Cytokine expression profile and blood parameter evaluation of patients undergoing cardiac surgery.

Cardiac surgery is accompanied by an important immune response that is poorly understood. This inflammatory response is caused by several stimuli: surgical trauma, cardiopulmonary bypass apparatus, aortic-cross clamping, reperfusion injury and hypothermia. The aim of the present study is to investigate the cytokine level profile involved in the inflammatory pathway of patients undergoing cardiac surgery. One hundred and two patients undergoing elective cardiac surgery utilizing cardiopulmonary bypass (CPB) apparatus were enrolled in the study. In the hematological and biochemical profiles investigated, we observed a significant increase of WBC and blood glucose concentration and a strong decrease of RBC, HB, HCT and PLT 24 h post-surgery compared to baseline and immediately after surgery groups. Furthermore, we found a modulation of cytokine levels mostly for IL-10 and an increase of IL-6, detected at 6 h post-surgery, IL-8 at 6 and 24 h, and TNFα only at 24 h post-surgery. In conclusion, these findings evidence a time course profile on cytokine levels and a balance between pro- and anti-inflammatory cytokine activation during and after cardiac surgery. In fact, IL-6 and IL-10, a pro- and an anti-inflammatory cytokine, respectively, increased immediately after surgery. The plasma level of TNF-α could be inhibited by the high concentration of IL-10 up to 6 h post-surgery. An IL-10 reduction at baseline level, after 24 h post-surgery, could explain a rise of TNF-α plasma concentration. On the other hand, considering the dual role of IL-6 on inflammation acting both as an activator of inflammatory cascade or an anti-inflammatory agent, the increased IL-6 levels 24 h after surgery could be related to the negative feedback action on TNFα activity.

Cytokine modulation in patients with idiopathic pulmonary fibrosis undergoing treatment with steroids, immunosuppressants, and IFN-γ 1b.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown etiology and pathogenic mechanisms. From an etiopathogenic point of view, alveolar macrophages play a key role in accumulation of fibroblasts and deposition of collagen and extracellular matrix by releasing specific cytokines and inflammatory mediators. IPF seems to be also associated with circulating fibrocytes, which might be involved with an abnormal pulmonary vascular repair and remodeling. Based on its hypothesized pathologic mechanisms, anti-inflammatory, anti-fibrotic and immunosuppressive therapies are often used. For these reasons, Interferon-g (IFN-g) has been used to exploit its activity on macrophages and fibroblasts. The aim of this study was to investigate the response to corticosteroids and/or IFN-g 1b treatments based on pulmonary function tests and on inflammatory cytokine patterns of expression on bronchoalveolar lavage (BAL), at baseline and during and after the therapies. Unlike previous studies, we analyzed a period of therapy longer than 1 year. Our results demonstrated the effectiveness of IFN-γ in a group of IPF patients in whom the treatment was prolonged for over a year. These data suggest a positive role of IFN-γ; treatment in patients in the initial stage of the disease.

Effect of low energy light irradiation by light emitting diode on U937 cells.

Photobiomodulation (PBM) can induce a set of different biological modulators either in vitro or in vivo. Experimental evidence has highlighted the role of light effects on the mechanisms related to inflammation, apoptosis and autophagy. The goal of this project was the evaluation of PBM on U937, an established cell line of histiocytic lymphoma origin. Several aspects of modulation of proinflammatory pathways were analyzed and autophagic and proapoptotic mechanisms related to low laser light exposure of cells were studied. As a source of low energy light emission, we used an NIR-LED device, characterized by an 880 nm-wavelength as light source. Flow cytometry analysis was performed on supernatants of controls and treated U937 cells to detect inflammatory cytokine levels. In order to evaluate NF-kB and caspase3 expressions, Western blot analysis was performed according to standard procedures. In this report, we show the effect of PBM on a monocyte/macrophage established tumor cell line (U-937). We demonstrate that LED exposure, in the presence or absence of lipopolysaccharide (LPS), activates cell degranulation, increased expression of Interleukin-8 (IL-8) and modulation of beta galactosidase activity. Evidence shows that the well-known pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and the apoptotic marker (caspase3/cleaved-caspase3 ratio) are up-regulated in response to a proinflammatory biochemical pathway.

