable levels of trypsin-like protease, respectively, induced significantly greater IL-6 and IL-8 levels in gingival fibroblast cultures compared with the other strains. The ability of antibody to P. gingivalis to alter human gingival fibroblast production of pro-inflammatory mediators was tested using nonhuman primate antisera. Both immune and nonimmune sera altered the P. gingivalis-generated pattern of mediators from the gingival fibroblasts. We conclude that: (i) viable and killed P. gingivalis were capable of inducing various pro-inflammatory cytokines from human gingival fibroblasts; (ii) strain differences in cytokine induction were noted, and the expression of a trypsin-like protease activity was related to decreased extracellular levels of IL-6 and IL-8; and (iii) the presence of serum, particularly with specific antibody to P. gingivalis, significantly altered human gingival fibroblast cytokine production compared with P. gingivalis alone.     

Generation of gingival T cell lines/clones specific with Porphyromonas gingivalis pulsed dendritic cells from periodontitis patients

OBJECTIVES AND BACKGROUND: It is well documented that in periodontitis lesions, most infiltrated gingival T cells are antigen-specific memory T cells. These cells play an important role as regulators and effector cells in the pathogenesis of periodontitis. In this study, we used dendritic cells (DCs) as antigen-presenting cells to generate human gingival T cell lines and clones specific for Porphyromonas gingivalis from periodontitis patients. METHODS: Autologous DCs were derived from the patients' adherent monocytes using granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. Lymphocytes were isolated from gingival biopsies using collagenase enzyme digestion and the number was increased by subsequent culturing in IL-2-containing medium. T cells were then negatively sorted using flow cytometry, cocultured with P. gingivalis-pulsed DCs and subsequently expanded in the culture medium containing IL-2. T cells were kept viable and active by periodic exposure to antigen-pulsed DCs. The specificity of the T cell lines was tested against four plaque bacteria: P. gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia and Actinomyces viscosus. The established T cell lines were then cloned. Three P. gingivalis-specific T cell lines and 12 gingival T cell clones were generated. They all showed good specificity against P. gingivalis but not to other plaque bacteria. RESULTS: All T cell clones were positive for CD4 and the majority of them produced interferon gamma, but a minimal or negligible amount of IL-5. CONCLUSIONS: The data obtained clearly showed that monocyte-derived DCs could be used as powerful antigen-presenting cells to generate antigen-specific T cells from periodontitis tissues.     

Treponema denticola,Porphyromonas gingivalis,and Tannerella forsythia induce cell death and release of endogenous danger signals

ObjectiveThe aim of this study was to analyze whether periodontopathogens induced inflammatory cell death and the release of diverse endogenous danger molecules in THP-1-derived macrophages.MethodsThe macrophages were treated with Treponema denticola, Porphyromonas gingivalis, and Tannerella forsythia. Activation of caspase-1 and caspase-4 was detected by Western blotting. Cell death of bacteria-stimulated macrophages was examined using a lactate dehydrogenase (LDH) assay and propidium iodide (PI)/annexin V (AV) staining. Levels of endogenous danger signals, including adenosine triphosphate (ATP), uric acid, heat shock protein 60 (HSP60), high-mobility group box protein 1 (HMGB1), and fibronectin in the culture supernatants were determined using an ATP bioluminescence assay kit, a uric acid assay kit, and Western blotting, respectively.ResultsT. denticola, P. gingivalis, and T. forsythia induced activation of caspase-1 and caspase-4. The LDH assay and PI/AV staining showed that all three pathogens induced pyroptotic cell death. All three bacteria induced release of ATP, which is an important ligand for inflammasome activation; the increase in ATP ultimately leads to caspase-1 activation. T. denticola induced release of HSP60 and fibronectin, while T. forsythia induced release of HMGB1 in addition to HSP60 and fibronectin. None of the endogenous molecules except for fibronectin were detected in P. gingivalis-infected cells, possibly due to degradation of these factors by the proteolytic activity of the bacteria. Interestingly, P. gingivalis induced uric acid release.ConclusionInflammatory cell death and endogenous danger molecules released from cells infected with periodontopathogens may play critical roles in the pathogenesis and progression of periodontitis by augmenting immune and inflammatory responses.     

Distribution of Porphyromonas gingivalis fimA genotypes in patients affected by rheumatoid arthritis and periodontitis

Objective: To determine and compare the distribution of Porphyromonas gingivalis fimA genotypes in patients affected by Rheumatoid arthritis (RA) and periodontitis (PE).

