Publications (13)39.98 Total impact
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Article: Lipopolysaccharides mediate leukotoxin secretion in Aggregatibacter actinomycetemcomitans.
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ABSTRACT: We previously reported that lipopolysaccharide (LPS) -related sugars are associated with the glycosylation of the collagen adhesin EmaA, a virulence determinant of Aggregatibacter actinomycetemcomitans. In this study, the role of LPS in the secretion of other virulence factors was investigated. The secretion of the epithelial adhesin Aae, the immunoglobulin Fc receptor Omp34 and leukotoxin were examined in a mutant strain with inactivated TDP-4-keto-6-deoxy-d-glucose 3,5-epimerase (rmlC), which resulted in altered O-antigen polysaccharides (O-PS) of LPS. The secretion of Aae and Omp34 was not affected. However, the leukotoxin secretion, which is mediated by the TolC-dependent type I secretion system, was altered in the rmlC mutant. The amount of secreted leukotoxin in the bacterial growth medium was reduced nine-fold, with a concurrent four-fold increase of the membrane-bound toxin in the mutant compared with the wild-type strain. The altered leukotoxin secretion pattern was restored to the wild-type by complementation of the rmlC gene in trans. Examination of the ltxA mRNA levels indicated that the leukotoxin secretion was post-transcriptionally regulated in the modified O-PS containing strain. The mutant strain also showed increased resistance to vancomycin, an antibiotic dependent on TolC for internalization, indicating that TolC was affected. Overexpression of TolC in the rmlC mutant resulted in an increased TolC level in the outer membrane but did not restore the leukotoxin secretion profile to the wild-type phenotype. The data suggest that O-PS mediate leukotoxin secretion in A. actinomycetemcomitans.Molecular oral microbiology. 04/2012; 27(2):70-82. -
Article: Expression of receptor activator of nuclear factor-kappaB ligand by B cells in response to oral bacteria.
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ABSTRACT: We investigated receptor activator of nuclear factor-kappaB ligand (RANKL) expression by B lymphocytes during early and late aspects of the immune response to Aggregatibacter actinomycetemcomitans, a gram-negative, anaerobic bacterium associated with aggressive periodontal disease. Expression of messenger RNA transcripts (tumor necrosis factor-alpha, Toll-like receptors 4 and 9, interleukins 4 and 10, and RANKL) involved in early (1-day) and late (10-day) responses in cultured rat splenocytes was examined by reverse transcription-polymerase chain reaction (RT-PCR). The immune cell distribution (T, B, and natural killer cells and macrophages) in cultured rat splenocytes and RANKL expression in B cells were determined by flow cytometric analyses. B-cell capacity for induction of osteoclast differentiation was evaluated by coculture with RAW 264.7 cells followed by a tartrate-resistant acid phosphatase (TRAP) activity assay. The expression levels of interleukins 4 and 10 in cultured cells were not changed in the presence of A. actinomycetemcomitans until cultured for 3 days, and peaked after 7 days. After culture for 10 days, the percentages of B and T cells, the overall RANKL messenger RNA transcripts, and the percentage of RANKL-expressing immunoglobulin G-positive cells were significantly increased in the presence of A. actinomycetemcomitans. These increases were considerably greater in cells isolated from A. actinomycetemcomitans-immunized animals than from non-immunized animals. RAW 264.7 cells demonstrated significantly increased TRAP activity when cocultured with B cells from A. actinomycetemcomitans-immunized animals. The addition of human osteoprotegerin-Fc to the culture significantly diminished such increases. This study suggests that B-lymphocyte involvement in the immune response to A. actinomycetemcomitans through upregulation of RANKL expression potentially contribute to bone resorption in periodontal disease.Oral Microbiology and Immunology 07/2009; 24(3):190-6. · 2.81 Impact Factor -
Article: Cross-reactive adaptive immune response to oral commensal bacteria results in an induction of receptor activator of nuclear factor-kappaB ligand (RANKL)-dependent periodontal bone resorption in a mouse model.
