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T regulatory (Treg) cells have a key role in immune homeostasis and the pathogenesis of chronic inflammatory and autoimmune diseases. CD69 is an early leukocyte activation molecule that under steady state conditions is detected in a small proportion of lymphocytes in peripheral blood and lymphoid tissues. Although it has been reported that a subset of CD69 + T cells behaves as Treg lymphocytes, the possible relationship between CD69 + Treg cells and CD4 + NKG2D + T lymphocytes, which also exert immunosuppressive activity, has not been explored. In this study, we analyzed the expression of CD69 and NKG2D by T lymphocytes from the peripheral blood of twenty-five healthy subjects by multi-parametric flow cytometry analysis, and their suppressive activity by an assay of inhibition of lymphocyte activation (CD40L expression) and proliferation (carboxyfluorescein partition assay). We found a very small percentage of CD4 + CD69 + NKG2D + T cells (median 0.002%, Q 1 – Q 3 , 0.001–0.004%), which also expressed TGF-β (Latency Associated Peptide or LAP) and IL-10, in all samples analyzed. These cells exerted an important in vitro suppressive effect on both activation and proliferation of T effector cells. Our data suggest that at very small numbers, CD4 + CD69 + NKG2D + lymphocytes seem to exert a relevant functional immune-regulatory role in healthy subjects. [ABSTRACT FROM AUTHOR]

Purpose: Peripheral helper T (Tph) cells have an important role in the induction of humoral immune responses and autoantibody production. Accordingly, it is feasible that this lymphocyte subset has a relevant role in the pathogenesis of autoimmune thyroid diseases (AITD). In this study we aim to analyze the levels and function of Tph cells in blood samples from patients with AITD., Methods: We performed an observational study with cases and controls. Blood samples were obtained from nineteen patients with Hashimoto's thyroiditis (HT), twenty-four with Graves' disease (GD), and fifteen healthy controls. In addition, the levels of follicular T helper (Tfh) cells and Tph cells, the release of interleukin-21 (IL-21) by these lymphocytes and the number of plasmablasts were analyzed by multi-parametric flow cytometry analyses., Results: Increased percentages of Tfh and Tph lymphocytes were detected in patients with HT and GD. Furthermore, an enhanced synthesis of the cytokine IL-21 by these cells was observed. Accordingly, we detected significant higher percentages of plasmablasts in patients with GD, and these values tended to be also higher in HT patients. Moreover, significant positive associations were observed between the levels of Tfh or Tph and the number of plasmablast or anti-TSHR Ab titers in patients with AITD., Conclusion: Our data suggest that Tph lymphocytes may have a relevant role in the pathogenesis of AITD., (© 2024. The Author(s).)

Periapical lesions are common pathologies affecting the alveolar bone, often initiated by intraradicular lesions resulting from microbial exposure to dental pulp. These microorganisms trigger inflammatory and immune responses. When endodontic treatment fails to eliminate the infection, periapical lesions persist, leading to bone loss. The RANK/RANKL/OPG pathway plays a crucial role in both the formation and the destruction of the bone. In this study, the objective was to inhibit the RANK/RANKL pathway in vitro within exposed Thp-1 macrophages to endodontic microorganisms, specifically Enterococcus faecalis , which was isolated from root canals of 20 patients with endodontic secondary/persistent infection, symptomatic and asymptomatic, and utilizing an α-IRAK-4 inhibitor, we introduced endodontic microorganisms and/or lipoteichoic acid from Streptococcus spp. to cellular cultures in a culture plate, containing thp-1 cells and/or PBMC from patients with apical periodontitis. Subsequently, we assessed the percentages of RANK+, RANKL+, and OPG+ cells through flow cytometry and measured the levels of several inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) in the cellular culture supernatant through a CBA kit and performed analysis by flow cytometry. A significant difference was observed in the percentages of RANK+RANKL+, OPG+ RANKL+ cells in thp-1 cells and PBMCs from patients with apical periodontitis. The findings revealed significant differences in the percentages of the evaluated cells, highlighting the novel role of the IRAK-4 inhibitor in addressing this oral pathology, apical periodontitis, where bone destruction is observed.