Materials and methods: This study involved 394 subjects divided into four groups, RA, PE, RA and PE and healthy subjects. PE was diagnosed by using clinical attachment loss (CAL) and probing depth (PD) indexes. Presence of P. gingivalis and its genotypes was identified by polymerase chain reaction in subgingival biofilm.

Results: P. gingivalis was more frequent in patients with RA (82.69%), and fimA II genotype was the most frequent in all groups, especially in PE/RA (76.71%). There was statistical difference (p?P. gingivalis genotypes such as fimA Ib, II and III.

Conclusions: Distribution of P. gingivalis fimA II genotypes was different among groups, it could play a critical role in the presence of PE in RA patients.     

Human antibody responses to outer envelope antigens of Porphyromonas gingivalis serotypes

Immunological studies examining the homogeneity of the major antigenic components of P. gingivalis have suggested 3 serotypes and have indicated a limited distribution of the serotypes in an individual patient. These studies prompted us to define the immunodominant antigens and distribution of immune responses to P. gingivalis serotypes. Serum IgG antibody levels in periodontitis patients in the present study were most frequently elevated above the normal subjects when tested against P. gingivalis serotype A (i.e., 33277). Nearly 1/3 of the patients showed significantly elevated antibody to multiple serotypes of the P. gingivalis apparently resulting from cross-reacting antigens. We determined distinctive differences among outer envelope protein and antigen patterns obtained from the three serotypes. Moreover, the results identified considerable similarities in the qualitative and quantitative antigen response patterns among patients to a particular serotype. There was a strong positive correlation between IgG antibody levels (ELISA) and the total level of reactivity determined in the immunoblots, as well as a positive correlation to the proportion of antibody to particular antigens. These findings suggest that responses to these antigens comprised a major portion of the response to the intact microorganism. Additionally, the detection of antibody to particular antigen bands was indicative of early responses to each of the P. gingivalis serotypes. The results of our study indicate that a subpopulation of periodontitis patients develop an extensive serum antibody response often to multiple serotypes of P. gingivalis and may define a patient population with a P. gingivalis disease. Finally, our results indicate a more consistent antigenic composition for P. gingivalis which may enhance the potential for strategies to immunologically interfere with disease caused by this microorganism.     

Characterization of T lymphocyte clones derived from Porphyromonas gingivalis infected subjects

Porphyromonas gingivalis plays a major role in the pathogenesis of periodontal disease, however some individuals with P. gingivalis infection do not experience periodontal breakdown. The aim of this study was to investigate the proliferative responses of two highly defined groups of subjects and to establish and characterize peripheral blood and gingival cell T cell lines and clones from subjects from these groups, The two groups were selected on the basis of P. gingivalis in their plaque and the presence of serum anti-P. gingiralis antibodies. Both groups therefore were seen to have P. gingivalis and to have responded to it. They however differed only in their clinical susceptibility (adult periodontitis) or resistance (gingivitis) to periodontal breakdown. Dose responses of peripheral blood mononuclear cells extracted from the subjects showed a trend towards a lower response by the adult periodontitis group to P. gingivalis outer membrane (OM) antigens. Peripheral blood T cell lines and clones responsive to P. gingivalis OM were established from a high responding gingivitis subject and a low responding adult periodontitis subject. Gingival T cell lines and clones were also derived from cells extracted from the periodontal tissues of the same periodontitis subject. The majority of T cells in the peripheral blood T cell line from the gingivitis subject were CD4 while those from the adult periodontitis subject were CD8. The gingival T cell line was CD3+ve CD4-ve and CD8-ve. All lines and clones proliferated slowly to P. gingivalis OM but phytohaemagglutinin (PHA) induced an increase in DNA synthesis in those derived from the gingivitis subject with little to no effect on those established from the adult periodontitis subject. Furthermore. PHA inhibited the proliferative response of the CD8 clone derived from the adult periodontitis subject. Phenotypic analysis demonstrated that all the peripheral blood clones expressed the αβ TCR while the gingival T cell clones expressed the γδ TCR. All clones had the memory/primed CD45RO+ve phenotype and at least 80% of cells in each clone were HLA-DR + ve. A lower percent of gingival cells expressed CD45RA than the CD4 peripheral blood clones and the two CD8 clones also had a decreased CD45RA expression. The gingival T cell clones also expressed a low percent CD25 as did the CD8 clone derived from the adult periodontitis subject. The results suggest that clones derived from the gingivitis and adult periodontitis subject may be functionally different. The presence of γδ T cells in adult periodontitis remains to be confirmed and their function determined.