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ABSTRACT: The present study examined whether induction of an adaptive immune response to orally colonizing non-pathogenic Pasteurella pneumotropica by immunization with the phylogenetically closely related bacterium, Actinobacillus actinomycetemcomitans, can result in periodontal bone loss in mice. BALB/c mice harboring P. pneumotropica (P. pneumotropica(+) mice) in the oral cavity or control P. pneumotropica-free mice were immunized with fixed A. actinomycetemcomitans. The animals were sacrificed on day 30, and the following measurements were carried out: (i) serum immunoglobulin G and gingival T-cell responses to A. actinomycetemcomitans and P. pneumotropica; (ii) periodontal bone loss; and (iii) identification of receptor activator of nuclear factor-kappaB ligand (RANKL) -positive T cells in gingival tissue. Immunization with A. actinomycetemcomitans induced a significantly elevated serum immunoglobulin G response to the 29-kDa A. actinomycetemcomitans outer membrane protein (Omp29), which showed strong cross-reactivity with P. pneumotropica OmpA compared to results in the control non-immunized mice. The A. actinomycetemcomitans-immunized P. pneumotropica(+) mice developed remarkable periodontal bone loss in a RANKL-dependent manner, as determined by the abrogation of bone loss by treatment with osteoprotegerin-Fc. The T cells isolated from the gingival tissue of A. actinomycetemcomitans-immunized P. pneumotropica(+) mice showed an in vitro proliferative response to both A. actinomycetemcomitans and P. pneumotropica antigen presentation, as well as production of soluble(s)RANKL in the culture supernatant. Double-color confocal microscopy demonstrated that the frequency of RANKL(+) T cells in the gingival tissue of A. actinomycetemcomitans-immunized P. pneumotropica(+) mice was remarkably elevated compared to control mice. The induction of an adaptive immune response to orally colonizing non-pathogenic P. pneumotropica results in RANKL-dependent periodontal bone loss in mice.Oral Microbiology and Immunology 07/2007; 22(3):208-15. · 2.81 Impact Factor -
Article: Diminished forkhead box P3/CD25 double-positive T regulatory cells are associated with the increased nuclear factor-kappaB ligand (RANKL+) T cells in bone resorption lesion of periodontal disease.
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ABSTRACT: Periodontal disease involves multi-bacterial infections accompanied by inflammatory bone resorption lesions. The abundant T and B lymphocyte infiltrates are the major sources of the osteoclast differentiation factor, receptor activator for nuclear factor-kappaB ligand (RANKL) which, in turn, contributes to the development of bone resorption in periodontal disease. In the present study, we found that the concentrations of RANKL and regulatory T cell (T(reg))-associated cytokine, interleukin (IL)-10, in the periodontal tissue homogenates were correlated negatively, whereas RANKL and proinflammatory cytokine, IL-1beta, showed positive correlation. Also, according to the fluorescent-immunohistochemistry, the frequency of forkhead box P3 (FoxP3)/CD25 double-positive cells was diminished strikingly in the bone resorption lesion of periodontal disease compared to healthy gingival tissue, while CD25 or FoxP3 single positive cells were still observed in lesions where abundant RANKL+ lymphocytes were present. Very importantly, few or no expressions of FoxP3 by the RANKL+ lymphocytes were observed in the diseased periodontal tissues. Finally, IL-10 suppressed both soluble RANKL (sRANKL) and membrane RANKL (mRANKL) expression by peripheral blood mononuclear cells (PBMC) activated in vitro in a bacterial antigen-specific manner. Taken together, these results suggested that FoxP3/CD25 double-positive T(reg) cells may play a role in the down-regulation of RANKL expression by activated lymphocytes in periodontal diseased tissues. This leads to the conclusion that the phenomenon of diminished CD25+FoxP3+ T(reg) cells appears to be associated with the increased RANKL+ T cells in the bone resorption lesion of periodontal disease.Clinical & Experimental Immunology 06/2007; 148(2):271-80. · 3.36 Impact Factor -
Article: Innate immune peptide LL-37 displays distinct expression pattern from beta-defensins in inflamed gingival tissue.
