Biljana Bozic

Production of proinflammatory and immunoregulatory cytokines by inflammatory cells from periapical lesions in culture

BACKGROUND The role of cytokines in pathogenesis of periapical lesions is not well understood. The aim of this study was to study the correlation between proinflammatory and immunoregulatory cytokines in periapical lesions and their relationship with cellular composition and clinical presentation. METHODS Inflammatory cells were isolated from 67 human periapical lesions and cultivated for 24 h. The levels of proinflammatory cytokines: interleukin-1 beta (IL-1beta), IL-6, IL-8 and tumour necrosis factor alpha (TNF-alpha) and immunoregulatory cytokines: transforming growth factor-beta (TGF-beta) and IL-10 were determined in culture supernatants using a fluorescent bead immunoassay or ELISA. The phenotype of cells was analysed by immunocytochemistry. RESULTS Inflammatory cells from symptomatic lesions which contained higher proportion of granulocytes, secreted higher levels of IL-1, IL-6 and IL-8 compared with asymptomatic lesions. Large-size lesions contained lower percentages of mononuclear phagocytes, higher percentages of CD8(+) T cells and produced higher levels of TNF-alpha, IL-6 and IL-10 compared with small-size lesions. There were negative correlations between the concentrations of TGF-beta and proinflammatory cytokines. TGF-beta, added to cultures, downregulated the levels of proinflammatory cytokines more strongly than IL-10, independently of clinical presentation of the lesions. By contrast, exogenous IL-10 was mainly immunosuppressive in cultures of asymptomatic lesions. CONCLUSION Symptomatic lesions are characterized by higher production of proinflammatory cytokines. Immunoregulatory cytokines are more important for suppression of inflammation in asymptomatic lesions and in this context the effect of TGF-beta is more potent and different from IL-10.

Relationship of reduced cerebral blood flow and heart failure severity in elderly males

Introduction. Brain detrimental effects are under-recognised complication of chronic heart failure (CHF). One of the major causes may be cerebral hypoperfusion. This study was designed to investigate the relationship between cerebral blood flow (CBF) and severity of CHF as well as to evaluate its determinants among different parameters of cardiac dysfunction. Methods. Seventy-one CHF males with NYHA class II and III and 20 control subjects age ≥ 55 years were recruited. CBF was evaluated by colour duplex sonography of extracranial arteries. Echocardiography, 6-min walk test, quality of life and endothelial function were also assessed. Serum NT-pro-BNP and adipokines levels (adiponectin and leptin) were measured. Results. CBF was significantly reduced in elderly patients with CHF compared to healthy controls (677 ± 170 vs 783 ± 128 ml/min, p = 0.011). Reduced CBF was associated with reduced left ventricular ejection fraction (LVEF) (r = 0.271, p = 0.022), lower 6-min walk distance (r = 0.339, p = 0.004), deteriorated quality of life (r = −0.327, p = 0.005), increased serum adiponectin (r = −0.359, p = 0.002), and NT-pro-BNP levels (r = −0.375, p = 0.001). In multivariate regression analysis, LVEF and adiponectin were independently associated with reduced CBF in CHF patients (R2 = 0.289). Conclusion. CBF was reduced in elderly males with mild-to-moderate CHF, and was associated with factors that represent the severity of CHF including high serum adiponectin and NT-pro-BNP levels, decreased LVEF, impaired physical performance, and deteriorated quality of life.

Association of increased parathyroid hormone with neuroendocrine activation and endothelial dysfunction in elderly men with heart failure

High PTH levels have been reported in patients with chronic heart failure (CHF). Similarly, its levels increase with aging and are related to impaired survival in elderly adults. However, its relationship with neuroendocrine activation and endothelial dysfunction in CHF has not been previously studied. Seventy-three CHF males with New York Heart Association (NYHA) classes II and III and 20 control subjects aged >55 yr were recruited. PTH, 25-hydroxyvita-min D [25(OH)D], N-terminal pro-brain natriuretic peptide (NT-pro-BNP), adiponectin, and osteoprotegerin were measured. Endothelial function (brachial flow mediated dilation), echocardiography, physical performance, and quality of life were assessed, as well. CHF patients had markedly increased serum PTH (77±33 vs 40±11 pg/ml, p<0.0001), NT-pro-BNP [1809 (2742) vs 67 (74) pg/ml, p<0.0001], adiponectin (17±9 vs 10±2 µg/ml, p<0.0001), osteoprotegerin, whereas 25(OH)D levels were decreased compared to controls. Increased PTH is positively correlated with NT-pro-BNP (r=0.399, p<0.0001), adiponectin (r=0.398, p<0.0001), and osteoprotegerin, whereas negatively with 25(OH)D in CHF patients. Additionally, increased serum PTH was associated with endothelial dysfunction, echocardiographic variables of heart failure progression, impaired physical performance, and deteriorated quality of life. In a multivariate linear regression analysis, increased serum PTH was independently associated with neuroendocrine activation (NT-pro-BNP, adiponectin) and endothelial dysfunction in elderly CHF men (R2=0.455). Additionally, demonstrated relations with other well-established variables of heart failure severity suggest the potential role of serum PTH in the pathogenesis and non-invasive monitoring of heart failure progression. Future studies are needed to evaluate the predictive value of serum PTH for clinical outcomes as well as beneficial potential of PTH suppression in CHF patients.

