Assessment of sCD14 levels in patients with endodontic pathology requiring root canal treatment / Avaliação dos níveis de sCD14 em pacientes que necessitam de tratamento endodôntico

Objective: Dental caries is one of the most common microbial diseases. Because of the infectious nature of the disease, the immunologic response by the host plays an essential role in its development. Therefore, the aim of this study was to evaluate the sCD14 levels in patients exhibiting two to three teeth with caries involving pulp along with apical periodontitis requiring root canal treatment. Material and Methods: This study was carried out on 20 participants, of whom 10 were caries-free (Control) and 10 had two to three teeth with symptomatic irreversible pulpitis along with apical periodontitis requiring root canal treatment, within the ages of 20- 30 years. Unstimulated saliva of the participants was collected with disposable needle-less syringe from buccal and labial vestibules. The sCD14 levels in salivary samples were assessed before and following endodontic treatment. The results were analyzed by ELISA. Results: The obtained levels of sCD14 were analyzed statistically. Paired T test was performed to assess the significance. The results revealed that there was a significant difference in sCD14 levels with a P=0.0005, as it had drastically reduced once the inflammation has subsided. Conclusion: Higher values of sCD14 levels were seen in patients with symptomatic irreversible pulpitis along with apical periodontitis than in caries free group. The study also showed that sCD levels were significantly reduced following post endodontic treatment. Therefore, increased levels of sCD14 can be considered as a marker of inflammation. (AU)

Objetivo: A cárie dentária é uma das doenças microbianas mais comuns. Devido à natureza infecciosa da doença, a resposta imunológica do hospedeiro desempenha um papel essencial no seu desenvolvimento. Portanto, o objetivo deste estudo foi avaliar os níveis de sCD14 em pacientes que possuiam dois a três dentes com necessidade de tratamento endodôntico por apresentarem lesão de cárie envolvendo polpa e periodontite periapical. Material e Métodos: Este estudo foi realizado em 20 participantes, dos quais 10 estavam livres de cárie (controle) e 10 tinham dois a três dentes com pulpite irreversível sintomática e periodontite periapical com necessidade de tratamento endodôntico, nas idades de 20 a 30 anos. A saliva não estimulada das crianças foi coletada com seringa descartável sem agulha dos vestíbulos bucal e labial. Os níveis de sCD14 em amostras salivares foram avaliados antes e após o tratamento endodôntico. Os resultados foram analisados por ELISA. Resultados: Os níveis de sCD14 obtidos foram analisados estatisticamente. O teste T pareado foi realizado para avaliar a significância. Os resultados revelaram que houve uma diferença significativa nos níveis de sCD14 com um P = 0,0005, uma vez que reduziu drasticamente uma vez que a inflamação diminuiu. Resultados: Os níveis de sCD14 obtidos foram analisados estatisticamente. O teste T pareado foi realizado para avaliar a significância. Os resultados revelaram que houve uma diferença significativa nos níveis de sCD14 com um P = 0,0005, uma vez que reduziu drasticamente uma vez que a inflamação diminuiu. Conclusão: Valores mais elevados de níveis de sCD14 foram observados em pacientes com pulpite irreversível sintomática junto com periodontite periapical do que no grupo livre de cárie. O estudo também mostrou que os níveis de sCD foram significativamente reduzidos após o tratamento endodôntico. Portanto, níveis aumentados de sCD14 podem ser considerados um marcador de inflamação. (AU)

Association between soluble biomarkers - microbial translocation, inflammation and cardiovascular risk in HIV- infected individuals: a systematic review

Microbial translocation is associated with the increased risk of cardiovascular disease in HIV-infected individuals. There is scarce information regarding the possible associations between the biomarkers of microbial translocation, inflammation and cardiovascular risk that can be evaluated in clinical laboratories using plasma or serum samples. This systematic review was conducted according to the PRISMA protocol in order to verify the most used soluble biomarkers of microbial translocation, inflammation and cardiovascular risk, as well as possible associations between them, in HIV-infected individuals. A search was performed using the Medline, Scopus and Web of Science databases to identify existing studies regarding the relationship between microbial translocation biomarkers, inflammation and cardiovascular risk in HIV-infected patients. Eleven articles that presented soluble biomarkers of microbial translocation (LPS, rDNA, sCD14, LBP and EndoCAb) were selected. The most frequently evaluated soluble biomarker was sCD14, followed by LPS; the latter were associated with some lipid profile parameters. This systematic review considered soluble blood biomarkers that can be utilized in laboratory diagnosis. The aim was to identify the interconnection between microbial translocation, inflammation and cardiovascular risk. Despite the fact that a large number of inflammation and cardiovascular risk biomarkers have been previously reported, it was noted that important markers involved in the pathophysiology of cardiovascular diseases need to be included in future research.