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ABSTRACT: Anti-microbial peptides produced from mucosal epithelium appear to play pivotal roles in the host innate immune defence system in the oral cavity. In particular, human beta-defensins (hBDs) and the cathelicidin-type anti-microbial peptide, LL-37, were reported to kill periodontal disease-associated bacteria. In contrast to well-studied hBDs, little is known about the expression profiles of LL-37 in gingival tissue. In this study, the anti-microbial peptides expressed in gingival tissue were analysed using immunohistochemistry and enxyme-linked immunosorbent assay (ELISA). Immunohistochemistry revealed that neutrophils expressed only LL-37, but not hBD-2 or hBD-3, and that such expression was prominent in the inflammatory lesions when compared to healthy gingivae which showed very few or no LL-37 expressing neutrophils. Gingival epithelial cells (GEC), however, expressed all three examined anti-microbial peptides, irrespective of the presence or absence of inflammation. Moreover, as determined by ELISA, the concentration of LL-37 in the gingival tissue homogenates determined was correlated positively with the depth of the gingival crevice. Stimulation with periodontal bacteria in vitro induced both hBD-2 and LL-37 expressions by GEC, whereas peripheral blood neutrophils produced only LL-37 production, but not hBD-2, in response to the bacterial stimulation. These findings suggest that LL-37 displays distinct expression patterns from those of hBDs in gingival tissue.Clinical & Experimental Immunology 12/2006; 146(2):218-25. · 3.36 Impact Factor -
Article: Effect of adoptive transfer of antigen-specific B cells on periodontal bone resorption.
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ABSTRACT: Host immune responses to periodontal pathogens have been considered to contribute to the alveolar bone destruction in periodontitis. However, the role of B lymphocytes in the pathogenesis of periodontal bone loss is not clear. We examined the effect of adoptive transfer of antigen-specific B cells from rat spleens on experimental periodontal bone resorption. Donor rats were immunized intraperitoneally (i.p.) with formalin-killed Actinobacillus actinomycetemcomitans. Antigen-specific B cells were prepared from splenocytes by first binding CD43(+) cells to Petri dishes coated with anti-CD43 antibody to remove T cells, and non-binding cells were passed through a nylon wool column to deplete accessory cells. The retained cells were then collected and bound to A. actinomycetemcomitans-coated Petri dishes for enrichment of A. actinomycetemcomitans-binding B cells (AAB). A. actinomycetemcomitans non-binding B cells (ANB) and B cells from non-immunized donor rats (NIB) were also collected from these procedures. Each type of B cell was injected into a group of recipient rats that were then orally infected with live A. actinomycetemcomitans. At termination, the antibody levels to A. actinomycetemcomitans in serum and gingival wash fluids were significantly higher in the recipients transferred with AAB when compared to the recipients transferred with ANB or NIB. A markedly elevated number of antibody-forming cells were observed in the spleens of the recipients transferred with AAB, and these recipient rats also exhibited significantly increased bone resorption when compared to the other groups. It is suggested that B cells can contribute to periodontal bone resorption and that antigen-triggering of B cells is required for the bone resorption.Journal of Periodontal Research 05/2006; 41(2):101-7. · 1.69 Impact Factor -
Article: Potassium channel-blockers as therapeutic agents to interfere with bone resorption of periodontal disease.
