Boldine and Laurolitsine Protect against Periodontitis-Associated Bone Loss via RANKL Downregulation.

Nasal application of soluble Ags leads to Ag-specific suppression of systemic immune responses. This tolerance can be transferred to naive mice by CD4(+) regulatory T cells (T(R) cells) from the spleen, but little is known about the induction of mucosal T(R) cells in vivo. To investigate the induction of T(R) cells in the nose-draining cervical lymph node (CLN), CD4(+) T cells from DO11.10 OVA TCR transgenic mice were transferred to BALB/c recipients. Within 48 h after nasal OVA application, CD4(+) DO11.10 T cells in CLN, but not in the peripheral lymph node, had divided. Similarly, nonmucosal (i.m.) OVA application also induced CD4(+) DO11.10 T cells to proliferate in the draining inguinal lymph node (ILN), yet more vigorously and with different kinetics than the CD4(+) DO11.10 T cells in CLN. Functional analysis revealed that only proliferating CD4(+) DO11.10 T cells from CLN, and not ILN, could transfer tolerance to naive recipients. CD4(+) DO11.10 T cells from CLN were phenotypically similar to CD4(+) DO11.10 T cells from ILN, however, in CLN a higher percentage of CD25(+) proliferating CD4(+) DO11.10 T cells were detected compared with ILN. CD25 is not a discriminative marker for mucosal T(R) cells because both CD25(+) and CD25(-) CD4(+) DO11.10 T cells from the CLN could suppress delayed type hypersensitivity responses in adoptive transfer. These findings demonstrate that although striking similarities exist between the differentiation of T(R) and effector T cells, this does not include their function. We are the first to demonstrate that functional T(R) cells, which reside within both CD25(+) and CD25(-) subsets, can be isolated from CLN as early as 3 days after nasal OVA application.

Conclusion. B cells in cervical lymph nodes correspond to typical conventional B cells (B-2). Objective. The special status of cervical lymph nodes in relation to the oropharynx, and the need to maintain the integrity of the oropharnygeal mucosal barrier, suggest the possibility that cervical lymph node B cells located in the oropharynx may behave differently from B cells located elsewhere. In this study we examined the symmetry or lack thereof between cervical lymph node B cells and other B-cell subsets. Material and methods. We isolated B cells from murine cervical lymph node tissue and evaluated them in vitro according to several criteria. Results. We found that cervical lymph node B cells expressed typical B-cell phenotypic markers and proliferated normally in response to mitogenic stimulation. They did not spontaneously secrete immunoglobulin and, in keeping with this, did not express elevated levels of either CD138 (Syndecan-1), a marker for plasma cells, or BLIMP-1, a putative master regulator of B-cell differentiation.

This study aimed to investigate the contribution of myeloid differentiation primary-response gene 88 (MyD88) on the differentiation of T helper type 17 (Th17) and regulatory T (Treg) cells and the emerging subgingival microbiota dysbiosis in Porphyromonas gingivalis-induced experimental periodontitis. Alveolar bone loss, infiltrated inflammatory cells, immunostained cells for tartrate-resistant acid phosphatase (TRAP), the receptor activator of nuclear factor-kB ligand (RANKL), and osteoprotegerin (OPG) were quantified by microcomputerized tomography and histological staining between age- and sex-matched homozygous littermates (wild-type [WT, Myd88+/+] and Myd88-/- on C57BL/6 background). The frequencies of Th17 and Treg cells in cervical lymph nodes (CLNs) and spleen were determined by flow cytometry. Cytokine expression in gingival tissues, CLNs, and spleens were studied by quantitative polymerase chain reaction (qPCR). Analysis of the composition of the subgingival microbiome and functional annotation of prokaryotic taxa (FAPROTAX) analysis were performed. P. gingivalis-infected Myd88-/- mice showed alleviated bone loss, TRAP+ osteoclasts, and RANKL/OPG ratio compared to WT mice. A significantly higher percentage of Foxp3+CD4+ T cells in infected Myd88-/- CLNs and a higher frequency of RORγt+CD4+ T cells in infected WT mice was noted. Increased IL-10 and IL-17a expressions in gingival tissue at D14-D28 then declined in WT mice, whereas an opposite pattern was observed in Myd88-/- mice. The Myd88-/- mice exhibited characteristic increases in gram-positive species and species having probiotic properties, while gram-negative, anaerobic species were noted in WT mice. FAPROTAX analysis revealed increased aerobic chemoheterotrophy in Myd88-/- mice, whereas anaerobic chemoheterotrophy was noted in WT mice after P. gingivalis infection. MyD88 plays an important role in inflammation-induced bone loss by modulating the dynamic equilibrium between Th17/Treg cells and dysbiosis in P. gingivalis-induced experimental periodontitis.

