Increased IL-12p70 and IL-8 Produced by Monocytes in Response to Streptococcus spp. and Actinomyces spp. Causals of Endodontic Primary Infections.

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 × 105 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.

Chronic Bi-Maxillary Osteomyelitis Caused by Actinomycetes in a Patient with Severe COVID-19 / Osteomyelitis bi-maxilar crónica causada por Actinomycetes en un paciente con COVID-19 severo

Abstract Osteomyelitis is defined as the inflammation of the either medullary, cortical, or cancellous bone, including nerves and blood vessels, causing necrosis and bone sequestrum formation; this condition has become a rare pathology, and odontogenic infections are considered the most frequent causal factor. This case shows a patient with bi-maxillary osteomyelitis caused by Actinomyces spp, which was worsened for severe COVID-19 infection. Patient was submitted at surgery as, amplified total bilateral maxillectomy through the surgical technique Weber-Fergusson, and prolonged use of combination of antibiotics, achieved a good recovery. Two years later follow- up, the patient no show imaging or clinical evidence of the infection of osteomyelitis. The present case shows an interesting relationship between a rare infection and its association with COVID-19.

Resumen La osteomielitis se define como la inflamación del hueso medular, cortical o esponjoso, incluyendo nervios y vasos sanguíneos, causando necrosis y formación de secuestro óseo; esta condición es una patología rara, y las infecciones odontogénicas son consideradas como el factor causal más frecuente. En este caso, se muestra un paciente con osteomielitis bi-maxilar causada por Actinomyces spp, la cual empeoró por la infección de COVID-19 severo. El paciente fue sometido a una cirugía, maxilectomía bilateral total amplificada, a través de la técnica quirúrgica de Weber- Fergusson, y el uso prolongado de una combinación de antibióticos, logrando una buena recuperación. A los 2 años de seguimiento, el paciente no mostró evidencia clínica o imagenológica de la infección de osteomielitis. El presente caso muestra una interesante relación entre una infección rara y su asociación con COVID-19.

Elimination of E. faecalis and C. albicans Biofilm: A comparison between single and multiple file systems in an Ex Vivo study

To evaluate whether the WaveOne Gold and Reciproc single file instrumentation systems, are effective in reducing the microbial load of a mixed biofilm and the cleaning of apical third compared to the Twisted File Adaptive system (multiple- file system). Seventy mesial roots of the first and second molars were included and randomly divided into three experimental groups (n=20, n=10 controls). Biofilms were formed inside canals over 31 days. After instrumentation with the unique file systems, WaveOne Gold and Reciproc and the multiple file system Twisted File Adaptive, using 2.25% sodium hypochlorite as an irrigant in all cases, a count of colony forming units was performed using serial dilutions, cleaning of the apical third was evaluated using scanning electron microscopy. Comparisons amongst groups were made by using parametric and non-parametric statistics, according to a normal or non-normal data distribution, respectively. No significant differences in the reduction of the microbial load after employing a single-file system in comparison to the multiple-file system were found; in addition, the cleaning of the apical third was similar for the three different instrumentation systems. The single-file system is equal in effectiveness compared with the multiple-file system in reducing the microbial load.

Evaluar si los sistemas de instrumentación de lima única, como WaveOne Gold y Reciproc son efectivos para reducir la carga microbiana de un biofilm mixto y la limpieza del tercio apical, comparado con los sistemas de limas múltiples, como Twisted File Adaptive. Setenta raíces mesiales de primeros y segundos molares fueron incluidos y divididos de forma aleatoria en tres grupos experimentales (n=20, n=10 controles). El biofilm fue formado en el interior de los conductos durante 31 días. Después se instrumentó con los sistemas de lima única (WaveOne Gold y Reciproc) y el sistema de limas múltiples Twisted File Adaptive, usando hipoclorito de sodio al 2.5% en todos los casos. El conteo de unidades formadoras de colonias se realizó usando diluciones seriales, la limpieza del tercio apical se evaluó empleando el microscopio electrónico de barrido. La comparación entre grupos se realizó con pruebas paramétricas y no paramétricas, de acuerdo con la distribución normal y no normal de los datos, respectivamente. No hubo una diferencia significativa en la reducción de la carga microbiana después de emplear los sistemas de lima única en comparación a los de limas múltiples, además, la limpieza del tercio apical fue similar en los 3 diferentes sistemas de instrumentación. Los sistemas de lima única son igual de efectivos para reducir la carga microbiana comparados con los sistemas de limas múltiples.