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ABSTRACT: Inflammatory lesions of periodontal disease contain all the cellular components, including abundant activated/memory T- and B-cells, necessary to control immunological interactive networks and to accelerate bone resorption by RANKL-dependent and -independent mechanisms. Blockade of RANKL function has been shown to ameliorate periodontal bone resorption and other osteopenic disorders without affecting inflammation. Development of therapies aimed at decreasing the expression of RANKL and pro-inflammatory cytokines by T-cells constitutes a promising strategy to ameliorate not only bone resorption, but also inflammation. Several reports have demonstrated that the potassium channels Kv1.3 and IKCa1, through the use of selective blockers, play important roles in T-cell-mediated events, including T-cell proliferation and the production of pro-inflammatory cytokines. More recently, a potassium channel-blocker for Kv1.3 has been shown to down-regulate bone resorption by decreasing the ratio of RANKL-to-OPG expression by memory-activated T-cells. In this article, we first summarize the mechanisms by which chronically activated/memory T-cells, in concert with B-cells and macrophages, trigger inflammatory bone resorption. Then, we describe the main structural and functional characteristics of potassium channels Kv1.3 and IKCa1 in some of the cells implicated in periodontal disease progression. Finally, this review elucidates some recent advances in the use of potassium channel-blockers of Kv1.3 and IKCa1 to ameliorate the clinical signs or side-effects of several immunological disorders and to decrease inflammatory bone resorption in periodontal disease. ABBREVIATIONS: AICD, activation-induced cell death; APC, antigen-presenting cells; B(K), large conductance; CRAC, calcium release-activated calcium channels; DC, dendritic cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN-gamma, interferon-gamma; IP(3), inositol (1,4,5)-triphosphate; (K)ir, inward rectifier; JNK, c-Jun N-terminal kinase; I(K), intermediate conductance; LPS, lipopolysaccharide; L, ligand; MCSF, macrophage colony-stimulating factor; MHC, major histocompatibility complex; NFAT, nuclear factor of activated T-cells; RANK, receptor activator of nuclear factor-kappaB; T(CM), central memory T-cells; T(EM), effector memory T-cells; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; OPG, osteoprotegerin; Omp29, 29-kDa outer membrane protein; PKC, protein kinase C; PLC, phospholipase C; RT-PCR, reverse-transcriptase polymerase chain-reaction; S(K), small conductance; TCR, T-cell receptor; and (K)v, voltage-gated.Journal of Dental Research 07/2005; 84(6):488-99. · 3.49 Impact Factor -
Article: Involvement of T-lymphocytes in periodontal disease and in direct and indirect induction of bone resorption.
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ABSTRACT: Periodontal disease is a peripheral infection involving species of gram-negative organisms. T-lymphocytes can be found in the dense inflammatory infiltrate in this disease. CD4+ and CD8+ T-cells are present in periodontal lesions, as are memory/activated T-lymphocytes. In addition, Th1- and Th2-type T-lymphocytes and their associated cytokines with a subtle polarization to Th1 may be present. Th1-type T-cells up-regulate the production of pro-inflammatory cytokines IL-1 and TNF-alpha, which can induce bone resorption indirectly by promoting differentiation of osteoclast precursors and subsequently by activating osteoclasts. Such osteoclast differentiation is dependent on stimulation of osteoprotegerin ligand (OPG-L) production by osteoblastic cells. By contrast, activated T-cells, by virtue of direct production and expression of OPG-L, can directly promote osteoclast differentiation. OPG-L appears to be predominantly expressed on Th1-type cells. The direct and indirect T-cell involvement in periodontal bone resorption appears to be dependent on the degree of Th1-type T-cell recruitment into inflamed gingival tissues. This T-cell recruitment is regulated by adhesion molecules and chemokines/chemokine receptors. The adhesion molecules involved include alpha4 and alpha6 integrins, LFA-1, and ICAM-1. The Th1-type T-cells preferentially express CCR5 and CXCR3, which are found prominently in diseased gingivae. By contrast, little CCR4, expressed by Th2-type T-cells, can be detected. Also, the chemokine ligands RANTES, MIP1-alpha (both CCR5), and IP-10 (CXCR3 ligand) were elevated in inflamed periodontal tissues. The T-cell features in diseased periodontal tissues can be compared with those in rheumatoid arthritis, wherein bone resorption often attributed to Th1-type T-cell involvement has also been demonstrated.Critical Reviews in Oral Biology & Medicine 02/2001; 12(2):125-35. -
Article: T(h)1 transmigration anergy: a new concept of endothelial cell-T cell regulatory interaction.