Cyanidin reduces preadipocyte differentiation and relative ChREBP expression.

Adipogenesis is a continuous process even in adult adipose tissue for the presence of preadipocytes that, when subjected to appropriate stimuli can proliferate and differentiate. ChREBP, the essential transcription factor for lipogenesis, is expressed in all tissues, but mainly in lipogenic organs. In this study, we focused on ChREBP expression during preadipocytes differentiation. Since it was found that cyanidin-3 reduces body weight in mice even in the presence of a high-fat diet, by decreasing levels of blood glucose and by improving insulin sensitivity, we studied the effect of this substance on adipogenic differentiation. For this purpose we used preadipocytes obtained from subcutaneous and visceral human adipose explant tissue, characterized and stimulated to differentiate in selective media. On cytofluorimetric analysis these cells showed mesenchymal markers (CD29, CD90, CD44), whereas they were negative for hematopoietic markers (CD45, CD10, CD117,CD31). ChREBP expression levels were quantified by immunoelectron-microscopy and western blotting analysis. In this report we show that ChREBP is expressed in preadipocytes (both nuclear and cytoplasmic compartments); the cytoplasmic level of ChREBP increased by 50 percent on day seven of differentiation into mature adipocytes. Cyanidin reduced differentiation by 20 percent (as evaluated by red oil O staining) and the expression of ChREBP. In addition, cyanidin-treated cells showed abnormal morphology, a square shape with irregular size, probably due to the fact that cyanidin may interfere with the extracellular matrix. These findings suggest that dietary cyanidin, may have inhibitory effects on adipogenesis.

Changes in matrix extracellular phosphoglycoprotein expression before and during in vitro osteogenic differentiation of human dental papilla mesenchymal cells.

The purpose of this study is to characterise the expression of matrix extracellular phosphoglycoprotein (MEPE) in cultured mesenchymal cells isolated from human dental papilla (PaMCs) of impacted third molars either before or during differentiation of these cells into osteo/odontoblasts. PaMCs, like mesenchymal cells deriving from human dental pulp (DPMCs), resulted positive for a number of mesenchymal markers including CD146 and STRO-1. During the first week in culture they showed a faster proliferation rate than DPMCs, coupled to an earlier down-regulation of MEPE. Also when the cells were further cultured in osteogenic medium (containing beta-glycerophosphate, ascorbic acid and dexamethasone) for 40 days, MEPE down-regulation coupled to an increased expression of osteogenic markers, such as osteocalcin and alkaline phosphatase, occurred earlier in PaMCs than in DPMCs. Thus, our data, indicating that also in PaMCs MEPE expression is higher when cells proliferate, whereas it is downregulated as cells differentiated, are in favour of a role of MEPE as an early regulator of odontogenic differentiation. We also confirm the superior proliferative potential of PaMCs in comparison with DPMCs, coupled to a more rapid induction of osteogenic differentiation. Therefore, these cells represent an optimal source to be conveniently used for dental tissue engineering and tooth regeneration.

Early cytokine modulation after the rapid induction phase of sublingual immunotherapy with mite monomeric allergoids.

