Streptococci and Actinomyces induce antibodies which cross react with epithelial antigens in periodontitis

Perturbation of epithelial structure is a prominent but poorly understood feature of the immunopathological response to bacterial antigens which characterizes the destructive lesion of periodontitis. Western analysis of sera from 22 patients with periodontitis detected multiple antigens in extracts of epithelial cells whereas sera from 12 periodontally healthy subjects displayed only trace reaction with epithelial antigens. To investigate a possible relationship between the bacterial flora adjacent to diseased sites and the presence of antibodies reactive with epithelium, subgingival plaque samples were taken from deep periodontal pockets and cultured anaerobically. Gram positive bacteria containing antigens cross-reactive with epithelial cells were reproducibly isolated by probing membrane colony-lifts with affinity-isolated (epithelium-specific) antibodies and identified by 16S rDNA sequence homology as streptococci (S. mitis, S. constellatus and two S. intermedius strains) and Actinomyces (A. georgiae, and A. sp. oral clone). Conversely, when serum from patients with periodontitis was absorbed with the captured bacterial species the number of epithelial antigens recognized was specifically reduced. It was concluded that development of cross-reactive antibodies related to these organisms may contribute to perturbation of the epithelial attachment to the tooth and the progression of periodontitis. These autoreactive antibodies could also be a contributing factor in other diseases affecting epithelia.     

Interleukin-6 expression and gene polymorphism are associated with severity of periodontal disease in a sample of Brazilian individuals

Interleukin (IL)-6 is an inflammatory mediator involved in bone resorption. G/C polymorphism at position -174 of the IL-6 gene has been reported to influence IL-6 expression, with the G allele associated with higher expression levels. The aims of this study were to investigate the expression of IL-6 as well as the incidence of IL-6 (-174) gene polymorphism and their correlation to the severity of periodontitis in Brazilians. Peripheral blood mononuclear cells were collected from 12 non-smoker individuals with periodontitis for evaluation of IL-6 expression using flow cytometry. We observed a positive correlation between the mean clinical attachment loss and intensity of expression of IL-6, in which the greater the attachment loss, the higher the expression of IL-6 (P=0 x 007, R2=0 x 52). Also, patients with severe periodontitis displayed a higher intensity of IL-6 expression compared to moderate periodontitis (P=0 x 04). To determine the occurrence of IL-6 gene polymorphism, DNA was obtained from oral swabs of 209 Brazilian individuals with and without periodontitis. Polymerase chain reaction, restriction endonuclease digestion and electrophoresis were performed, allowing for detection of the IL-6 (-174) polymorphism. We observed that non-smokers with moderate periodontitis (P=0 x 05) and control (P=0 x 04) groups displayed a higher incidence of the G genotype when compared to severe periodontitis. This suggests that the G genotype may represent a protective role in severity of periodontitis. Thus, the increased expression of IL-6 and IL-6 (-174) polymorphism are associated with periodontal disease severity in Brazilian individuals.     

Identification of an epitope from the epithelial cell adhesion molecule eliciting HLA-A*2402-restricted cytotoxic T-lymphocyte responses

Because the epithelial cell adhesion molecule (Ep-CAM) is expressed in almost all carcinomas and human leucocyte antigen (HLA)-A*2402 is the most common allele in many ethnic groups, including Japanese, the identification of peptide sequences, which elicit HLA-A*2402-restricted Ep-CAM-specific cytotoxic T-lymphocyte (CTL) responses, would facilitate specific immunotherapy for various histological types of carcinomas. An epitope was identified through the following steps: (i) computer-based epitope prediction from the amino acid sequence of Ep-CAM, (ii) major histocompatibility complex (MHC) stabilization assay to determine the affinity of the predicted peptide with HLA-A*2402 molecules, (iii) stimulation of CD8+ T cells with peptide-pulsed dendritic cells and (iv) testing the CTL specificity by means of enzyme-linked immunospot (ELISPOT) assays, CTL assays and MHC/peptide-tetramer staining. Peripheral CD8+ T cells of four of five healthy donors after three rounds of stimulation with the peptide Ep-CAM173-181 (RYQLDPKFI) secreted interferon-gamma in ELISPOT assays when exposed to the peptide. A CTL clone specific to the peptide efficiently lysed Ep-CAM-expressing cancer cell lines in an HLA-A*2402-restricted fashion. Endogenous processing and presentation of the peptide in a lung cancer cell line were confirmed by means of cold target inhibition assays. The CTL clone was also lytic to normal bronchial epithelial cells but to a lesser extent at low effector: target ratios. All these data suggest that the peptide-specific CTL responses may play some roles both in anti-cancer and autoimmune reactions. The peptide should prove useful to study anti-Ep-CAM CTL responses among population possessing HLA-A*2402.     