We sought to evaluate the effect of endodontic-causative microorganisms of primary infections on mononuclear cells such as CD14 + , CD4 + , CD8 + , CD19 + and Tregs Foxp3 + . Facultative anaerobic microorganisms were isolated from radicular conducts and peripheral blood samples, which were taken from patients with primary infections. Cellular cultures were performed with peripheral blood mononuclear cells (PBMC) with and without Actinomyces spp. and Streptococcus spp. during 48, 72, and 96 h of contact in culture (concentration 5 × 10 5 cells/well) in a round plate bound with 48 wells. Later, PBMC was collected for analysis by flow cytometry, with the monoclonal antibodies αCD14, αCD4, αCD8, αCD19 and αFoxp3, and acquired using an FACSCanto II cytometer. The supernatant of cellular cultures was analyzed for the quantification of inflammatory cytokines. Data analysis was performed in FlowJo v10.8.2 and FCAPArray software, and statistical analysis was performed using GraphPad v5.0. software. We observed an increase in the percentage of CD14 + cells in patients at different hours of cellular culture in the presence of both Actinomyces spp. and Streptococcus spp. microorganisms, compared to healthy controls. This study demonstrates the role played by the innate immune system in the pathogeny of endodontic primary infections, explaining the effects that generate the more common microorganisms in this oral pathology.

Rheumatoid arthritis (RA) is a chronic autoimmune condition characterized, among others, by tissue damage and activation/differentiation of proinflammatory lymphocytes. Accordingly, several studies have concluded that type 17 T helper (Th17) cells seem to have an important role in the pathogenesis of this condition. However, the strategy for the identification and analysis of proinflammatory Th17 cells in those studies has not been consistent and has usually been different from what was originally described. Therefore, we decided to evaluate the levels of Th17 cells in patients with RA employing an extended immune phenotype by using an eight-color multiparametric flow cytometry analysis. For this purpose, blood samples were obtained from 30 patients with RA and 16 healthy subjects, and the levels of Th17 and type 22 helper (Th22) lymphocytes were analyzed as well as the in vitro differentiation of peripheral blood mononuclear cells into Th17 lymphocytes induced by interleukin-23 (IL-23) and IL-1 β . We found significant enhanced levels of total Th17 lymphocytes (defined as CD4 + IL-17 + ) as well as enhanced numbers of their pathogenic (defined as CD4 + CXCR3 + IL-17 + IL-22 + CD243 + CD161 + IFN- γ + IL-10 - ) and nonpathogenic (CD4 + CXCR3 + IL-17 + IL-22 - CD243 - CD161 - IFN- γ - IL-10 + ) cell subsets in patients with RA. Likewise, the number of Th22 (CD4 + CXCR3 +/- IL-17 - IL-22 + ) was also increased in these patients compared to healthy controls. However, the in vitro induction/differentiation of pathogenic Th17 cells showed similar results in controls and patients with RA. Likewise, no significant associations were detected in patients with RA between the levels of Th17 or Th22 cells and clinical or laboratory parameters. Our data indicate that patients with RA show enhanced blood levels of the different subsets of Th17 cells and Th22 lymphocytes tested in this study and suggest that these levels are not apparently associated with clinical or laboratory parameters., Competing Interests: MV-N, BH-C, DA-H, LB, SB-S, CA-M, PN-M, and RG-A have nothing to declare., (Copyright © 2022 Marlen Vitales-Noyola et al.)