CD14 genotype and functional dichotomy of CD14+ and CD14- cells are associated with activated immune response and development of Chagas dilated cardiomyopathy

We aimed to investigate the association of CD14 -260C/T (rs2569190) polymorphism and Chagas cardiomyopathy and the functional characteristics of CD14+ and CD14- monocytes upon infection with Trypanosoma cruzi. We observed an association between the T- genotype (absence of allele -260T) related to low CD14 expression and the dilated cardiomyopathy type of Chagas disease. Furthermore, we observed that CD14- monocytes showed a more activated profile upon in vitro infection with T. cruzi than CD14+ monocytes. Our findings suggest that T- genotype is associated with susceptibility to develop Chagas dilated cardiomyopathy, likely linked to the T. cruzi-induced inflammatory profile of CD14- monocytes.

CD4+CD69+ T cells and CD4+CD25+FoxP3+ Treg cells imbalance in peripheral blood, spleen and peritoneal lavage from pristane-induced systemic lupus erythematosus (SLE) mice

Abstract Background: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. Methods: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. Results: Compared with the controls, SLE-induced animals presented increased numbers of CD4+CD69+ T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4+ CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038). Conclusion: Increased numbers of CD4+CD69+ T cells and reduced numbers of CD4+CD25+FoxP3+ Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

Clinical and hematological changes in sheep induced by Escherichia coli lipopolysaccharide / alterações clínicas e hematológicas induzidas pela administração de lipopolisscarídeo de Escherichia coli em ovelhas / Alteraciones clínicas y hematológicas inducidas por la administración de lipopoliscárido de Escherichia coli en ovejas

Organic response to infection is characterized by a systemic reaction known as acute phase response (APR). In order to know the effect of the administration of Escherichia coli lipopolysaccharide (LPS) on physiological, hematological and biochemical variables, 10 sheep weighing 45 ± 5 kg were divided in two groups: Experimental group treated with 3 doses of 1 µg.kg-1 LPS and control group treated with saline solution (SS) at the same frequency as experimental group. Body temperature (BT°), heart rate (HR) and respiratory rate (RR) were monitored. Blood samples for hemogram and enzyme activity for aspartate amino transferase (AST) and gamma glutamyl transferase (GGT) were collected between 1 and 24 h post-LPS. LPS-treated sheep presented mean values of BT (41.2 ± 0.4°C), HR (132 ± 12.3 beats/min) and RR (107.2 ± 25 cycles/min) higher than those observed in control sheep (39.8 ± 0.2°C, 88.8 ± 8.7 beats/min and 53.6 ± 17.1 cycles/min respectively). Between 4 and 8 hours post-injection (hpi) of LPS the leukocyte count was associated with lymphopenia, followed by leukocytosis at 24 hours. No changes were observed in the activity of AST and GGT enzymes. The results characterize APR induced by LPS in sheep, representing a useful model to study cardiovascular, hematological and biochemical responses to infection. (AU)

A resposta orgânica à infecção é caracterizada por uma reação sistêmica conhecida como resposta de fase aguda (RFA). Para conhecer o efeito da administração de lipopolissacárideo (LPS) de Escherichia coli sobre as variáveis fisiológicas, hematológicas e bioquímicas, 10 carneiros pesando 45 ± 5 kg foram divididos em dois grupos: o grupo experimental tratado com três doses de 1 µg.kg -1 de LPS e grupo controle tratado com solução salina (SS) na mesma frequência que o grupo experimental. Temperatura corporal (T°C), frequência cardíaca (FC) e frequência respiratória (FR) foram monitoradas. As amostras de sangue foram tomadas para hemograma e atividade da enzima aspartato aminotransferase (AST) e gama- -glutamiltransferase (GGT) entre uma e 24 h pós-LPS. Ovelhas tratadas com LPS apresentaram valores médios de T°C (41,2 ± 0,4°C), FC (132 ± 12,3 batimentos/min) e FR (107,2 ± 25 ciclos/min) acima dos observados em ovelhas do tratamento controle (39,8 ± 0,2°C, 88,8 ± 8,7 batimentos/min e 53,6 ± 17,1 ciclos/min, respectivamente). Entre 4 e 8 horas após a injeção de LPS, a contagem de leucocitos foi asociado com linfopenia, seguida de leucocitose as 24 horas. Nenhuma mudança na atividade das enzimas AST e GGT foi observada. Os resultados caracterizam uma resposta de fase aguda induzida por LPS em ovelhas, o que representa um modelo útil para estudar os sistemas cardiovascular, hematológico e bioquímico em resposta à infecção.(AU)

