Dietary supplementation with low-dose omega-3 fatty acids reduces salivary tumor necrosis factor-α levels in patients with chronic periodontitis: a randomized controlled clinical study.

BACKGROUND AND OBJECTIVE: Recent studies have demonstrated the beneficial effects of omega-3 polyunsaturated fatty acids (PUFAs) on physiological processes and on a variety of chronic inflammatory diseases, including periodontal diseases. In this study, we evaluated the impact of omega-3 PUFAs in conjunction with scaling and root planing (SRP) on salivary markers in patients with chronic periodontitis. MATERIAL AND METHODS: Thirty systemically healthy subjects with chronic periodontitis were enrolled and randomly allocated into two groups. The control group (n = 15) was treated with SRP + placebo whereas the test group was treated with SRP and dietary supplementation of low-dose omega-3 PUFAs (6.25 mg eicosapentaenoic acid and 19.19 mg docosahexaenoic acid). Clinical parameters were taken at baseline, 1, 3 and 6 mo following therapy. Saliva samples were obtained at the same time intervals and analyzed for tumor necrosis factor-α (TNF-α) and superoxide dismutase (SOD). RESULTS: Both groups showed significant changes in clinical parameters in response to treatment compared to baseline with no significant difference between groups. Salivary TNF-α levels showed a statistically significant decrease in the test group at 6 mo compared to the control group. Salivary SOD levels increased significantly at 3 and 6 mo in the test group and at 6 mo in placebo groups compared to baseline with no statistically significant differences between the groups. CONCLUSION: The results demonstrated that dietary supplementation with low-dose omega-3 PUFAs improves salivary TNF-α without any significant impact on clinical parameters in patients with chronic periodontitis, suggesting that the systemic benefits of dietary omega-3 PUFAs may not be translated to periodontal health. (ClinicalTrials.gov ID NCT02719587).

Salivary infectious agents and periodontal disease status.

BACKGROUND AND OBJECTIVES: The potential of salivary microorganisms to diagnose periodontal disease and to guide periodontal treatment is a research topic of current interest. This study aimed to determine whether the salivary counts of periodontopathic microbes correlated with the periodontal pocket counts of the same infectious agents, and whether the salivary counts of the test infectious agents could distinguish among individuals with periodontal health and various types of periodontal disease. MATERIAL AND METHODS: The study included 150 systemically healthy adults, of whom 37 were periodontally healthy, 31 had gingivitis, 46 had chronic periodontitis and 36 had aggressive periodontitis. Each study subject contributed microbial samples from the two deepest periodontal pockets of the dentition and from whole saliva. Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Epstein-Barr virus were identified using the TaqMan real-time PCR methodology. Statistical analysis was performed using the Mann-Whitney U-test and the receiver operating characteristic statistics. RESULTS: C. rectus, F. nucleatum, P. gingivalis, P. intermedia and T. forsythia occurred with significantly higher copy-counts in salivary samples from patients with gingivitis, chronic periodontitis and aggressive periodontitis than from periodontally healthy individuals. A. actinomycetemcomitans only showed higher salivary copy-counts in subjects with aggressive periodontitis compared with subjects with healthy periodontium, and the salivary copy-counts of Epstein-Barr virus did not reveal any significant difference among the four subject groups studied. The diagnostic sensitivity for periodontitis was 89.19 for P. gingivalis and for T. forsythia and 86.49 for P. intermedia, with specificities ranging from 83.78 to 94.59. The optimal copy-counts per mL saliva for identifying periodontitis were 40,000 for P. gingivalis, 700,000 for T. forsythia and 910,000 for P. intermedia. CONCLUSION: Salivary copy-counts of P. gingivalis, T. forsythia and P. intermedia appear to have the potential to identify the presence of periodontitis, whereas the salivary level of the other test infectious agents may possess little or no diagnostic utility. Longitudinal studies are warranted to determine the ability of salivary copy-counts of major periodontopathic bacteria to predict future periodontal breakdown.

Efficient presentation of both cytosolic and endogenous transmembrane protein antigens on MHC class II is dependent on cytoplasmic proteolysis.