A low-grade inflammatory phenomenon is a feature of overweight and metabolic syndrome. The involvement of a pro-inflammatory Th17 lymphocyte subset and the CD69+ T regulatory (Treg) cell subtype in patients with metabolic dysfunction associated with or without overweight has not been fully elucidated. The aim of this study was to perform a quantitative and functional analysis of pathogenic Th17 lymphocytes and CD69+ Treg cells in patients with metabolic dysfunction (insulin resistance and dyslipidemia). The number of pathogenic Th17 cells and the levels and function of CD69+ Treg cells were analyzed in blood samples from individuals with metabolic dysfunction, associated with or without overweight. Pathogenic and non-pathogenic Th17 lymphocytes as well as Th22 cells were determined by eight-color flow cytometry analysis, whereas the levels and suppressive function of CD69+ Treg cells were also analyzed by multiparametric flow cytometry. We detected increased levels of pro-inflammatory Th17 pathogenic cells and Th22 lymphocytes in overweight unhealthy individuals (P < 0.001, compared to normal weight healthy). Conversely, diminished numbers of CD69+ Treg lymphocytes were observed in metabolically unhealthy individuals, with or without overweight. Likewise, the immunosuppressive function of CD69+ Treg cells was also defective in these patients. The increased levels of pathogenic Th17 cells along with a diminished number and function of CD69+ Treg lymphocytes may significantly contribute to the low-grade inflammatory phenomenon of metabolically unhealthy patients., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Secondary/persistent infections are present in teeth with previous root canal treatment, and a great variety of anaerobic microorganisms has been observed in these infections. The antibiotics prescription is occasional; however, in some cases, it is essential, particularly when the general health condition of the patient is compromised. The aims of this study were to identify the associated microorganisms in diagnosis of persistent or secondary apical periodontitis and to evaluate the susceptibility level to the antibiotics most used. Fifteen patients with persistent or secondary apical periodontitis requiring endodontic re-treatment were included. Microbiological samples were taken from the root canals and incubated in thioglycollate under anaerobic conditions. Anaerobic CDC agar (formulated by the Center for Disease Control and Prevention) was employed as a culture medium and the microorganisms were identified using the API system. The microorganisms were subjected to antibiograms with three different antibiotics. Twenty-six microorganisms were identified, the most common genus was Enterococcus (26.8%), Streptococcus (19.22%), Aerococcus (19.1%), and Clostridium (11.4%). 48% of them were susceptible to amoxicillin, with 28% of resistance. For clavulanic acid/amoxicillin, the susceptibility occurred in 32%, with 28% of resistance; and for clindamycin, the susceptibility was present in 40%, with 52% of resistance. The most frequently detected associated microorganism for secondary infections was genus Enterococcus, which exhibited high resistance to the studied antibiotics.

This study sought to evaluate biofilm elimination using the HBW Ultrasonic Ring based on continuous ultrasonic irrigation. Forty-five premolars and molars with complex curvatures were included. An Enterococcus faecalis biofilm was established for 30 days on the extracted teeth. The teeth were then stratified into three experimental groups for instrumentation and irrigation (i.e. HBW Ultrasonic Ring, conventional irrigation, and passive ultrasonic irrigation). Pre- and post-instrumentation samples were collected, and reductions of bacterial load were evaluated by McFarland's scale, counting of colony-forming units, and scanning electronic microscopy. The HBW Ultrasonic Ring promoted a higher reduction in bacterial load relative to conventional irrigation (P < 0.05) and a similar reduction compared with passive ultrasonic irrigation (P > 0.05). These results suggest the HBW Ultrasonic Ring is a promising alternative modality for simultaneous instrumentation and irrigation during root canal treatment, achieving an appropriate level of bacterial reduction and allowing the passage of the irrigating solution throughout the entire working length.

This study aimed to identify the aerobic and anaerobic causal microorganisms of odontogenic infections and their antibiotic sensitivity. Purulent exudates were taken from patients with odontogenic infections by transdermal puncture, and aerobic and anaerobic microorganisms were identified using biochemical tests. Susceptibility to antibiotics was tested using the Kirby-Bauer method; the inhibition halos were measured according to NCCLS, and based on the results, the microorganisms were classified as susceptible, intermediate or resistant to each antibiotic. Frequencies of species and percentages of resistance were calculated. The microorganisms associated with odontogenic infections were principally anaerobic (65.3% anaerobic vs. 35.7% aerobic), and the susceptibility to antibiotics was higher in anaerobic than in aerobic microorganisms. The majority of isolated microorganisms (82%) showed susceptibility to amoxicillin/clavulanic acid. The causal agents of odontogenic infections were anaerobic microorganisms, which exhibited high resistance to antibiotics.

Context: T regulatory type 1 (Tr1) cells are a subpopulation of T lymphocytes (CD4+CD49+LAG-3+IL-10+) that exert a considerable immunosuppressive effect. However, their possible role in autoimmune thyroid disease (AITD) has not been explored so far., Purpose: To analyze the levels and function of Tr1 cells in peripheral blood and thyroid tissue of patients with AITD., Design: Cases and controls, observational study., Setting: Department of Endocrinology, Hospital Universitario de la Princesa, Madrid, Spain., Patients: Thirty-eight patients with AITD (23 with Graves disease and 15 with Hashimoto thyroiditis) and 26 controls., Intervention: Multiparametric flow cytometry and immunofluorescence techniques were used to analyze the levels in peripheral blood (n = 38) and thyroid mononuclear cells (n = 5). An in vitro assay of suppression of cellular activation and cytokine release was performed to study the function of Tr1 cells., Main Outcome Measure: Levels and function of Tr1 cells in patients with AITD and controls., Results: Levels of Tr1 cells were significantly diminished in peripheral blood from patients with AITD. Functional studies showed that Tr1 cells from patients with AITD exhibit a diminished suppressive function compared with healthy controls. Tr1 levels were associated with disease severity, including longer duration of the disease and ophthalmopathy activity, and with autoantibody titers., Conclusions: The low levels of Tr1 cells and their diminished function may have a relevant role in the defective immune-regulatory function characteristic of patients with AITD.