Therapeutic intervention with inhibitors of co-stimulatory pathways in autoimmune disease

Many humanized antibodies and fusion proteins targeting T-cell co-stimulatory molecules are now in late-stage clinical development (phase II, phase III) or have recently completed phase III clinical trials. Both Amevive, an LFA-3-Ig fusion protein targeting CD2, and Xanelim, a humanized anti-CD11a antibody, have shown efficacy in pivotal phase III trials in patients with plaque psoriasis. These new medicines are poised to enter clinical use in 2002.     

Local production of IgA- and IgM-rheumatoid factors in adult periodontal disease

The enzyme-linked immunospot assay was used to enumerate both the number and the frequency of spontaneous IgG, IgA, and IgM immunoglobulin-secreting cells and IgA- and IgM-rheumatoid factor (RF)-producing cells present in the gingivae and peripheral blood of adult periodontitis patients. Cells from 29 patients were incubated on plates coated with human IgG, Fc, or F(ab)₂ fragments and on plates coated with class-specific antihuman antibodies and secreted antibodies were subsequently visualized by means of an immunoenzymatic procedure. The data indicate that (1) IgA-RF- and IgM-RF-secreting cells are frequently present in the gingiva of adult periodontitis patients; (2) production of RF in gingivae of adult periodontitis patients occurs in the absence of demonstrable RF production by simultaneously obtained peripheral blood mononuclear cells, suggesting that local autoimmune reactions may occur in this disease; and (3) lack of correlation between IgA-RF and IgM-RF production in diseased gingiva suggests that the two RF isotypes are regulated independently of each other.     

Differential expression of costimulatory molecules in chronic inflammatory periodontal disease tissue

Although B cell activation and subsequent immunoglobulin production are the immunopathological features of chronic inflammatory periodontal disease, in situ expression of costimulatory molecules in humoral immunity has not been investigated. In the present study we examined the expression of CD40, CD40 ligand (CD40L), CD80, CD86, CD28 and cytolytic T lymphocyte-associated antigen-4 (CTLA-4) on lymphocytes immunohistochemically. Cryostat sections were prepared from the gingival tissue samples of 14 patients with moderate to advanced adult periodontitis. In vitro kinetics of the expression of CD40L and CTLA-4 by peripheral blood T cells and that of CD80 and CD86 by peripheral blood B cells were also investigated by flow cytometry. Positive percentage expression of CD40L, CD28 and CTLA-4, and CD40, CD80 and CD86 was calculated for the number of CD3⁺ and CD19⁺ cells, respectively. Flow cytometric analysis demonstrated that the expression of CD40L and CTLA-4 on T cells, and CD80 and CD86 on B cells of peripheral blood was up-regulated upon activation. While most T cells and B cells expressed CD28, and CD80 and CD86, respectively, in gingival tissues, the expression of CD40L and CTLA-4 was lower but highly variable between specimens. Furthermore, these two molecules seemed to be expressed reciprocally in the lesion. As both CD40L and CTLA-4 expression are induced transiently by stimulation, variability in the expression of the molecules may reflect immunological activities and participation in the regulation of B cell activation of the lesion.     

Effect of non surgical periodontal therapy on gingival crevicular fluid and serum visfatin concentration in periodontal health and disease

