α-IRAK-4 Suppresses the Activation of RANK/RANKL Pathway on Macrophages Exposed to Endodontic Microorganisms.

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-1ß, 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.

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.

Peripheral Blood Mitochondrial DNA Levels Were Modulated by SARS-CoV-2 Infection Severity and Its Lessening Was Associated With Mortality Among Hospitalized Patients With COVID-19.

Introduction: During severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the virus hijacks the mitochondria causing damage of its membrane and release of mt-DNA into the circulation which can trigger innate immunity and generate an inflammatory state. In this study, we explored the importance of peripheral blood mt-DNA as an early predictor of evolution in patients with COVID-19 and to evaluate the association between the concentration of mt-DNA and the severity of the disease and the patient's outcome. Methods: A total 102 patients (51 COVID-19 cases and 51 controls) were included in the study. mt-DNA obtained from peripheral blood was quantified by qRT-PCR using the NADH mitochondrial gene. Results: There were differences in peripheral blood mt-DNA between patients with COVID-19 (4.25 ng/µl ± 0.30) and controls (3.3 ng/µl ± 0.16) (p = 0.007). Lower mt-DNA concentrations were observed in patients with severe COVID-19 when compared with mild (p= 0.005) and moderate (p= 0.011) cases of COVID-19. In comparison with patients with severe COVID-19 who survived (3.74 ± 0.26 ng/µl) decreased levels of mt-DNA in patients with severe COVID-19 who died (2.4 ± 0.65 ng/µl) were also observed (p = 0.037). Conclusion: High levels of mt-DNA were associated with COVID-19 and its decrease could be used as a potential biomarker to establish a prognosis of severity and mortality of patients with COVID-19.

Cytomegalovirus glycoprotein B genotypes in Mexican children and women.

OBJECTIVE: Cytomegalovirus (CMV) is widely distributed and constitutes the main cause of congenital infections worldwide. CMV transmission during pregnancy represents one of the major impacts of this virus on public health. This study aimed at assessing glycoprotein B (gB) CMV genotypes in Mexican children and pregnant women, since there is limited information regarding CMV genomic diversity in Mexico. METHODS: We analyzed CMV strains detected in Mexican children (n = 38) and women (n = 38) between 2001 and 2012. A fragment of the gB gene was amplified and sequenced, and genotypes were defined based on prototype sequences. RESULTS: The gB1 genotype was detected more frequently in children (68.4%) compared to women (31.6%; p = 0.0028), while genotype 2 was more common in women (65.8%) compared to children (26.3%, p = 0.0012). Genotype 3 was uncommon in both groups (5.3 and 2.6%). Nucleotide sequences exhibited a high degree of similarity to prototype strains. However, we identified 17 distinct sequences that resulted in changes in the encoded amino acid sequence in four strains. CONCLUSIONS: gB1 and gB2 are the most common strains associated with CMV infection in Mexican children and women. In addition, we found that the frequency for each genotype differed amongst them, possibly due to variability in transmission or reactivation dynamics.

Assessment of exposure to organochlorine pesticides and levels of DNA damage in mother-infant pairs of an agrarian community.

Exposure to organochlorine pesticides was studied in a group of mother-infant pairs living in a rural area where agriculture is the main economic activity. Fumigation in this zone is performed with airplanes, thus affecting the inhabited areas around them, including schools. Heparinized venous blood of mothers and umbilical cords was used to evaluate the olive tail moment in the comet assay, and micronuclei, chromatin buds, and nucleoplasmic bridges in peripheral blood lymphocytes. Cord blood samples were taken at the moment of birth only from natural and normal parturitions. Determinations of hexachlorobenzene, aldrin, heptachlor epoxide, oxichlordane, t and c-chlordane, cis-nonachlor, mirex, alpha and beta-endosulfan, alpha, beta and gamma hexachlorocyclohexane, and p'p'-DDT, p'p'-DDE were conducted to establish the differential distribution of the toxicants between compartments, i.e., mother and umbilical cord. Significantly higher pesticide levels were found in umbilical cord plasma than in mothers' plasma for almost all compounds tested, except DDE and oxychlordane. Significantly higher olive tail moments were found in umbilical cords than in mothers, whereas micronuclei frequencies were higher in mothers than in umbilical cords. However, neither the levels of micronuclei nor the olive tail moment were correlated with pesticide levels. Given that no other exposure to toxic compounds has been identified in this region, the lack of correlation between genotoxicity biomarkers and pesticide levels may be due to the variability of the exposure and to endogenous processes related to lipid mobility during pregnancy, the metabolism of the compounds, and individual susceptibilities.

KIR gene diversity in Mexican mestizos of San Luis Potosí.

Natural killer (NK) cell function is regulated by different types of membrane-bound receptors of which killer-cell immunoglobulin-like receptors (KIRs) are the most complex and diverse. KIRs are encoded by 17 different genes located within the leukocyte receptor complex (19q13.4). The frequency with which KIR gene features are present in different human populations differs. Here, we present our results on the KIR gene diversity observed in a large group of mestizos from the central Mexican city of San Luis Potosí. In total, 53 different KIR genotypes were observed, 47 with previously described gene profiles and six harboring novel KIR gene combinations. Group A homozygous haplotypes were seen in 102 individuals (34%), while group B homozygous haplotypes were present in 45 (15%). Heterozygous combinations of groups A and B haplotypes were seen in 153 individuals (51%). Haplotype frequency estimations based on a true content of 600 chromosomes showed a relatively balanced proportion of group A (59.5%) and group B (40.5%) haplotypes in our study population. A homozygous combination of the cA01|tA01 haplotype was present in 33% of the population with other frequent combinations being cA01|tA01, cB03|tB01 in 14.7% and cA01|tA01, cB02|tA01 in 12%. The dendrogram derived from activating KIR gene phylogenetic analysis revealed five clearly distinct clades corresponding to African, East Asian, Arab/Caucasoid, Mexican mestizo/Amerindian and South Asian populations. Our results illustrate the genetic contribution that Caucasoid and Amerindian populations have made toward present-day Mexicans and suggest an important Southeast Asian genetic contribution to native Amerindian populations.