We assessed different immune parameters in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) with low (LSI) and high (HSI) sodium intake. Thirty-eight patients with RA, thirty-seven with SLE, and twenty-eight healthy subjects were studied and classified as LSI or HSI. Levels and suppressive function of CD4 + CD25 + Foxp3 + and CD4 + CD69 + Foxp3 - Treg cells were determined by flow cytometry in blood samples. Levels and in vitro differentiation of Th17 cells were also assessed. Similar levels of CD4 + CD25 + Foxp3 + and CD4 + CD69 + Foxp3 - Treg cells were observed in LSI and HSI patients or controls. However, a positive correlation was detected between sodium intake and levels of CD4 + CD25 + Foxp3 + Treg cells in SLE and a negative association between CD4 + CD69 + Foxp3 - Treg cells and sodium intake in RA. No other significant associations were detected, including disease activity and sodium intake. Moreover, the suppressor activity of CD4 + CD25 + Foxp3 + and CD4 + CD69 + Foxp3 - Treg cells was similar in LSI and HSI patients or controls. The levels and in vitro differentiation of Th17 cells were also similar in LSI and HSI individuals. Our results suggest that, in the population studied (Mexican mestizo), the level of sodium intake is not apparently associated with different relevant immune parameters in healthy subjects or patients with SLE or RA.

Purpose: To study the levels of pathogenic and non-pathogenic Th17 and Th22 cells in autoimmune thyroid disorders patients. Although Th17 cells seem to play an important role in the pathogenesis of thyroid autoimmune disorders, the specific subsets of these lymphocytes have not been analyzed in this condition., Methods: We assessed the levels of Th17 (pathogenic and non-pathogenic) and Th22 cells in peripheral blood and thyroid glands of autoimmune thyroid disorders patients (n   =  26, 16 with Graves' disease and 10 with Hashimoto's thyroiditis) and 15 healthy controls by multi-parametric flow cytometry and immunofluorescence microscopy., Results: We found increased levels of pathogenic Th17 lymphocytes and Th22 cells in peripheral blood from autoimmune thyroid disorders patients. In addition, these cells were detected in thyroid glands from HT patients. Furthermore, we found significant correlations between the levels of these cells and disease activity, disease duration, and the presence of ophthalmopathy., Conclusions: The increased levels of pathogenic Th17 lymphocytes and Th22 cells in autoimmune thyroid disorders suggest their involvement in the pathogenesis of this condition.

Background: Periodontal disease is chronic inflammatory process that affects the attachment structures of the teeth and constitutes a significant cause of tooth loss in adults. Although different bacteria play an important role in the triggering of this condition, the progression and severity of the disease are strongly affected by the host immune response, which is under the control of different immune regulatory mechanisms, including T regulatory (Treg) cells. The aim of this study was to assess the frequency and function of CD69 + Treg lymphocytes in patients with chronic periodontal disease., Methods: Peripheral blood samples (n = 33) and gingival tissue (n = 9) were obtained from patients with chronic periodontal disease. Blood samples from 25 healthy individuals were also studied. Levels of CD69 + Treg lymphocytes in peripheral blood and gingival tissue were determined by six-color multiparametric flow cytometry, immunofluorescence, and immunohistochemistry. The immune regulatory function of CD69 + Treg cells was tested by an in vitro assay of inhibition of lymphocyte activation., Results: Percentages of CD69 + Treg cells were significantly higher in the peripheral blood from patients with active periodontal disease compared to healthy controls, and these percentages inversely correlated with the periodontal attachment loss. Increased numbers of these Treg cells were detected in the gingival tissue from active PD patients compared to their peripheral blood. However, the suppressive function of CD69 + Treg cells was significantly diminished in patients with periodontal disease compared to healthy controls., Conclusions: Our data suggest that CD69 + Treg cells seem to be another important piece in the complex immunopathogenesis of periodontal disease., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Context: Signaling lymphocytic activation molecule family 1 (SLAMF1) is a costimulatory receptor expressed by most immune cells. Its role in autoimmune thyroid disease (AITD) is not well known., Objective: To analyze the expression and function of the costimulatory receptor SLAMF1 in lymphocytes of patients with AITD., Design: Cross-sectional, prospective, single-center study., Setting: Department of Endocrinology, Hospital Universitario de la Princesa, Madrid., Patients: Twenty-eight patients with AITD (17 with Graves disease and 11 with Hashimoto thyroiditis) and 21 controls., Intervention: Multiparametric flow cytometry and immunofluorescence techniques to analyze the expression of SLAMF1 in peripheral blood (n = 28) and thyroid tissue (n = 5) mononuclear cells. Assay of inhibition of cellular proliferation to study the function of SLAMF1 in CD4+CD25+ T regulatory (Treg) cells., Main Outcome Measure: Expression levels and the function of SLAMF1 in lymphocytes in AITD patients and controls., Results: Expression of SLAMF1 was significantly increased in peripheral blood CD4+, T helper 17, and CD19+ B cells from AITD patients. Immunofluorescence microscopy detected the presence of SLAMF1+ lymphocytes in thyroid inflammatory cell infiltrate. Functional studies showed that SLAMF1 engagement in Treg cells increased their suppressive function in healthy controls but not in AITD patients., Conclusions: The altered expression of SLAMF1, as well as its defective function observed in patients with AITD, may have a relevant role in the defective immune-regulatory function observed in this condition., (Copyright © 2017 by the Endocrine Society)