Visfatin is a pleiotropic mediator which acts as growth factor, cytokine, enzyme involved in energy including nicotinamide adenine dinucleotide metabolism and has been recently demonstrated to exert several pro-inflammatory functions. The purpose of this study is to evaluate the Visfatin concentration in gingival crevicular fluid (GCF) and serum in patients with chronic periodontitis, and to evaluate the effect of non-surgical periodontal therapy on the GCF and serum visfatin concentration. 30 subjects (age range: 25 to 52 years) were selected and divided into two groups based on the gingival index, probing depth, periodontal attachment level, and radiologic parameters (bone loss): group 1 (15 subjects with healthy periodontium), group 2 (15 subjects with chronic periodontitis), while, Group 2 patients after 8 weeks of the treatment (scaling and root planning, SRP) constituted group 3. GCF samples (by microcapillary pipettes) and serum samples (by venipuncture) were collected to estimate the levels of Visfatin using enzyme linked immunosorbent assay kit. The mean Visfatin concentration in GCF and serum was observed to be the highest in group 2 and lowest in group 1. While concentration in group 3 was similar to group 1. The concentration of Visfatin in GCF and serum decreased after SRP. The Visfatin concentration in GCF and serum found to be highest in chronic periodontitis group and decreases after treatment. Hence Visfatin values can be considered as an "inflammatory marker" can be explored in future as a potential therapeutic target in the treatment of periodontal disease.     

The immune responses to human and microbial heat shock proteins in periodontal disease with and without coronary heart disease

The human 60 kDa and microbial 65 kDa heat shock proteins (HSP) have been implicated in the pathogenesis of chronic periodontitis (P) and coronary heart disease (CHD). We have studied four male non-smoking cohorts of 81 subjects, matched for age. Group (a) consisted of a healthy group with minimal gingivitis (n = 18), group (b) were patients with P (n = 23), group (c) patients with CHD and minimal gingivitis (n = 20) and group (d) patients with CHD and P (n = 20). T cells separated from peripheral blood were found to be primed to both microbial HSP65 and human HSP60 but significant CD4, human leucocyte antigen (HLA) class II-restricted proliferative responses were found only with the human HSP60 in patients with P (P < 0.001) and CHD without (P < 0.001) or with (P < 0.00001) periodontitis. Dose-dependent inhibition of T cell proliferative responses was carried out to determine the receptors involved in recognition of HSP60 and HSP65. Monoclonal antibodies to CD14 showed inhibition of T cell proliferation stimulated by both HSP60 and HSP65, consistent with the role of CD14 as a receptor for these HSPs in P and CHD. The toll-like receptor 2 (TLR-) and TLR-4 were then studied and these showed that TLR-4 was recognized by microbial HSP65, whereas TLR-2 was recognised by human HSP60 in both P and CHD. However, a dissociation was found in the HSP60 and TLR4 interaction, as TLR4 appeared to have been recognized by HSP60 in P but not in CHD. The results suggest an autoimmune or cross-reactive CD4(+) class II-restricted T cell response to the human HSP60 in P and CHD. Further studies are required to determine if there is a common epitope within HSP60 that stimulates T cell proliferation in P and CHD.     

Immunolocalization of CD1d in human intestinal epithelial cells and identification of a beta2-microglobulin-associated form

In order to better understand the role of intestinal CD1d, we sought to define the cellular localization and further characterize the biochemical structure of CD1d in human intestinal epithelial cells (IEC). Using a CD1d-specific rabbit anti-gst-CD1d antibody, immunoprecipitation of radiolabeled cell surface proteins detected a previously identified 37 kDa protein as well as a 48-50 kDa protein which were confirmed by Western blotting with a CD1d-specific mAb, D5. Immunoprecipitation of protein lysates with the CD1d-specific mAb, D5 and 51.1.3, and the beta2-microglobulin (beta2m)-specific mAb, BBM.1, followed by N-glycanase digestion and Western blotting with the D5 mAb showed that the 48-50 kDa protein was a beta2m-associated, CD1d glycoprotein. CD1d was immunolocalized to the apical and lateral regions of native small and large intestinal IEC as defined by confocal laser microscopy using the D5 mAb and the rabbit anti-gst-CD1d antibody. In addition, a large apical intracellular pool of CD1d was identified. Identical observations were made with polarized T84 cells. Selective biotin labeling of apical and basolateral cell surfaces followed by immunoprecipitation with the D5 mAb, N-glycanase digestion and avidin blotting confirmed the presence of glycosylated CD1d on both cell surfaces and immunolocalization of the 37 kDa non-glycosylated form of CD1d to the apical cell surface. These studies show that CD1d is located in an ideal position for luminal antigen sampling and presentation to subjacent intraepithelial lymphocytes.     