La respuesta orgánica a la infección se caracteriza por una reacción sistémica conocida como respuesta de fase aguda (RFA). Para conocer el efecto de la administración de lipopolisacárido (LPS) de Escherichia coli sobre las variables fisiológicas, hematológicas y bioquímicas, 10 ovejas con un peso de 45 ± 5 kg se dividieron en dos grupos: grupo experimental tratado con 3 dosis de 1 µg.kg-1de LPS y grupo control tratado con solución salina (SS) en la misma frecuencia que el grupo experimental. Temperatura corporal (T°C), frecuencia cardíaca (FC) y frecuencia respiratoria (FR) fueron monitoreadas. Se tomaron muestras de sangre para hemograma y actividad enzimática para el aspartato amino transferasa (AST) y gamma glutamil transferasa (GGT) entre 1 y 24 h post-LPS. Las ovejas tratadas con LPS presentaron valores medios de T°C (41.2 ± 0.4°C), FC (132 ± 12.3 latidos / min) y FR (107.2 ± 25 ciclos/min) por encima de los observados en ovinos controles (39.8 ± 0.2°C, 88.8 ± 8.7 latidos/min y 53.6 ± 17.1 ciclos/min respectivamente). Entre las 4 y 8 horas después de la inyección de LPS, el recuento de leucocitos se asoció a linfopenia, seguida de leucocitosis a las 24 horas. No se observaron cambios en la actividad de las enzimas AST y GGT. Los resultados caracterizan una respuesta de fase aguda inducida por LPS en ovinos, representando un modelo útil para estudiar los sistemas cardiovascular, hematológico y bioquímico en respuesta a la infección.(AU)

Effects of triethylene glycol dimethacrylate (TEGDMA) on the odontoclastic differentiation ability of human dental pulp cells

Abstract Objectives: The primary purpose of this study was to examine the effects of triethylene glycol dimethacrylate (TEGDMA) on odontoclastic differentiation in the dental pulp tissue. Material and Methods: The effects of different TEGDMA dosages on the odontoclastic differentiation capability of dental pulp cells were analyzed in vitro using the following methodologies: i) flow cytometry and tartrate-resistant acid phosphatase (TRAP) staining; ii) apoptotic effects using Annexin V staining; iii) mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor (NF)-kB ligand (RANKL) genes by quantitative Real-time PCR (qRT-PCR); and iv) OPG and RANKL protein expression by enzyme-linked immunosorbent assay (ELISA). Results: TEGDMA caused relatively less odontoclastic differentiation in comparison with the control group; however, odontoclastic differentiation augmented with increasing doses of TEGDMA (p<0.05). The mRNA and protein expression of OPG was lower in TEGDMA treated pulp cells than in the control group (p<0.05). While the mRNA expression of RANKL remained unchanged compared to the control group (p>0.05), its protein expression was higher than the control group (p<0.05). In addition, TEGDMA increased the apoptosis of dental pulp cells dose dependently. Conclusions: TEGDMA reduced the odontoclastic differentiation ability of human dental pulp cells. However, odontoclastic differentiation ratios increased proportionally with the increasing dose of TEGDMA.

Classical and alternative macrophages have impaired function during acute and chronic HIV-1 infection

Abstract Objectives: Three decades after HIV recognition and its association with AIDS development, many advances have emerged – especially related to prevention and treatment. Undoubtedly, the development of Highly Active Antiretroviral Therapy (HAART) dramatically changed the future of the syndrome that we know today. In the present study, we evaluate the impact of Highly Active Antiretroviral Therapy on macrophage function and its relevance to HIV pathogenesis. Methods: PBMCs were isolated from blood samples and monocytes (CD14+ cells) were purified. Monocyte-Derived Macrophages (MDMs) were activated on classical (MGM-CSF+IFN-γ) or alternative (MIL-4+IL13) patterns using human recombinant cytokines for six days. After this period, Monocyte-Derived Macrophages were stimulated with TLR2/Dectin-1 or TLR4 agonists and we evaluated the influence of HIV-1 infection and Highly Active Antiretroviral Therapy on the release of cytokines/chemokines by macrophages. Results: The data were obtained using Monocyte-Derived Macrophages derived from HIV naïve or from patients on regular Highly Active Antiretroviral Therapy. Classically Monocyte-Derived Macrophages obtained from HIV-1 infected patients on Highly Active Antiretroviral Therapy released higher levels of IL-6 and IL-12 even without PAMPs stimuli when compared to control group. On the other hand, alternative Monocyte-Derived Macrophages derived from HIV-1 infected patients on Highly Active Antiretroviral Therapy released lower levels of IL-6, IL-10, TNF-α, IP-10 and RANTES after LPS stimuli when compared to control group. Furthermore, healthy individuals have a complex network of cytokines/chemokines released by Monocyte-Derived Macrophages after PAMP stimuli, which was deeply affected in MDMs obtained from naïve HIV-1 infected patients and only partially restored in MDMs derived from HIV-1 infected patients even on regular Highly Active Antiretroviral Therapy. Conclusion: Our therapy protocols were not effective in restoring the functional alterations induced by HIV, especially those found on macrophages. These findings indicate that we still need to develop new approaches and improve the current therapy protocols, focusing on the reestablishment of cellular functions and prevention/treatment of opportunistic infections.