T regulatory (Treg) cells have a key role in the pathogenesis of chronic inflammatory and autoimmune diseases. A CD4 + CD69 + T cell subset has been described that behaves as Treg lymphocytes, exerting an important immune suppressive effect. In this study, we analyzed the levels and function of CD4 + CD69 + Treg cells in patients with systemic lupus erythematosus (SLE). Blood samples were obtained from 22 patients with SLE and 25 healthy subjects. Levels of CD4 + CD69 + Treg cells were analyzed by multiparametric flow cytometry, and their function was measured by an assay of suppression of lymphocyte activation and through the inhibition of cytokine synthesis. We found an increased percent of CD4 + CD25 var CD69 + TGF- β + IL-10 + Foxp3 - lymphocytes in patients with SLE compared to controls. In addition, a significant diminution in the suppressive effect of these cells on the activation of autologous T lymphocytes was observed in most patients with SLE. Accordingly, CD69 + Treg cells from SLE patients showed a defective capability to inhibit the release of IL-2, IL-6, IL-10, and IL-17A by autologous lymphocytes. Our findings suggest that while CD4 + CD69 + Treg lymphocyte levels are increased in SLE patients, these cells are apparently unable to contribute to the downmodulation of the autoimmune response and the tissue damage seen in this condition.

Regulatory T (Treg) cells play an important role in the pathogenesis of autoimmune thyroid disorders (AITD). New subsets of CD4(+)CD69(+) and CD4(+)NKG2D(+) T lymphocytes that behave as regulatory cells have been recently reported. The role of these immunoregulatory lymphocytes has not been previously explored in AITD. We analyzed by multi-parametric flow cytometry different Treg cell subsets in peripheral blood from 32 patients with AITD and 19 controls, and in thyroid tissue from seven patients. The suppressive activity was measured by an assay of inhibition of lymphocyte activation. We found a significant increased percentage of CD4(+)CD69(+)IL-10(+), CD4(+)CD69(+)NKG2D(+), and CD4(+)CD69(+)IL-10(+)NKG2D(+) cells, in peripheral blood from GD patients compared to controls. The increase in CD4(+)CD69(+)IL-10(+) and CD4(+)CD69(+)IL-10(+)NKG2D(+) T cells was especially remarkable in patients with active Graves' ophthalmopathy (GO), and a significant positive correlation between GO activity and CD4(+)CD69(+)IL-10(+) or CD4(+)CD69(+)IL-10(+)NKG2D(+) cells was also found. In addition, these cells were increased in patients with a more severe and/or prolonged disease. Thyroid from AITD patients showed an increased proportion of CD69(+) regulatory T cells subpopulations compared to autologous peripheral blood. The presence of CD69(+), NKG2D(+), and IL-10(+) cells was confirmed by immunofluorescence microscopy. In vitro functional assays showed that CD69(+) Treg cells exerted an important suppressive effect on the activation of T effector cells in controls, but not in AITD patients. Our findings suggest that the levels of CD69(+) regulatory lymphocytes are increased in AITD patients, but they are apparently unable to down-modulate the autoimmune response and tissue damage.