Down-regulation of Pax6 is associated with abnormal differentiation of corneal epithelial cells in severe ocular surface diseases

Pax6 is the universal master control gene for eye morphogenesis. Other than retina and lens, Pax6 also expressed in the ocular surface epithelium from early gestation until the postnatal stage, in which little is known about the function of Pax6. In this study, corneal pannus tissues from patients with ocular surface diseases such as Stevens-Johnson syndrome (SJS), chemical burn, aniridia and recurrent pterygium were investigated. Our results showed that normal ocular surface epithelial cells expressed Pax6. However, corneal pannus epithelial cells from the above patients showed a decline or absence of Pax6 expression, accompanied by a decline or absence of K12 keratin but an increase of K10 keratin and filaggrin expression. Pannus basal epithelial cells maintained nuclear p63 expression and showed activated proliferation, evidenced by positive Ki67 and K16 keratin staining. On 3T3 fibroblast feeder layers, Pax6 immunostaining was negative in clones generated from epithelial cells harvested from corneal pannus from SJS or aniridia, but positive in those from the normal limbal epithelium; whereas western blots showed that some epithelial clones expanded from pannus retained Pax6 expression. Transient transfection of an adenoviral vector carrying EGFP-Pax6 transgenes into these Pax6(-) clones increased both Pax6 and K12 keratin expression. These results indicate that Pax6 helps to maintain the normal corneal epithelial phenotype postnatally, and that down-regulation of Pax6 is associated with abnormal epidermal differentiation in severe ocular surface diseases. Reintroduction of activation of the Pax6 gene might be useful in treating squamous metaplasia of the ocular surface epithelium.     

B-lymphocyte contributions to human autoimmune disease

Summary: Autoimmunity results from abnormal B- and T-cell recognition of self-antigens, which leads to autoantibody production in many cases. Autoantibodies produced by B-cell-derived plasma cells provide diagnostic markers for autoimmunity but also contribute significantly to disease pathogenesis. As discussed in this review, the therapeutic benefit of depleting B cells in mice and humans has refocused attention on B cells and their role in autoimmunity beyond autoantibody production. B cells specifically serve as cellular adjuvants for CD4⁺ T-cell activation, while regulatory B cells, including those that produce interleukin-10 (B10 cells), function as negative regulators of inflammatory immune responses. The emerging picture is that B cells, autoantibodies, and T cells are all important components of abnormal immune responses that lead to tissue pathology unique to each autoimmune disease, with their relative contributions changing during disease progression. Autoimmune diseases where B-cell functions are closely correlated with disease activity include systemic lupus erythematosus, rheumatoid arthritis, scleroderma, type 1 diabetes, and multiple sclerosis. Understanding the overlapping roles of B cells as mediators of autoimmune disease will facilitate the development of more precisely directed therapies and combination therapies with broader clinical efficacy than current depletion strategies that remove all B cells.     

New insights into the emerging role of oral spirochaetes in periodontal disease

Spirochaetes are prominent in the polymicrobial infections that cause periodontal diseases. Periodontitis is a chronic inflammatory condition of the periodontium, characterized by proinflammatory soft tissue damage and alveolar bone loss. Treponema denticola is the most well-understood oral spirochaete, expressing a wealth of virulence factors that mediate tissue penetration and destruction as well as evasion of host immune responses. This review focuses on emerging knowledge of virulence mechanisms of Treponema denticola as well as mechanisms of other less-studied oral treponemes.     

Identification of Bacteroides species from adult periodontal disease

Samples from deep (4-7 mm) periodontal pockets were collected from 17 patients with adult-type periodontal disease and one with the juvenile form of the disease. They were streaked immediately on selective and non-selective media and incubated anaerobically for 96 h. There was a heavy growth of Bacteroides spp. from most samples and 10 representative colonies from each sample were sub-cultured for identification. In a total of 149 isolates from patients with adult-type disease, the commonest species were B. oralis (40), B. asaccharolyticus (35), B. intermedius (31), B. fragilis (12) and B. ureolyticus (10); B. gingivalis was not detected. The distribution of species was not distorted by multiple identical isolates from individual patients. There was a heavy growth of a single species, B. ureolyticus, from the patient with juvenile-type disease.     