Peptides from extracellular proteins presented on MHC class II are mostly generated and loaded in endolysosomal compartments, but the major pathways responsible for loading peptides from APC-endogenous sources on MHC class II are as yet unclear. In this study, we show that MHC class II molecules present peptides from proteins such as OVA or conalbumin introduced into the cytoplasm by hyperosmotic pinosome lysis, with efficiencies comparable to their presentation via extracellular fluid-phase endocytosis. This cytosolic presentation pathway is sensitive to proteasomal inhibitors, whereas the presentation of exogenous Ags taken up by endocytosis is not. Inhibitors of nonproteasomal cytosolic proteases can also inhibit MHC class II-restricted presentation of cytosolically delivered protein, without inhibiting MHC class I-restricted presentation from the same protein. Cytosolic processing of a soluble fusion protein containing the peptide epitope I-Ealpha(52-68) yields an epitope that is similar to the one generated during constitutive presentation of I-Ealpha as an endogenous transmembrane protein, but is subtly different from the one generated in the exogenous pathway. Constitutive MHC class II-mediated presentation of the endogenous transmembrane protein I-Ealpha is also specifically inhibited over time by inhibitors of cytosolic proteolysis. Thus, Ag processing in the cytoplasm appears to be essential for the efficient presentation of endogenous proteins, even transmembrane ones, on MHC class II, and the proteolytic pathways involved may differ from those used for MHC class I-mediated presentation.

Ligation of CD27 on murine B cells responding to T-dependent and T-independent stimuli inhibits the generation of plasma cells.

B cells can be stimulated either allogenically with the Th cell clone D10G4.1 and bone marrow-derived dendritic cells or polyclonally with LPS to proliferate and undergo terminal differentiation to Ig-secreting plasma cells in vitro. The addition of anti-CD27 to such cultures inhibits Ig secretion, and inhibition is more marked in T-dependent cultures than in T-independent cultures. Both IgM and secondary isotypes are affected, and addition of anti-CD27 even 4 days after culture initiation inhibits Ig secretion. Anti-CD27 does not affect B cell proliferation or the acquisition of activation markers by B cells, and no marked loss of B cell viability is detected in cells cultured in the presence of anti-CD27, suggesting that the inhibition of Ig secretion is not due to inhibition of early activation events or to death of activated cells in vitro. However, the presence of anti-CD27 significantly inhibits the induction of Blimp-1 and J chain transcripts, which are turned on in cells committed to plasma cell differentiation. Furthermore, mice immunized under cover of anti-CD27 make less Ag-specific IgM and IgG, but have equivalent T cell responses when compared with control mice. These data suggest that ligation of CD27, a member of the TNFR family, on the B cell surface may prevent terminal differentiation of activated B cells into Ig-secreting plasma cells.

Expression of an immunoglobulin heavy chain transgene in macrophage as well as lymphocyte lineages in vivo.

A rearranged immunoglobulin heavy chain (IgH) transgene-encoded protein is expressed in macrophage lineage cells, in addition to B and T lineages, in transgenic mouse bone marrow. Peripheral macrophages also express transgenic IgH protein. Mature T cells express lower levels than immature thymocytes. Almost all B220+ cells in the bone marrow express transgenic IgH protein, and this early expression in the B lineage is accompanied by a reduction of cell frequency even in the early B220+ CD43+ BP-1- stages, although it is more prominent in BP-1+ pre-B cells. Thus, an IgH transgene can be expressed not only in lymphoid but also in myeloid cells, although its developmental effects are restricted to the B cell lineage.

Presence of pentoxifylline during T cell priming increases clonal frequencies in secondary proliferative responses and inhibits apoptosis.