The influence of different treatment schedules of sublingual immunotherapy (SLIT) in activating IL-10-producing T-cells, crucial in inducing allergen-specific tolerance, is not completely understood. The present work was designed to evaluate allergen driven interleukin release by mononuclear cells in the early phase of SLIT, after application of different induction schemes. Twenty mite-allergic patients were enrolled, 10 (group A) treated with a traditional 98 day induction scheme and 10 (group B) with a 16 day scheme with monomeric allergoid vaccine. At the end of the induction phase, the cumulative doses taken by group A and group B patients were equivalent to 50.5 and 50.3 microg of mite group 1 allergens, respectively. The release of Th1-, Th2- and Treg-related interleukins was assessed in culture supernatants of 5 microg/ml Der-p1-stimulated mononuclear cells, isolated before and after the induction phases. No relevant treatment-related side effects were observed. Interleukin release was similar in the two groups at the enrolment. Non-stimulated and Der p 1 stimulated release of studied cytokines was similar in the two groups at enrolment. Der p 1 stimulation significantly increased IL-10 release (p<0.0002) after treatment in group B patients, and this effect was higher (p=0.05) compared to group A patients. Furthermore, at the end of SLIT induction TNF-alpha, IL-4 and IFN-gamma production were reduced in group B patients (p<0.05, p=0.062 and p=0.060, respectively). The rapid induction scheme of sublingual immunotherapy induces an early immune suppression more effectively than the slower one. The rapid induction scheme should be the preferential way to start sublingual immunotherapy, particularly when monomeric allergoids are utilized.

Morphostructural analysis of human follicular stem cells on highly porous bone hydroxyapatite scaffold.

In this study we investigated the in vitro behaviour, morphostructure and extracellular matrix synthesis of human dental follicular stem cells (hDFSCs) isolated from human dental bud, which resulted to be positive for mesenchymal markers (CD29, CD90, CD146 and CD166) by FACS analysis. Cells were analysed by light and electronic microscopy to evaluate their biological response either at week 1, that is before differentiation, or at weeks 3-6, when they had been cultured in osteogenic medium onto a highly porous natural scaffold material (Bio-Oss). Microscopy analysis of primary culture cells showed they had a mesenchymal stem cell-like morphostructure, spindle shaped, similar to the culture of mesenchymal stem cells derived from adult bone marrow. Also, after osteogenic differentiation, these analyses indicate typical osteoblast morphostructure and reveale a tri-dimensional organization of the cells and deposition of extracellular matrix (ECM) in close contact with biomaterial. This approach would allow to personalize the scaffold for bone tissue engineering in order to accelerate the process of osteogenesis.

Cobalt nano-particles modulate cytokine in vitro release by human mononuclear cells mimicking autoimmune disease.

The use of particles from micro to nanoscale provides benefits to diverse scientific fields, but because a large percentage of their atoms lie on the surface, nanomaterials could be highly reactive and pose potential risks to humans. Due to their wide range of application, Cobalt nano-particles are of great interest both in industry and in life-science. To date, there are few studies on Co nano-particle toxicology. In this respect, this study aims at evaluating in vitro the potential interference of Co nano-particles on the production of several cytokines (IL-2, IL-4, IL-6, IL-10, IFNgamma and TNFalpha) by PBMCs, comparing their effects to those of Co micro-particles and Co solution (CoCl2). Cells were cultured in Opticell flasks with escalating concentrations (10-5, 10-6 and 10-7 M), of Co nano and micro-particles and CoCl2 or without metal. Cytokines were quantified in the supernatants using a human Th1/Th2 cytokine cytometric bead array. Co micro-particles showed a greater inhibitory effect compared to other Co forms. Its inhibitory activity was detected at all concentrations and towards all cytokines, whereas Co solutions selectively inhibited IL-2, IL-10 and TNF-alpha at maximal concentration. Co nano-particles induced an increase of TNF-alpha and IFN-gamma release and an inhibition of IL-10 and IL-2: a cytokine pattern similar to that detected in the experimental and clinical autoimmunity. On the basis of the obtained data, immune endpoints should be sought in the next series of studies both in vitro and in vivo in subjects exposed to cobalt nano-particles.

Follow up study on the immune response to low frequency electromagnetic fields in men and women working in a museum.