Reactivity to alloantigens and polyclonal mitogens and CD4 +/CD8 + cell ratio shifts of cervical lymph node and spleen cells during pregnancy in rats

We investigated the characteristic features of cervical lymph node B cells to determine whether their behavior differs from that of B cells located elsewhere, because cervical lymph nodes may be exposed to continual antigenic stimulation from the naso- and/or oropharynx. B cells were isolated from cervical lymph nodes, spleen and peritoneal fluid of mice, cultured in medium, and exposed to various stimuli. The expression of various surface molecules characteristic of lymphoid B cells was assayed by flow cytometry, and immunoglobulin secreted into the culture supernatants was evaluated by enzyme-linked immunosorbent assay. B220+ cells were cultured in medium alone or with lipopolysaccharide, and their entrance into S phase in response to stimuli was measured by proliferative assays. Phenotypic characteristics of cervical lymph node B cells included CD5low, CD23high, CD43low, B7.1low, B7.2low, and Syndecan-1low. Unstimulated lymphoid B cells did not secrete immunoglobulin, but, upon stimulation, secretion of IgM was increased more than secretion of IgA and IgG. B cells actively entered S phase after 48 hr stimulation. These results show that B cells in cervical lymph nodes are conventional B2 cells, like splenic B cells.

Therapeutic activity against oral candidiasis of orally administered bovine lactoferrin (LF), a multifunctional milk protein, was shown in a previous report using an immunosuppressed murine model. In the present study, the influence of orally administered LF on immune responses relevant to this therapeutic effect was examined. Because mice were immunosuppressed with prednisolone 1 day before and 3 days after the infection with Candida, the numbers of peripheral blood leukocytes (PBL) and cervical lymph node (CLN) cells were reduced. LF feeding prevented the reduction in the numbers of PBL on day 1 and CLN cells on days 1, 5 and 6 in the Candida-infected mice. The number of CLN cells of individual mice on days 5 and 6 was inversely correlated with the Candida c.f.u. in the oral cavity. Increased production of IFN-gamma and TNF-alpha by CLN cells stimulated with heat-killed Candida albicans on day 6 was observed in LF-treated mice compared with non-treated mice. Concanavalin A (ConA)-stimulated CLN cells from LF-treated mice also showed a significant increase in the production of IFN-gamma and IL12 on day 5 and a tendency for increased production of IFN-gamma and TNF-alpha on day 6. The levels of cytokine production by ConA-stimulated CLN cells on day 6 were inversely correlated with the Candida c.f.u. in the oral cavity. In conclusion, the alleviation of oral candidiasis by LF feeding in this model may correlate with the enhancement of the number of leukocytes and their cytokine responses in regional lymph nodes against Candida infection.

Evaluation of morphological, histological, and immune-related cellular changes in ligature-induced experimental periodontitis in mice

ALVEOLAR BONE LOSS AND TOOTH LOSS IN MALE CIGAR AND PIPE SMOKERS

The detection of disseminated tumor cells in differentiated (DTC) and medullary thyroid carcinomas (MTC) is one of the main topics in current thyroid cancer research. Immunocytochemistry and polymerase chain reaction (PCR) provide the tools for the identification of a small number of thyroid cancer cells in peripheral blood and cervical lymph nodes. Thyroid-specific markers, such as thyroglobulin (Tg) mRNA and thyroid peroxidase (TPO) mRNA, have been detected with RT-PCR in blood samples of tumor patients and healthy control subjects. To prevent false-positive results, quantitative PCR systems were established. Tumor-specific markers, such as telomerase activity and cytokeratin 20 (CK20), have been detected in various epithelial tumors. Amplification products of these markers were found in blood samples and in fine-needle aspiration (FNA) biopsies of patients with thyroid carcinomas. Using molecular detection of disseminated tumor cells in cervical lymph nodes with CK20 RT-PCR, a higher percentage of involved lymph nodes was detected compared to immunohistochemistry. The results of the presented studies may help researchers to develop more sensitive methods for early tumor cell dissemination, and refine risk groups that might benefit from more extensive surgical procedures or adjuvant therapy. However, the prognostic value of minimal residual disease (MRD) in thyroid carcinoma has to be confirmed in large or multicenter prospective studies.

The purpose of this paper is two-fold: (1) to review the evidence that osteoporosis and post-menopausal estrogen deficiency are associated with progressive alveolar bone loss and an elevated risk of tooth loss; and (2) to propose the use of tetracyclines, specifically low-dose doxycycline (LDD) (and, perhaps in the future, the chemically modified tetracyclines), to mitigate alveolar bone loss in post-menopausal osteoporotic/osteopenic women. Design concepts for a randomized clinical trial to study the effects of LDD on progressive alveolar bone loss in this patient population are reviewed. Since osteoporosis affects over 20 million people in the United States, progressive alveolar bone loss in this patient group represents a potentially significant public health problem unique from common adult periodontitis. Stopping progressive alveolar bone loss is essential to prevent both tooth loss and micro-architectural deterioration of alveolar bone.