Levels of Pathogenic Th17 and Th22 Cells in Patients with Rheumatoid Arthritis.

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-1ß. 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.

Increased levels of pathogenic Th17 cells and diminished function of CD69+ Treg lymphocytes in patients with overweight.

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.

Root canal microorganisms and their antibiotic susceptibility in patients with persistent endodontic infections, with and without clinical symptoms.

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.

Evaluation of Apical Transportation by Three Rotary Systems in Extracted Teeth with Curved Canals / Evaluación de la transportación apical con tres sistemas rotatorios en dientes extraídos con conductos curvos

Abstract The aim of this study was to compare the apical transportation in extracted teeth using three different rotary nickel-titanium systems (Mtwo, Hyflex CM, and Typhoon). Sixty extracted first molars were randomly divided into three groups (n=20 in each group) with similar root canal curvatures (25-52 degrees). All root canals were prepared to size 30 using a crown-down preparation technique for each rotary system. Loss of working length and apical transportation were determined by X-ray evaluation. Comparisons between the groups were performed using one-way analysis of variance (ANOVA) or Kruskal-Wallis tests, and the post hoc analyses employed were Dunn´s or Tukey´s multiple comparison tests. No significant differences were detected between the different rotary systems in the loss of working length and apical transportation. This in vitro study showed that the use of these three rotary systems are safe and useful for instrumentation in curved canals; however, further in vivo research is essential to compare the effectiveness and safety of these systems for use in curved canals.

Resumen El objetivo del presente estudio fue comparar la transportación apical en dientes extraídos utilizando tres sistemas rotatorios de níquel-titanio (Mtwo, Hyflex CM y Typhoon). Sesenta primeros molares extraídos se dividieron aleatoriamente en tres grupos (n=20 en cada grupo) con curvaturas del conducto radicular similares (25-52 grados). Todos los conductos radiculares se prepararon usando una técnica corono apical para cada sistema rotatorio. La pérdida de la longitud de trabajo y la transportación apical se determinaron mediante evaluación radiográfica. Las comparaciones entre los grupos se realizaron utilizando análisis de varianza de una vía (ANOVA) o Kruskal-Wallis, y los análisis post hoc empleados fueron las pruebas de comparación múltiple de Dunn o Tukey. No se detectaron diferencias significativas entre los diferentes sistemas rotatorios en la pérdida de la longitud de trabajo y transportación apical. Este estudio demostró que el uso de estos tres sistemas es seguro y útil para su instrumentación en conductos curvos; sin embargo, investigación in vivo es necesaria para comparar la efectividad y seguridad de estos sistemas para su uso en conductos curvos.

Evaluation of in vitro biofilm elimination of Enterococcus faecalis using a continuous ultrasonic irrigation device.

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.

Elimination of an intraradicular biofilm composed of enterococcus faecalis and candida albicans using a negative apical pressure irrigation system / Eliminación de una biopelícula intraradicular compuesta por enterococcus faecalis y candida albicans utilizando un sistema de irrigación de presión apical negativa