Seven women and eight men, exposed to low frequency (50 Hz) electromagnetic fields (EMFs) in a museum for 20 hours a week, were investigated in the years 1999 and 2005. During the first study, the mean EMF exposure in the working place was 1.7 microT and 1.1 microT, respectively. In the first investigation, the EMF-exposed men showed reduced blood NK lymphocytes in relation to controls, while EMF-exposed women presented reduced PHA-stimulated IFN-gamma release from peripheral blood mononuclear cells (PBMC). In the year 2005, blood cytotoxic activity, state and trait anxiety (STAI I and II, respectively) and occupational stress were also investigated. The scores of STAI I and II of the control women were slightly higher than those of the control men. EMF-exposed men showed higher occupational stress but normal immune parameters. EMF-exposed women showed, in relation to controls, lower PHA-stimulated IFN-gamma release from PBMC and reduced blood cytotoxic activity/CD45+-CD16+-56+ NK lymphocytes (but not per ml of blood). One of the women exposed to EMF, who worked a night shift, showed marked lymphopenia with very low NK lymphocytes and reduced IFN-gamma release; these immune parameters returned to normal following a change of work site. This study suggests that low frequency EMFs affect the immune functions of women more than those of men. Moreover, the determination of immune parameters seems to be a useful marker of the health effects of exposure to EMFs.

The immune response of women with prolonged exposure to electromagnetic fields produced by radiotelevision broadcasting stations.

Twelve women, five of them housewives, exposed in their residences to electromagnetic fields (EMFs)emitted by radio-television broadcasting stations for a mean period of 13 years, were investigated. The EMFs in the balconies of the homes were (mean + S.D.) 4.3 + 1.4 V/m in the year 2000 and 3.7 + 1.3 V/m in 2005, while the exposure in the nearby area was <2.0 V/m. The EMF exposed women showed in 2000 reduced blood NK lymphocytes as well as PHA stimulated PBMC proliferation and IL-2 and IFN-gamma release. In the year 2005, the EMF exposed women and 48 control women with similar ages(mean 43 years), smoking habits, atopy and social level were investigated. State (temporary) and trait(tendency of the personality) anxiety were determined by STAI I and II, respectively. Blood cytotoxic activity and lymphocyte subsets were also determined. The ratio STAI I/STAI II of the EMF exposed group was lower than that of the control group. The blood cytotoxic activity of the exposed women was lower (p<0.01), percent of B CD45+-CD19+ lymphocytes higher and percent of CD45+-CD3+-CD8+ cells lower (p<0.05). Moreover, cytotoxic activity/CD45+-CD16+-56+ NK lymphocytes of the controls was negatively correlated with STAI I and STAI II (p<0.001). In conclusion, this study demonstrates reduced blood cytotoxic activity and increased trait anxiety in relation to state anxiety in EMF exposed women. An effect of EMFs on immune functions, in part mediated by nervous mechanisms, may be hypothesized. However, the influence of lifestyle may not be excluded.

Effect of occupational stress and anxiety on natural killer lymphocyte activity of men and women employed in a university.

The aim of this study is the immune response of the staff of a university and a museum (referent group). 46 women and 40 men with similar mean age (44 years) were investigated. The first group of women (A) worked in libraries, B in offices in contact with students, C as secretaries and D in a museum. One group of men (E) was composed of employees of the university, one worked in a library temporarily located underground (F) and the last group (G) in a museum. STAI I and II (state and trait anxiety, respectively), and occupational stress were measured by questionnaires. Blood samples were collected for determining NK cytotoxic activity vs human erythroleukaemia cells and the lymphocyte subsets CD45+, CD45+-CD3+, CD45+-CD3+-CD4+, CD45+-CD3+-CD8+, CD45+-CD3-CD8+, CD3+-CD16+-56+ and CD3+-CD19+. Group B showed significantly higher score of occupational stress and STAI I (but not of STAI II) than the other groups of women. Group F of men showed higher levels of occupational stress and both STAI I and II than groups E and G. Group B of women and F of men also showed significantly lower values of blood cytotoxic activity. The scores of STAI I and II were negatively correlated with the cytotoxic activity expressed per ml of blood and/or total lymphocytes. and/or NK CD45+-CD16+-CD56+ cells. In conclusion, this study shows that occupational stress may increase anxiety which is negatively correlated with blood cytotoxic activity. This is a useful tool for determining the health effects of stress.