Composite-derived monomers affect cell viability and cytokine expression in human leukocytes stimulated with Porphyromonas gingivalis

Abstract Objectives: Dental composites release unreacted resin monomers into the oral environment, even after polymerization. Periodontal cells are, therefore, exposed to substances that potentially elicit the immune inflammatory response. The underlying molecular mechanisms associated with the interaction between resin monomers and human immune cells found in the gingival crevicular fluid are not fully understood yet. This study investigated the ability of bisphenol A-glycidyl methacrylate (BISGMA), urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) to induce apoptosis and cytokine release by human leukocytes stimulated with a periodontal pathogen. Methodology: Peripheral blood mononuclear cells (PBMC) from 16 healthy individuals were included in this study. To determine the toxicity, the PBMC were incubated for 20 hours, with monomers, for the analysis of cell viability using MTT assay. To evaluate cell death in the populations of monocytes and lymphocytes, they were exposed to sub-lethal doses of each monomer and of heat-inactivated Porphyromonas gingivalis (P. gingivalis) for 5 hours. Secretions of IL-1β, IL-6, IL-10 and TNF-α were determined by ELISA after 20 hours. Results: UDMA and TEGDMA induced apoptosis after a short-time exposure. Bacterial challenge induced significant production of IL-1β and TNF-α (p<0.05). TEGDMA reduced the bacterial-induced release of IL-1β and TNF-α, whereas UDMA reduced IL-1β release (p<0.05). These monomers did not affect IL-10 and IL-6 secretion. BISGMA did not significantly interfere in cytokine release. Conclusions: These results show that resin monomers are toxic to PBMC in a dose-dependent manner, and may influence the local immune inflammatory response and tissue damage mechanisms via regulation of bacterial-induced IL-1β and TNF-α secretion by PBMC.

Efecto del extracto vegetal JV-001 sobre elementos sanguíneos concapacidad fagocítica / Effect of the plant extract JV-001 on blood elements with phagocytic capacity

INTRODUCCIÓN: La respuesta inmune combina una serie de señales y mecanismos complejos para producirse, las que pueden ser moduladas y potenciadas. Esto, según el conocimiento popular es posible empleando extractos vegetales, siendo uno el extracto vegetal JV-001. Éste demostró, en estudios previos, la capacidad de activar y mejorar la respuesta inmune celular de origen murino, pero su efecto a nivel leucocitario humano es desconocido; debido a esto, el objetivo de la investigación fue estudiar el efecto del JV-001 en leucocitos humanos. MATERIAL Y MÉTODO: Para este trabajo se extrajo sangre heparinizada a varones sanos, ésta fue tratada, en primera instancia, con NH-4Cl 64 por ciento para obtener leucocitos totales, y en otra, separada su fracción mononuclear mediante gradiente de Histopaque®. Las células obtenidas se ajustaron a 8x106; alicuotas de 100 uL de JV-001 o PBS. La placa se incubó a 37ºC por 30 minutos, luego se lavaron y la peroxidasa remanente en cada pocillo se determinó revelándola con Ortofenilendiamina y H2O2, expresando lo obtenido como absorbancia leída a 450 nm. RESULTADOS: Se identificó un aumento en la adhesión de leucocitos totales entre un 44,35 por ciento y 63,3 por ciento expresado mediante la obtención de un delta positivo de absorbancia con respecto a los controles. DISCUSIÓN: Se desconoce el mecanismo que incrementa la adhesión, pero probablemente obedece al aumento en la expresión de integrinas. Es relevante destacar que este aumento no ocurre al incubar las células mononucleares o polimorfonucleares por separado, sugiriendo que el principio activo de JV-001 no actúa directa ni únicamente sobre células adherentes.

INTRODUCTION: The immune response combines a series of signals and complex mechanisms to occur, which can be modulated and enhanced. This one according the popular knowledge is possible using plant extracts, being one of them the plant extract JV-001. This showed, in previous studies, the ability to activate and enhance the cellular immune response of murine origin, but its effect on human leukocyte level is unknown, so the objective of this work was to study the effect of JV-001 in human leukocytes. MATERIAL AND METHOD: In this paper was used heparinized blood of healthy males, at first instance, this was treated with NH4Cl 64 percent to obtain total leukocytes, and in another, separate the mononuclear fraction by Histopaque®gradient. The cells obtained were adjusted to 8x106; aliquots of 100 uL of JV-001 or PBS. The plate was incubated at 37ºC for 30 minutes and then washed, the peroxidase remaining in each recipient was revealed and determined with Orthophenylenediamine and H2O2, expressing the proceeds as absorbance read at 450 nm. RESULTS: The results showed an increase in total leukocyte adhesion between 44.35 percent and 63.3 percent expressed by obtaining a positive delta absorbance with respect to controls. DISCUSSION: The mechanism that increases the adhesion is unknown, but probably due to increased expression of integrins. It is important to note that this increase does not occur upon incubation of mononuclear or polymorphonuclear cells separately, suggesting that the active principle of JV-001 not only does not act directly on adherent cells.