Naive T cells appear to be primed by specific Ag to differentiate into either effectors or memory cells. We have been analyzing the factors involved in this differential commitment in the priming of alloresponsive human T cells in vitro and have shown that the presence of a phosphodiesterase inhibitor, pentoxifylline (POX), during priming results in a decrease in the primary response and enhancement in the secondary proliferative response. We now show that the POX-mediated effect can be mimicked by dibutyryl cAMP. The secondary response enhancement is due to the effects of POX on the T cells rather than the APCs, because even fixed APCs can prime T cells in the presence of POX. POX affects T cells directly by increasing clonal frequency rather than the burst size of the secondary responders. The known inhibition of IL-2 production by POX is not responsible for this effect, because exogenous IL-2 supplementation does not block it. The presence of POX during priming alters the outcome of T cell activation, resulting in a lower frequency of cells expressing IL-2R alpha (CD25) and a decrease in their subsequent apoptosis, and this antiapoptotic effect is consistent with the enhanced commitment of T cells to secondary responsiveness by POX.

Modulation of T cell cytokine profiles and peptide-MHC complex availability in vivo by delivery to scavenger receptors via antigen maleylation.

We have previously shown that conversion of proteins to scavenger receptor (SR) ligands by maleylation increases their immunogenicity. We now show that maleyl-Ag-immune spleen cells make relatively more IFN-gamma and less IL-4 or IL-10 than native Ag-immune cells. This is also reflected in the IgG1:IgG2a ratios in Abs generated in vivo. SR engagement on macrophages does not alter their surface levels of the adhesive/costimulatory molecules CD11a/CD18, CD11b/CD18, CD24, CD54, or CD40, nor does it enhance their ability to support anti-CD3-driven proliferation of naive T cells in vitro. Costimulatory molecules implicated in differential Th1/Th2 commitment--CD80, CD86, and IL-12--are not inducible by SR ligation. In addition to macrophages and dendritic cells, B cells also show receptor-mediated uptake and enhanced presentation of maleyl-Ags. Using a monoclonal T cell line to detect peptide-MHC complexes expressed on spleen cells in Ag-injected mice, we find that higher levels of these complexes are generated in vivo from maleyl-proteins and they persist longer than those generated from the native protein. Together, these data suggest that in certain situations, the levels of cognate ligand available and/or the time course of their availability may play a major role in determining the cytokine profiles of the responding T cells in addition to the costimulatory signals implicated so far.

Disruption of T cell tolerance by directing a self antigen to macrophage-specific scavenger receptors.

Breakdown of immune self tolerance is speculated to cause autoimmune diseases, but most studies on tolerance use foreign molecules as targets. In this study, we show another approach using delivery of a maleylated self protein to macrophage-specific scavenger receptors. Mice generate Abs against the maleylated form of a ubiquitous self Ag, mouse serum albumin (MSA), although native MSA is nonimmunogenic. This generation of anti-maleyl MSA Abs depends on binding of maleyl MSA to scavenger receptors in vivo, since coinjection of a serologically unrelated scavenger receptor ligand inhibits it, suggesting that the Ab response is T cell dependent. Spleen cells as well as nylon adherence-purified splenic T cells from maleyl MSA-immune mice proliferate in response to both maleyl MSA and MSA; this response is blocked by anti-MHC class II mAbs, and the autoimmune cells can recognize at least five 15-mer peptides from the MSA sequence, establishing that T cell tolerance to MSA has been broken in these mice. Maleyl MSA and MSA are recognized equally well, provided the scavenger receptor-specific delivery of maleyl MSA is blocked during stimulation in vitro, indicating that maleyl MSA-specific non-self peptides are unlikely to play a major role in the observed disruption of T cell tolerance. Thus, delivery of some self molecules to scavenger receptors may lead to disruption of immune tolerance. These results are relevant to mechanisms of immune tolerance and the etiopathogenesis of autoimmunity.

Differential regulation of T cell activation for primary versus secondary proliferative responses.