BackgroundSublingual immunotherapy (SLIT) is an established efficacious approach for the treatment of allergic rhinitis (AR). However, SLIT requires a long administration period to establish stable and adequate responses. This study investigated the efficacy of the sublingual administration of an allergen with liposomes enclosing α-GalCer (α-GC-liposome) as a potential adjuvant in mice with AR. MethodsMice with AR induced by OVA received the sublingual administration of OVA, α-GC-liposomes, or OVA plus α-GC-liposomes for 7 days. After nasal re-challenge with OVA, nasal symptoms were evaluated. The serum levels of OVA-specific Ig, the cytokine production of CD4+ T cells in the cultures of cervical lymph node (CLN) cells, and the gene expression of CLNs were analyzed. ResultsAlthough IL-4, IL-5 and IL-13 production from CD4+ T cells in CLN cells was significantly inhibited by the sublingual administration of OVA alone in mice with AR induced by OVA, their nasal symptoms were not significantly diminished. However, the combined sublingual administration of α-GC-liposomes and OVA completely suppressed nasal symptoms, downregulated Th2 and Th17 type cytokine production in CD4+ T cells as well as Th2 and Th17 gene expressions, and upregulated Th1 type cytokine production as well as Th1 gene expressions in CLN cells. Additionally, the serum levels of specific IgG2a were promoted, and specific IgE and IgG1 were inhibited. ConclusionsOur findings suggest that the sublingual administration of an allergen with α-GC-liposomes as an adjuvant might increase the therapeutic efficacy and effectiveness of this treatment method.

Automatic methods for alveolar bone loss degree measurement in periodontitis periapical radiographs

Intranasal (i.n.) immunization with bacterial protein antigens coupled to cholera toxin B subunit (CTB) effectively induces mucosal, especially salivary immunoglobulin A (IgA), and nonmucosal antibody responses in mice. To examine the regional distribution of antigen-specific B and T cells after i.n. immunization, antibody-secreting cells and antigen-responsive T cells in cervical lymph nodes (CLN) were compared with those found after intraoral or subcutaneous (in the neck) administration of the same antigen and with T cells found in mesenteric lymph nodes (MLN) and spleen after intragastric immunization. The i.n. immunization induced predominantly IgA antibody-secreting cells in salivary glands and IgA and IgG antibody-secreting cells in the superficial and central CLN; these responses were quantitatively enhanced if the antigen was coupled to CTB. Intraoral immunization also induced IgA and IgG antibody-secreting cells in the superficial and central CLN, but only if intact cholera toxin was included as an adjuvant. In contrast, subcutaneous (neck) immunization induced IgG antibody-secreting cells mainly in the draining facial lymph nodes. CLN cell populations resembled those of MLN, except that CLN lymphocytes had higher proportions of T cells and lower proportions of B cells and a slightly higher CD4+/CD8+ ratio among T cells than the MLN lymphocytes did. T cells that proliferated in response to antigen in vitro were found especially in central CLN 2 days after i.n. immunization and persisted for up to 6 months, whereas after intragastric immunization, responsive T cells were not found in the MLN for up to 14 days. After culture with antigen in vitro, T cells from the superficial CLN of i.n. immunized mice secreted both gamma interferon and interleukin-4. Therefore, after i.n. immunization, superficial and central CLN represent sites of regional lymphocyte development, and the central CLN in particular appear to be sites where memory T cells persist.

MMP-9 deficiency accelerates the progress of periodontitis

Introduction: Periodontitis is an inflammation affecting the supporting tissue of the teeth. It is characterized by periodontal ligament destruction and progressive alveolar bone loss. Periodontitis is often induced in laboratory animal models, such as Wistar rats used for experimental models. This study was conducted to develop a reproducible technique for induction of periodontitis in the mandible of Wistar rats using a modified ligation technique with an additional injection of Aggregatibacter actinomycetemcomitans. Context: Periodontitis is an inflammation affecting the supporting tissue of the teeth. It is characterized by periodontal ligament destruction and progressive alveolar bone loss. Aims: The aim of this study was to develop a reproducible technique for induction of periodontitis in the mandible of Wistar rats using a modified ligation technique with an additional injection of A. actinomycetemcomitans. Settings and Design: The study was conducted at LPPT Unit IV Universitas Gadjah Mada, Yogyakarta. Subjects and Methods: Ten 2-month-old male adult Wistar rats were used as experimental subjects with an average weight of 150–200 g. Nonresorbable silk ligature wire (4/0) was used as ligatures and placed with an “8”-shaped knot technique. The ligatures were placed in between both central incisors of the mandible for 7 days with additional injections of A. actinomycetemcomitans on the 1st day. The procedure was performed under ketamine anesthesia. After 7 days, ligatures were removed and the animal subjects were euthanized with cervical dislocation method and samples of mandibles were preserved in formalin solution and processed histologically and radiographically. Results: Modified induction technique of periodontitis with ligatures and additional injection of A. actinomycetemcomitans showed significant clinical inflammation, periodontal ligament widening, attachment loss, and alveolar bone loss. Conclusions: We demonstrated clinical, radiographic, and histological evaluation from this modified induction technique and concluded that it has several advantages: shorter period of induction time with more significant clinical changes and advanced bone loss, simplifying the technique for induction of periodontitis.