Determinación de cobre, magnesio y zinc en leucocitos mononucleares mediante espectrometría de absorción atómica con llama / Determination of copper, magnesium and zinc in mononuclear leukocytes by flame atomic absorption spectrometry

En este trabajo se evaluó un nuevo microinyector de inyección en flujo para la determinación de Cu, Mg y Zn en células mononucleares de la sangre. Este dispositivo permitió analizar muestras en el orden de microlitros mediante espectrometría de absorción atómica con llama, es fácil de construir y adaptar al inyector convencional del espectrofotómetro de absorción atómica. En la determinación de Cu, Mg y Zn se obtuvieron límites de detección de 106, 65 y 37 µg L-1,respectivamente. En las pruebas de recuperación de estos elementos en leucocitos mononucleares se encontraron porcentajes de recuperación entre 98 y 110%.

In this paper we evaluated a new micro-flow injector for the determination of the concentrations of Cu, Mg and Zn in mononuclear blood cells. This device analyzed sample volumes in the order of microliters by flame atomic absorption spectrometry; it is inexpensive, and easy to build and to adapt to the conventional injector of the atomic absorption spectrophotometer. Detection limits of 106, 65 and 37 µg L-1 for Cu, Mg and Zn were obtained, respectively. The percentages of recovery tests were found between 98 and 110%.

TNF receptor-associated factor 6 expression in peripheral blood mononuclear cells of patients with primary sjögren's syndrome and its cinical significance / 第二军医大学学报

Objeciive To investigative the expressoin of TNF receptor-associated factor 6 (TRAF6) in the peripheral blood mononuclear cells (PBMCs) of primary Sjögren syndrome (pSS) patients and its clinical significance. Methods The expression of TRAF6 mRNA was determined by quantified reverse transceipsional PCR in the PBMCs of 23 pSS patients and 23 heaithy controls. The correlation 0f TRAF6 mRNA level with the laboratory findings, including serum rheumatoid factor (RF), globulin, anli-SSA and anli-SSA antibody, were analyzed by Spearman test. Nine of the pSS patients were followed up and their TRAF6 mRNA expression was detected after glucocorlicoid treatment for 1-2 months. Results The TRAF6 mRNA level in pSS patients was 2. 77 times higher than that in the healthy controls (P< 0. 01). The TRAF6 mRNA level in pSS patients was positively correlated with serum RF (r=0. 45, P = 0. 03) and globidln (r=0. 43, P = 0, 04), but was not assosiated the antibody. TRAF6 mRNA level was decreased by aboijt 50% n the palients who were treated with glucocorlicoids for 1-2 mcmths (P<0. 05). Conclusion TRAF6 n PBMCs is involved in the pathogenesiss of pSS and it may be a potential biomarker for pSS.

Irradiation of Donor Mononuclear Cells for Treatment of Chemorefractory Metastatic Solid Cancers: A Community-Based Immune Transplant Pilot Study / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Chemotherapy has demonstrated ability to generate tumor antigens secondary to induction of apoptosis, against which human leukocyte antigen-compatible, irradiated, related donor mononuclear cells may be administered with immune stimulation to activate antigen presenting and cytotoxic T cells, while minimizing risk of graft-versus-host disease (GVHD). The present study endeavours to describe feasibility and efficacy of this treatment, specifically in the community setting. MATERIALS AND METHODS: Eligible patients had rapidly progressive, chemorefractory metastatic solid tumors. Treatment consisted of intravenous etoposide and cyclosporine for three days followed by granulocyte-macrophage colony-stimulating factor for 5 days. The following week, 5x10(7) haploidentical or more closely matched irradiated donor mononuclear cells were given weekly for 10 weeks along with interleukin-2. RESULTS: Three patients were enrolled, and the regimen was well-tolerated, with no GVHD observed. All patients had clinical response, despite advanced and heavily pretreated disease. CONCLUSION: The above-outlined protocol demonstrates favorable tolerability and efficacy, and appears to be feasible in the community setting. While the optimal chemotherapy, immunostimulation, and irradiation regimens may be further optimized, future investigation appears warranted, and may include community oncology programs.