T cell activation in vivo results in proliferation and generation of effector cytokine-secreting cells, as also in development of memory cells that mount enhanced responses upon restimulation. However, differences in the signals promoting generation of effector vs memory T cells are not yet characterized. In this study, using various strategies to modulate an allorecognition system for priming human T cells in vitro, we show that there are indeed differences between the signaling requirements for a first proliferative response and those for priming T cells for enhanced recall proliferative responses. Using APCs fixed with varying concentrations of paraformaldehyde, we show that the loss of ability of these APCs to generate a first response is not matched by a similar loss in their ability to prime responder T cells for recall responses. Prevention of DNA replication during T cell priming with aphidicolin, a DNA polymerase inhibitor, is not inimical to successful T cell priming. Thus, clonal expansion during priming is less crucial than the primed activation status of T cells for the enhanced recall response. We also show that pentoxifylline, a phosphodiesterase inhibitor, inhibits the primary proliferative response, but its presence during priming enhances the recall response capabilities of T cells. On the other hand, the presence of the calcineurin inhibitor cyclosporin A during priming reduces the efficiency of priming, but at low concentrations it induces, like pentoxifylline, enhancement in recall response capability. These findings have significant implications in designing immunosuppressive therapy and in the analysis of signals for T cell memory commitment.

Modulation of immunogenicity and antigenicity of proteins by maleylation to target scavenger receptors on macrophages.

We have maleylated proteins to target macrophage-specific scavenger receptors and have used this system to study changes in the epitopes and immunogenicity of such proteins. We show that maleylation of diphtheria toxoid (DT) induces targeting to macrophage scavenger receptors and enhances its immunogenicity. DT does not evoke detectable serum Ab responses upon injection as soluble protein. However, maleylated DT (mDT) does generate a significant Ab response. Furthermore, immunization with soluble mDT leads to a better T cell proliferative response in vitro than immunization with DT can generate, thereby demonstrating that maleylation leads to enhanced T cell immunogenicity in vivo. We also find that maleylation disrupts the native B cell epitopes of DT and creates new epitopes, because antisera to DT and mDT do not cross-react. At least some of the new epitopes generated are maleylation specific, because antisera against various maleylated proteins do cross-react. In contrast, maleylation does not significantly modify the repertoire of T cell epitopes generated from DT, because T cells generated by either DT or mDT immunization are cross-reactive, and both DT and mDT can stimulate T cells that are specific for single synthetic DT peptide. Maleylated proteins are better presented in vitro than are their native counterparts, and this enhancement of presentation is blocked by unrelated maleylated proteins. These results suggest that Ags targeted to scavenger receptors on macrophages by maleylation are better presented to T cells and are immunogenic in vivo without adjuvant.

Molecular mimicry by major histocompatibility complex molecules and peptides accounts for some alloresponses.

One explanation offered for the uniquely high precursor frequencies of T cells which recognize allogeneic major histocompatibility complex (MHC) molecules, and their lack of self-MHC restriction, is that the alloreactive cells are polyclonal populations the primary specificity of which is self-MHC plus peptide X1, X2, ... Xn. These are postulated to cross-react with allo-MHC plus peptides Y1, Y2, ... Yn. It has been further suggested that the structural basis for the crossreactivity between different MHC alleles is the similarity in amino acid sequence of that part of the molecule predicted to make contact with the T cell receptor (TcR). In order to test this concept, T cells were obtained with dual specificity for influenza haemagglutinin (HA), restricted by HLA-DR1Dw1, and for DR4Dw4/Dw14 expressed on allogeneic human B cell lines, and the specificity of one clone was studied in detail. The exposed, TcR-contacting surfaces of these two DR molecules are predicted to be identical. Although the HA-specific response was stimulated by DR1-expressing mouse DAP.3 transfectants, DAP.3 cells expressing the alloantigen DR4Dw4 were unable to stimulate, possibly because of a failure to present the necessary human peptide for anti-DR4 allorecognition. Therefore, the effects of pulsing the DR4Dw4-expressing DAP.3 cells with the HA peptide were examined. This peptide is known to bind to both DR1 and DR4. Addition of the HA peptide restored the anti-DR4Dw4 response. These data support the concept that allorecognition in some responder/stimulator combinations can be explained by cross-reactivity at the level of the MHC molecule and the peptide.

Structural analysis of a peptide--HLA class II complex: identification of critical interactions for its formation and recognition by T cell receptor.