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ABSTRACT: Stimulation of endothelial cells (EC) with IFN-gamma generates selective enhancement of T(h)1 cell transmigration and induction of MHC class II expression on EC. In the present study, we tested whether antigen presentation by EC could influence transmigrating T cells in an in vitro system. Bacterial antigen presentation by EC from primary culture and after cloning induced antigen-specific anergy of transmigrating T(h)1 clone cells in a MHC class II-dependent manner as characterized by non-responsiveness to subsequent antigen presentation and inability to produce IL-2. This T cell transmigration anergy induced by EC was abrogated by anti-rat CD28 mAb, suggesting that lack of B7 co-stimulatory signals by EC might be related to the induction of anergy. While MHC class II expression on primary and cloned EC was observed after IFN-gamma stimulation, these cells never expressed B7. B7-1 gene-transfected endothelial clone cells (ECC/B7-1) were developed to elucidate the influence of B7 co-stimulation by EC. ECC/B7-1 induced proliferation of T(h)1 clone cells, whereas ECC did not induce proliferation in co-culture of T(h)1 clone cells and EC stimulated with IFN-gamma and antigen. In the transmigration assay, ECC/B7-1 did not induce transmigration anergy of T(h)1 clones or T(h)1 lines unless anti-rat B7-1 blocking mAb was added. Therefore, in rats, the T cell anergy induced during transmigration across a layer of EC seemed to be due to antigen presentation in the absence of B7 on the EC. We introduce the concept of transmigration anergy in this manuscript. Thus, EC can play a critical immune regulatory role in the context of antigen presentation by MHC class II to transmigrating T cells.International Immunology 07/2000; 12(6):937-48. · 3.41 Impact Factor -
Article: Requirement of B7 costimulation for Th1-mediated inflammatory bone resorption in experimental periodontal disease.
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ABSTRACT: The CD28 costimulation at TCR signaling plays a pivotal role in the regulation of the T cell response. To elucidate the role of T cells in periodontal disease, a system of cell transfer with TCR/CD28-dependent Th1 or Th2 clones was developed in rats. Gingival injection of specific Ag, Actinobacillus actinomycetemcomitans 29-kDa outer membrane protein, and LPS could induce local bone resorption 10 days after the transfer of Ag-specific Th1 clone cells, but not after transfer of Th2 clone cells. Interestingly, the presence of LPS was required not only for the induction of bone resorption but also for Ag-specific IgG2a production. LPS injection elicited the induction of expression of both B7-1 and B7-2 expression on gingival macrophages, which otherwise expressed only MHC class II when animals were injected with Ag alone. The expression of B7 molecules was observed for up to 3 days, which corresponded to the duration of retention of T clone cells in gingival tissues. Either local or systemic administration of CTLA4Ig, a functional antagonist of CD28 binding to B7, could abrogate the bone resorption induced by Th1 clone cells combined with gingival challenge with both Ag and LPS. These results suggest that local Ag-specific activation of Th1-type T cells by B7 costimulation appeared to trigger inflammatory bone resorption, whereas inhibition of B7 expression by CTLA4Ig might be a therapeutic approach for intervention with inflammatory bone resorption.The Journal of Immunology 03/2000; 164(4):2102-9. · 5.79 Impact Factor -
Article: Selective diapedesis of Th1 cells induced by endothelial cell RANTES.