Evaluation of serum resistin levels in periodontal health and disease and effects of non surgical periodontal therapy on its levels

Background: Resistin and adiponectin are the adipokines secreted by adipocytes and various inflammatory cells. These adipokines are known to play an important role in insulin resistance. The aim of this study was to determine the serum resistin levels in periodontal health and disease and also, to determine the effect of nonsurgical periodontal therapy on its levels. Methods: A total of 40 patients (20 Males and 20 Females; age range 20–50 years) participated in the study. Subjects were categorized as healthy (group 1; Controls) and chronic periodontitis (group 2; Study) groups based on their periodontal status. Periodontal parameters (Plaque index (PI), Gingival index (GI), Bleeding index (BI), Probing pocket depth (PPD), Clinical attachment loss (CAL)) together with serum resistin levels were assessed at baseline and between 6–8 weeks following nonsurgical periodontal therapy for subjects in group~2 and only at baseline in group 1. Sera were tested in duplicate (single run), and the results were averaged. Results: Study group showed higher (1.89 ± 1.83 ng/ml) serum resistin levels, compared to control group (1.35 ± 0.70 ng/ml). However, this difference was not statistically significant (P=0.227). Also, resistin levels decreased following nonsurgical periodontal therapy but, this decrease failed to show any statistical significance, with pretreatment levels being 1.89 ± 1.83 ng/ml and post treatment levels being 1.59 ± 1.01 ng/ml (P=0.386). Conclusion: Observations of the present study revealed that there was not much difference in the serum resistin levels between the cases and the controls. Also the decrease in the resistin levels following nonsurgical periodontal therapy did not show any statistical significance.     

Sialic acids and autoimmune disease

An important underlying mechanism that contributes to autoimmunity is the loss of inhibitory signaling in the immune system. Sialic acid-recognizing Ig superfamily lectins or Siglecs are a family of cell surface proteins largely expressed in hematopoietic cells. The majority of Siglecs are inhibitory receptors expressed in immune cells that bind to sialic acid-containing ligands and recruit SH2-domain-containing tyrosine phosphatases to their cytoplasmic tails. They deliver inhibitory signals that can contribute to the constraining of immune cells, and thus protect the host from autoimmunity. The inhibitory functions of CD22/Siglec-2 and Siglec-G and their contributions to tolerance and autoimmunity, primarily in the B lymphocyte context, are considered in some detail in this review. The relevance to autoimmunity and unregulated inflammation of modified sialic acids, enzymes that modify sialic acid, and other sialic acid-binding proteins are also reviewed.     

Corneal epithelial stem cells in health and disease

The cornea on the front surface of the eye provides our window to the world. Maintenance of corneal transparency is dependent on the integrity and functionality of the outermost corneal epithelium which itself is maintained throughout life by a population of limbal epithelial stem cells (LESC). If this adult stem cell population is depleted by injury or disease, the transparency of the cornea and therefore vision is threatened. LESC deficiency results in corneal opacification, inflammation, vascularization, and severe discomfort. Cultured LESC therapy is one of only several examples of the successful use of an adult stem cell therapy in patients. Hence, the ready accessibility of a transparent stem cell niche and the clinical precedence for use of stem cell therapy make the cornea a unique and excellent model for the study of adult stem cells in health and disease. This review will discuss our current understanding of LESC biology, pathology, and therapeutic application.     

不同类型乳腺组织中CD44~+/CD24~-细胞的数量与分布

目的检测CD44+/CD24-细胞在乳腺正常组织及良恶性肿瘤中的数量与分布特点,探讨其在乳腺癌发生、发展中的作用。方法采用免疫组化SP双染法,检测了30例乳腺正常组织(normal tissue,NT),30例乳腺纤维腺瘤(fibroadenoma,FA),60例乳腺浸润性导管癌(invasive ductal carcinoma,IDC)中的CD44及CD24的共表达情况。结果在乳腺NT及FA中,可见CD44+/CD24+,CD44+/CD24-,CD44-/CD24-3种表型,而在IDC中,除上述3种表型外,尚可见CD44-/CD24+表型。从NT,FA到IDC,CD44+/CD24-细胞的阴性率(-,阳性率1%)依次降低(40.0%→36.7%→35.0%);而强阳性率(,阳性率60%)依次升高(0.0→6.7%→21.7%)。结论 CD44+/CD24-可能作为一种阶段性祖细胞标记,参与了正常乳腺的分化及乳腺肿瘤的发展过程。