Increased Reactive Oxygen Species Production by Peripheral Blood Mononuclear Leukocytes, not by Polymorphonuclear Leukocytes, in Atopic Dermatitis / 소아알레르기및호흡기학회지

PURPOSE: Reactive oxygen species (ROS) are known as a potential mediators that sustain chronic inflammation in atopic dermatitis (AD). To determine the role of peripheral blood mononuclear leukocytes (MO) and polymorphonuclear leukocytes (PMN) in prolonged inflammation, ROS generation of those cells in AD was examined. METHODS: Seventeen AD patients and 10 healthy controls were enrolled. MO and PMN were stimulated with the reagents: phobol ester (PMA), adenosine triphosphate (ATP), and chemotactic peptide (f-MLP). ROS levels were measured using chemiluminescence assay. RESULTS: In AD, chemiluminescence response of unstimulated MO was higher than that of normal controls. MO from AD patients produced 1.58-1.80 higher ROS for up to 30 minutes than the controls. When the cells were treated with the reagents (PMA, ATP, f-MLP), all the stimuli enhanced chemiluminescence activity of MO. When MO were treated with PMA, the ratio of ROS produced by MO of patients to that of the controls decreased. When the cells were treated with either ATP or f-MLP, the quantity of ROS generated by MO from the controls was greater than the controls. PMN from both AD patients and the controls generated ROS for 30 min similarly. As treated with the reagents, PMN from AD patients produced a smaller ROS than the controls. CONCLUSION: These results indicate MO but not PMN from AD patients were primed and ready for activation in vivo, and a reduced function of PMN from AD patients was observed. In conclusion, enhanced respiratory burst activity of MO is implicated in the prolonged inflammation of AD.

Inhibition of Platelet Aggregation by Mononuclear Leukocytes / 대한혈액학회지

BACKGROUND: Although normal vascular endothelium prevents adhesion and aggregation of platelets by the release of nitric oxide (NO) and prostacyclin, circulating blood cells, such as polymorphonuclear leukocytes (PMNs) and mononuclear leukocytes (ML) may be considered to be also important in modulating platelet aggregation. Recently, nitric oxide synthase (NOS) activity was found in PMNs and ML, so these cells can also release NO to inhibit platelet aggregation. We studied platelet-ML interactions using an experimental model in which isolated ML were placed in the aggregometer in contact with human platelets, stimulated by collagen. METHODS: Platelet count in platelet-rich plasma (PRP) was adjusted to approximately 300x109/L. ML were separated using Ficoll-Hypaque (specific gravity 1.077) and finally resuspended at 1, 3 and 5x109/L, in Tyrode albumin buffer (TAB), respectively. Platelet aggregation was measured with Chrono-Log Aggregometer (USA) after adding variable numbers of the ML, stimulating with 2.5, 5 and 10 microgram/mL of collagen. Mechanisms of ML to inhibit the platelet aggregation were evaluated after incubating the ML with 10 micrometer indomethacin and 300 micrometer NG-monomethyl-L-arginine (L-NMMA). RESULTS: Non-stimulated ML (3x109/L) inhibited (43.2 +/- 19.6 versus TAB control 69.2 +/- 10.7% transmission) the platelet aggregation induced by 2.5 microgram/mL of collagen. The inhibition was not attenuated by increasing the concentration of collagen from 5.0 microgram/ mL (50.1 +/- 18.0% versus TAB control 75.5 +/- 13.1%, P<0.001) to 10 microgram/mL (62.9 +/- 17.3% versus TAB contol 82.3 +/- 12.6%, P<0.01). In addition, it was dependent on the number of ML and incubation time. While preincubation of the ML with indomethcin did not affect the antiaggregating capacity of the ML (63.4 +/- 11.1 versus TAB control 73.3 +/- 7.3%), preincubation of the ML with L-NMMA slightly inhibit the antiaggregating capacity of the ML (86.6 +/- 6.8 versus TAB control 73.3 +/- 7.3%). CONCLUSION: These findings suggest that blood ML inhibited the collagen-induced platelet aggregation, of which mechanism appears to be only partly dependent on NO and to be independent on prostaglandins. Release of other substances affecting platelet aggregation from ML requires to be clarified. Using our experimental model, it has been demonstrated that cell-cell contact may facilitate the exchange of a wide array of mediators between platelets and ML which may influence the cellular responses. This experimental model thus allows to study interactions between platelets and ML.