Endodontic treatment consists of the cleaning and disinfecting the root canal system, which is achieved using adequate mechanical instru-mentation and chemical irrigation. Endodontic microorganisms are present in root canals in the form of a biofilm, and their elimination ensures the success of endodontic treatment. Irrigation is a key factor contributing to the elimination of this intraconduct biofilm, and different irrigator agents and irrigation techniques, such as irrigation with negative apical pressure, a novel automated irrigation mechanism based on suction intraconduct, have been used. In this study, we evaluated the ability of a negative apical pressure system with different concentrations of sodium hypochlorite and durations to reduce the microbial load. Materials and Methods: An intraradicular biofilm composed of Enterococcus faecalis and Candida albicans was generated during twenty-one days of static culture on one hundred mesio-vestibular roots of upper molars with complex curvatures greater than 30°C, and the roots were classified in six groups with different concentrations and contact times of sodium hypochlorite. Subsequently, the reduction in the microbial load was measured with McFarland scale and the enumeration of colony forming units and was evaluated with scanning electronic microscopy. Results: We observed a significant difference in the reduction of the microbial load prior to instrumentation compared with postinstrumentation between the groups treated with 2.25% and 5.25% NaOCl for 30, 60 and 90 seconds of contact time (p<0.05), but we did not observe differences in the reduction of microbial load between different contact times and concentrations of sodium hypochlorite employed (p>0.05). Conclusion: Negative apical pressure is a good option for irrigation in endodontics, as it allows the passage of the irrigation fluid along the total length of the root canal and produces a better antimicrobial effect.

El tratamiento de endodoncia consiste en la limpieza y desinfección del sistema de conducto radicular, lo que se logra utilizando instrumentación mecánica adecuada y riego químico. Los microorganismos endodónticos están presentes en los conductos radiculares en forma de una biopelícula, y su eliminación asegura el éxito del tratamiento endodóntico. La irrigación es un factor clave que contribuye a la eliminación de esta biopelícula intraconductora, y se han utilizado diferentes agentes irrigadores y técnicas de irrigación, como la irrigación con presión apical negativa, un nuevo mecanismo automatizado de irrigación basado en la intraconducción de succión. En este estudio, evaluamos la capacidad de un sistema de presión apical negativa con diferentes concentraciones de hipoclorito de sodio y duraciones para reducir la carga microbiana. Material y Métodos: Se generó una biopelícula intraradicular compuesta por Enterococcus faecalisy Candida albicans durante veintiún días de cultivo estático en cien raíces mesio-vestibulares de molares superiores con curvaturas complejas superiores a 30°C, y las raíces se clasificaron en seis grupos con diferentes concentraciones y tiempos de contacto de hipoclorito de sodio. Posteriormente, la reducción en la carga microbiana se midió con la escala de McFarland y la enumeración de las unidades formadoras de colonias y se evaluó con microscopía electrónica de barrido. Resultado: Observamos una diferencia significativa en la reducción de la carga microbiana antes de la instrumentación en comparación con la postinstrumentación entre los grupos tratados con NaOCl 2.25% y 5.25% durante 30, 60 y 90 segundos de tiempo de contacto (p<0.05), pero lo hicimos No se observan diferencias en la reducción de la carga microbiana entre los diferentes tiempos de contacto y las concentraciones de hipoclorito de sodio empleado (p>0.05). Conclusión:La presión apical negativa es una buena opción para el riego en endodoncia, ya que permite el paso del líquido de riego a lo largo de todo el conducto radicular y produce un mejor efecto antimicrobiano.

[Células T reguladoras en lupus eritematoso generalizado].

El lupus eritematoso generalizado (LEG) es una enfermedad autoinmune crónica caracterizada por la pérdida de la tolerancia a los antígenos propios y la síntesis de diferentes autoanticuerpos con la formación y depósito de complejos inmunes y el daño de múltiples órganos. Las células T reguladoras (Treg) desempeñan un papel esencial en el mantenimiento de la tolerancia periférica, controlan el estado de activación del sistema inmune y limitan las respuestas autoinmunes. El estudio del número y la función de las diferentes subpoblaciones de células Treg en LEG ha sido objeto de una intensa investigación. Dependiendo del fenotipo de las células Treg analizado se ha reportado que la frecuencia de estas células en pacientes con LEG se encuentra disminuida, aumentada o sin alteraciones. Además, diferentes grupos han descrito que la función supresora de las células Treg de los pacientes con LEG se encuentra reducida o no se ve afectada. En conjunto, lo datos reportados sugieren que las células Treg desempeñan un papel relevante en la patogénesis del LEG y que estos linfocitos pueden ser considerados blancos potenciales para el diseño de nuevas estrategias terapéuticas para esta enfermedad.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by a loss of tolerance to self-antigens and synthesis of different autoantibodies, with the formation and deposition of immune complexes and damage to multiple organs. T regulatory cells (Tregs) play a crucial role in maintaining peripheral tolerance, controlling the state of activation of the immune system and limiting autoimmune responses. The study of the number and function of the different Treg cell subpopulations in SLE has been the subject of intense research. Depending on the analyzed Treg cell phenotype, the frequency of these cells has been reported to be reduced, increased or unaltered in patients with SLE. In addition, different groups have described that Treg cells suppressive function is reduced or unaffected in patients with SLE. Taken together, the reported data suggest that Treg cells play a relevant role in the pathogenesis of SLE and that these lymphocytes can be considered potential targets for the design of new therapeutic strategies for this condition.