Immune development in HIV-exposed uninfected children born to HIV-infected women

ABSTRACT Immunological and clinical findings suggestive of some immune dysfunction have been reported among HIV-exposed uninfected (HEU) children and adolescents. Whether these defects are persistent or transitory is still unknown. HEU pediatric population at birth, 12 months, 6-12 years were evaluated in comparison to healthy age-matched HIV-unexposed controls. Plasma levels of LPS, sCD14, cytokines, lymphocyte immunophenotyping and T-cell receptor excision circles (TREC) were assessed. HEU and controls had similar LPS levels, which remained low from birth to 6-12 years; for plasma sCD14, IL-2, IL-6, IL-7, IL-10, IL-12p70, IL-13, IL-17, IFN-γ, TNF-α, G-CSF, GM-CSF and MCP-1, which increased from birth to 12 months and then decreased at 6-12 years; and for TREC/106 PBMC at birth in HEU and controls. By contrast, plasma MIP-1β levels were lower in HEU than in controls (p=0.009) at 12 months, and IL-4 levels were higher in HEU than controls (p=0.04) at 6-12 years. Immune activation was higher in HEU at 12 months and at 6-12 years than controls based on frequencies of CD38+HLA-DR+CD8+T cells (p=0.05) and of CD38+HLA-DR+CD4+T cells (p=0.006). Resting memory and activated mature B cells increased from birth to 6-12 years in both groups. The development of the immune system in vertically HEU individuals is comparable to the general population in most parameters, but subtle or transient differences exist. Their role in influencing clinical incidences in HEU is unknown.

Immune response of the Antarctic sea urchin Sterechinus neumayeri: cellular, molecular and physiological approach / Respuesta inmune del erizo de mar Antártico Sterechinus neumayeri: un enfoque celular, molecular y fisiológico

Abstract In the Antarctic marine environment, the water temperature is usually between 2 and - 1.9 °C. Consequently, some Antarctic species have lost the capacity to adapt to sudden changes in temperature. The study of the immune response in Antarctic sea urchin (Sterechinus neumayeri) could help us understand the future impacts of global warming on endemic animals in the Antarctic Peninsula. In this study, the Antarctic sea urchins were challenged with lipopolysaccharides and Vibrio alginolitycus. The cellular response was evaluated by the number of coelomocytes and phagocytosis. A significant increase was observed in red sphere cells and total coelomocytes in animals exposed to LPS. A significant rise in phagocytosis in animals stimulated by LPS was also evidenced. Moreover, the gene expression of three immune related genes was measured by qPCR, showing a rapid increase in their expression levels. By contrast, these immune genes showed a depression in their expression by a thermal effect at 5 and 10 °C. In addition, during bacterial injection, the oxygen consumption was higher in challenged animals. Our results showed that the immune response in the Antarctic sea urchin may be affected by acute thermal stress and that this immune response has a metabolic cost. Rev. Biol. Trop. 63 (Suppl. 2): 309-320. Epub 2015 June 01.

Resumen En el medio ambiente de la Antártica la temperatura del agua es de entre 2 y - 1.9 °C. Por consecuencia ciertas especies han perdido la capacidad de adaptarse a los cambios repentinos de la temperatura del agua. El estudio de la respuesta inmune del erizo antártico (Sterechinus neumayeri) podría ayudar a comprender los futuros impactos en los animales endémicos del cambio climático en la Península Antártica. En este estudio nosotros hemos evaluado la respuesta inmunitaria de S. neumayeri respecto de estimulaciones con bacterias (Lipopolisacáridos y Vibrio alginolitycus) asi como durante el estrés térmico a 5 y 10 °C. La respuesta del erizo fue evaluada en relación al número de celomocitos circulantes, capacidad fagocítica de estos y por el análisis de la expresión de tres genes inmunitarios. Después de la estimulación con LPS un aumento significativo de células esferoidales rojas, de amebocitos fagocíticos y de celomocitos totales fue observado después de las primeras horas de estimulación, de la misma manera que la capacidad fagocítica. Por otra parte los tres genes inmunes medidos mostraron un aumento significativo de su expresión por qPCR después de la estimulación con LPS. El estrés térmico de 5 °C produjo un aumento de la expresión de estos tres genes inmunitarios, por el contrario a una temperatura más alta (10 °C) se produce la reducción de dos de entre ellos. Adicionalmente un aumento del consumo de oxígeno fue observado durante la estimulación bacteriana. Nuestros resultados muestran que la respuesta inmunitaria en el erizo antártico puede ser afectada por el estrés térmico agudo y que la respuesta inmune en invertebrados antárticos tendría un costo metabólico.

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.