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ABSTRACT: Differentiated CD4 T cells can be divided into Th1 and Th2 types based on the cytokines they produce. Differential expression of chemokine receptors on either the Th1-type or the Th2-type cell suggests that Th1-type and Th2-type cells differ not only in cytokine production but also in their migratory capacity. Stimulation of endothelial cells with IFN-gamma selectively enhanced transmigration of Th1-type cells, but not Th2-type cells, in a transendothelial migration assay. Enhanced transmigration of Th1-type cells was dependent on the chemokine RANTES produced by endothelial cells, as indicated by the findings that Ab neutralizing RANTES, or Ab to its receptor CCR5, inhibited transmigration. Neutralizing Ab to chemokines macrophage-inflammatory protein-1alpha or monocyte chemotactic protein-1 did not inhibit Th1 selective migration. Whereas anti-CD18 and anti-CD54 blocked basal levels of Th1-type cell adherence to endothelial cells and also inhibited transmigration, anti-RANTES blocked only transmigration, indicating that RANTES appeared to induce transmigration of adherent T cells. RANTES seemed to promote diapedesis of adherent Th1-type cells by augmenting pseudopod formation in conjunction with actin rearrangement by a pathway that was sensitive to the phosphoinositol 3-kinase inhibitor wortmannin and to the Rho GTP-binding protein inhibitor, epidermal cell differentiation inhibitor. Thus, enhancement of Th1-type selective migration appeared to be responsible for the diapedesis induced by interaction between CCR5 on Th1-type cells and RANTES produced by endothelial cells. Further evidence that CCR5 and RANTES play a modulatory role in Th1-type selective migration derives from the abrogation of this migration by anti-RANTES and anti-CCR5 Abs.The Journal of Immunology 10/1999; 163(6):3269-78. · 5.79 Impact Factor -
Article: Cloning of the gene encoding the Actinobacillus actinomycetemcomitans serotype b OmpA-like outer membrane protein.
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ABSTRACT: 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.Infection and Immunity 03/1999; 67(2):942-5. · 4.16 Impact Factor -
Article: Antigen direction of specific T-cell clones into gingival tissues.
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ABSTRACT: This study was performed to investigate T-cell traffic to periodontal tissues during infection with a periodontal pathogen Actinobacillus actinomycetemcomitans (Aa). Rowett rat T-cell clones, A3 (CD4+ CD8-, alpha beta TCR+, NKRP-1-, specific to Aa) and G2 (CD4- CD8-, alpha beta TCR+, NKRP-1+, which reacts to Aa, Gram-negative and -positive bacteria), both expressed the same prominent adhesion molecules (LFA-1, VLA-4) to the same extent. Binding of both T-cell clones to rat endothelial cells in vitro was blocked by antibody to VLA-4. Rowett rats were infected with Aa and infused with Aa-stimulated, isogenic T-clone lymphocytes that had been labelled in vitro with 125IUdR. Radioactivity associated with recovery of clone A3, but not G2, was significantly elevated in the gingivae of infected rats, suggesting migration to infected animals' gingival tissues. Migration of radioactive Aa-specific A3 clone cells traced by autoradiography reached a maximum at 24 hr (1.2% of total lymphocytes as radiolabelled cells in infected gingiva versus 0.6% in noninfected), indicating an apparent antigen-directed retention in infected rats' gingival tissues. The G2 clone was not retained in the gingival tissues (0.20% of total lymphocytes as radiolabelled cells in infected gingiva versus 0.26% in non-infected). However, the possibility of A3 retention directed by inflammation or tissue-selective homing could not be excluded. In further experiments, other adoptively transferred T-clone lymphocytes [clones G23 (Th1) and F13 (Th2)] with specificity for the 29,000 MW outer membrane protein of Aa with the same prominent adhesion molecules could be recovered from rat gingivae previously challenged with this antigen. However, transferred T-clone lymphocytes [clone G26 (Th1)] with specificity for a different Aa antigen were not recovered. Therefore, the dynamics of cell entry into periodontal lesions vary for activated T lymphocytes with different antigenic specificities, indicating the significance of antigen in lymphocyte traffic to periodontal tissues.Immunology 01/1998; 93(1):11-9. · 3.32 Impact Factor
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1999–2009
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The Forsyth Institute
Cambridge, MA, USA
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2005
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Tufts University
Boston, GA, USA
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