Aerobic and anaerobic microorganisms and antibiotic sensitivity of odontogenic maxillofacial infections.

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.

Células T reguladoras en lupus eritematoso generalizado / Regulatory T cells in systemic lupus erythematosus

Resumen El lupus eritematoso generalizado (LEG) es una enfermedad autoinmune crónica caracterizada por la pérdida de la tolerancia a los antígenos propios y la síntesis de diferentes autoanticuerpos con la formación y depósito de complejos inmunes y el daño de múltiples órganos. Las células T reguladoras (Treg) desempeñan un papel esencial en el mantenimiento de la tolerancia periférica, controlan el estado de activación del sistema inmune y limitan las respuestas autoinmunes. El estudio del número y la función de las diferentes subpoblaciones de células Treg en LEG ha sido objeto de una intensa investigación. Dependiendo del fenotipo de las células Treg analizado se ha reportado que la frecuencia de estas células en pacientes con LEG se encuentra disminuida, aumentada o sin alteraciones. Además, diferentes grupos han descrito que la función supresora de las células Treg de los pacientes con LEG se encuentra reducida o no se ve afectada. En conjunto, lo datos reportados sugieren que las células Treg desempeñan un papel relevante en la patogénesis del LEG y que estos linfocitos pueden ser considerados blancos potenciales para el diseño de nuevas estrategias terapéuticas para esta enfermedad.

Abstract Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by a loss of tolerance to self-antigens and synthesis of different autoantibodies, with the formation and deposition of immune complexes and damage to multiple organs. T regulatory cells (Tregs) play a crucial role in maintaining peripheral tolerance, controlling the state of activation of the immune system and limiting autoimmune responses. The study of the number and function of the different Treg cell subpopulations in SLE has been the subject of intense research. Depending on the analyzed Treg cell phenotype, the frequency of these cells has been reported to be reduced, increased or unaltered in patients with SLE. In addition, different groups have described that Treg cells suppressive function is reduced or unaffected in patients with SLE. Taken together, the reported data suggest that Treg cells play a relevant role in the pathogenesis of SLE and that these lymphocytes can be considered potential targets for the design of new therapeutic strategies for this condition.

Analysis of Sodium Chloride Intake and Treg/Th17 Lymphocytes in Healthy Individuals and Patients with Rheumatoid Arthritis or Systemic Lupus Erythematosus.

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.

Patients With Autoimmune Thyroiditis Show Diminished Levels and Defective Suppressive Function of Tr1 Regulatory Lymphocytes.

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.

Patients with Systemic Lupus Erythematosus Show Increased Levels and Defective Function of CD69+ T Regulatory Cells.

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+CD25varCD69+TGF-ß+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.

Pathogenic Th17 and Th22 cells are increased in patients with autoimmune thyroid disorders.

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.

Quantitative and functional analysis of CD69+ T regulatory lymphocytes in patients with periodontal disease.

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.

Expression and Function of the Costimulatory Receptor SLAMF1 Is Altered in Lymphocytes From Patients With Autoimmune Thyroiditis.

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.

Levels of regulatory T cells CD69(+)NKG2D(+)IL-10(+) are increased in patients with autoimmune thyroid disorders.

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.