An assay for the binding of peptides to major histocompatibility complex (MHC) class II proteins on the surface of cells has been used to determine the relative importance of the amino acids composing an influenza haemagglutinin T cell determinant in binding. The important contact residues were identified by the effect substitution of each residue with biotinylated lysine had on the ability of the peptide to bind. The spacing of the critical residues within the peptide sequence was consistent with the central core, of approximately eight amino acids, adopting a helical conformation. The terminal residues were less constrained and might not be part of a regular conformation. Increasing the helical propensity of the determinant, by simply acetylating and amidating the peptide, resulted in an analogue that was able to stimulate a specific T cell clone at significantly lower concentrations than the natural sequence. A potential location for the peptide in the binding site was postulated based on the presence of complementary amino acids in the class II molecule and supported by screening a large number of peptide analogues for their ability either to bind the restriction element or to stimulate T cell proliferation.

Reversal of HLA restriction by a point mutation in an antigenic peptide.

The HLA restriction of a human T cell clone specific for residues 307-319 of influenza haemagglutinin was changed by the introduction of a point substitution in the peptide. Cells expressing HLA Dw1, DR4 Dw4, Dw13, Dw14, or Dw15 could present the natural haemagglutinin peptide to varying extents to the clone, but DR4 Dw10 could not. Replacement of glutamine-312 of the peptide with arginine produced an analogue that was recognized by the T cell clone only in the context of DR4 Dw10; neither DR1 nor any of the other DR4 alleles could present this peptide to the clone. The inability to bind either the natural or modified peptide was shown not to be the cause of the non-responsiveness. Rather, the differential stimulation of the clone appeared to arise from sequence variations between the DR alleles in residues comprising the beta-chain helix, which either affected recognition by directly contacting the T cell receptor or modified the conformation of the bound peptide. The latter effects were sufficiently subtle to modulate recognition by changing the fine details of the bound peptide but not to eliminate the capacity of the restriction element to bind the peptide.

The phenotypic and molecular characterization of Nb2 lymphoma cells activated with IL-2 and human growth hormone.

The Nb2 rat lymphoma cell line has the unique property that its growth is dependent on lactogenic pituitary hormones. Cell surface staining with monoclonal antibodies showed expression of class I MHC alloantigens of the RT1u haplotype, but no expression of class II MHC antigens. Staining for differentiation markers was strongly positive with antibodies OX52, W3/13 and OX44. Partial and weaker staining was obtained with CD2, P4/16 and the transferrin receptor. Nb2 cells were negative with CD5, OX40 and CD4, whilst CD8 stained only a minor fraction (1%) and certain variant clones of the cell line. This general pattern of staining is consistent with the phenotype of a small subpopulation of immature T cells. Nb2 cells proliferated in response to recombinant human IL-2, although they did not stain with antibodies against the IL-2 receptor. Enhancement of the stimulation by IL-2 in the presence of a submitogenic concentration of hGH indicated a synergism between these two hormones, and responses were suppressed by a similar dose of cyclosporin A (ID50=2 microg/ml). Although IL-2 could not be identified in culture supernatants, the presence of mRNA for IL-2, IL-2R and IL-4 was demonstrated by dot blot analysis. Finally, evidence that the Nb2 lymphoma is of T-cell lineage was given by Northern blot detection of mRNA for the alpha and beta chains of the T-cell receptor.

Prediction and identification of an HLA-DR-restricted T cell determinant in the 19-kDa protein of Mycobacterium tuberculosis.

An allele-specific motif has been identified in the sequence of several peptides which are recognized by T cells in association with HLA-DR1. In order to test the predictive values of such a motif we analyzed the 19-kDa antigen from Mycobacterium tuberculosis and identified a sequence containing a pattern characteristic of DR1 restriction. Peripheral blood mononuclear leukocytes from every DR1 and 4 individual tested responded to the corresponding synthetic peptide. Nine other donors, constituting seven different DR alleles, failed to recognize this sequence. Recognition of the peptide in association with DR1 and DR4 was confirmed using T cell clones and transfected murine L cell lines expressing DR molecules.