Association of adiponectin with peripheral muscle status in elderly patients with heart failure

BACKGROUND Reduced peripheral muscle mass was demonstrated in patients with chronic heart failure (HF). Adipokines may have potent metabolic effects on skeletal muscle. The associations between adipokines, peripheral muscle mass, and muscle function have been poorly investigated in patients with HF. METHODS We measured markers of fat and bone metabolism (adiponectin, leptin, 25-hydroxy vitamin D, parathyroid hormone, osteoprotegerin, RANKL), N-terminal pro B-type natriuretic peptide (NT-pro-BNP) in 73 non-cachectic, non-diabetic, male patients with chronic HF (age: 68 ± 7 years, New York Heart Association class II/III: 76/26%, left ventricular ejection fraction 29 ± 8%) and 20 healthy controls of similar age. Lean mass as a measure of skeletal muscle mass was measured by dual energy X-ray absorptiometry (DEXA), while muscle strength was assessed by hand grip strength measured by Jamar dynamometer. RESULTS Serum levels of adiponectin, parathyroid hormone, osteoprotegerin, RANKL, and NT-pro-BNP were elevated in patients with chronic HF compared to healthy controls (all p<0.0001), while no difference in serum levels of leptin, testosterone or SHBG was noted. Levels of 25-hydroxy vitamin D were reduced (p=0.002) in HF group. Peripheral lean mass and hand grip strength were reduced in patients with HF compared to healthy subjects (p=0.006 and p<0.0001, respectively). Using backward selection multivariable regression, serum levels of increased adiponectin remained significantly associated with reduced arm lean mass and muscle strength. CONCLUSIONS Our findings may indicate a cross-sectional metabolic association of increased serum adiponectin with reduced peripheral muscle mass and muscle strength in non-cachectic, non-diabetic, elderly HF patients.

Relationship Between High Circulating Adiponectin With Bone Mineral Density and Bone Metabolism in Elderly Males With Chronic Heart Failure

BACKGROUND The aim of the study was to investigate the associations of adiponectin and leptin to bone mass and bone specific surrogates in elderly males with chronic heart failure (CHF). METHODS AND RESULTS Seventy-three males (mean age 68 +/- 7 years) with stable mild to moderate CHF and 20 healthy individuals age- and body mass index-matching underwent dual energy x-ray absorptiometry measurements (bone mineral density (BMD) at hip and lumbar spine, total bone mineral content, and body composition); echocardiography; 6-minute walk test; grip strength; and biochemical assessment including adiponectin, leptin, bone specific surrogates (osteocalcin, beta-CrossLaps, osteoprotegerin [OPG], receptor activator of nuclear factor kappaB ligand [RANKL]), parathyroid hormone, 25-hydroxy vitamin D, testosterone, sex hormone-binding globulin, and NT-pro-BNP. Serum adiponectin, osteocalcin, beta-CrossLaps, OPG, RANKL, and parathyroid hormone were significantly increased in CHF patients, whereas 25-hydroxy vitamin D was significantly lower compared to healthy controls. The significant positive association was found between adiponectin level with osteocalcin, beta-CrossLaps, OPG, and RANKL among CHF patients. In multivariate regression analysis, adiponectin was a significant determinant of total hip BMD, although the variance was small (r(2) = 0.239), whereas leptin was determinant for total bone mineral content (r(2) = 0.469) in patients with CHF. CONCLUSIONS Serum adiponectin is an independent predictor of BMD in elderly males with mild to moderate CHF, and showed a positive correlation to bone specific surrogates. Adiponectin, as cardioprotective hormone, seems to be able to exert a negative effect on bone mass in chronic heart failure. Further research is needed to confirm the potential for adipokines in the crosstalk between bone and energy metabolism in CHF patients.

Loxoribine, a selective Toll-like receptor 7 agonist, induces maturation of human monocyte-derived dendritic cells and stimulates their Th-1- and Th-17-polarizing capability.

Recently, a guanosine analog, 7-allyl-7,8-dihydro-8-oxo-guanosine (loxoribine), has been identified as a selective Toll-like receptor (TLR)7 agonist. Bearing in mind the controversy regarding the expression of TLR7 by human myeloid dendritic cells (DCs) and its significance for functions of these cells, the goal of this study was to investigate the effect of loxoribine on differentiation, maturation and functions of human monocyte-derived (Mo)DCs. Immature MoDCs were obtained by cultivation of monocytes for 6 days with recombinant granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. These cells were stimulated with loxoribine (250 μM) for an additional 48 h. Phenotypic properties of MoDCs were determined by flow cytometry, cytokine production was assayed by ELISA, whereas their allostimulatory capability was tested using a mixed leukocyte reaction. We showed that loxoribine up-regulated the expression of TLR7, CD40, CD54, CD80, CD83 and CCR7 and stimulated the production of IL-12, IL-23, IL-27 and IL-10 by MoDCs, whereas the level of interferon (IFN)-β was not modulated. Allogeneic CD4(+)T cells in co-culture with loxoribine-treated MoDCs proliferated more strongly, at lower DC/CD4(+)T-cell ratio (1:80), and secreted significantly higher levels of IL-17 and IFN-γ compared to the cultures with control MoDCs. The stimulatory effect of loxoribine on T helper (Th)1 polarization capability of MoDCs was further potentiated by ligation of CD40. In conclusion, our results show that loxoribine stimulated differentiation, maturation, allostimulatory as well as Th1 and Th17 polarization capability of human MoDCs and suggests that these effects might be associated with up-regulation of TLR7 expression, but not increased IFN-β production.

Differences in T-helper polarizing capability between human monocyte-derived dendritic cells and monocyte-derived Langerhans'-like cells

Langerhans’ cells (LCs) represent a specific subset of dendritic cells (DCs) which are important for detecting and processing pathogens that penetrate the skin and epithelial barriers. The aim of our study was to explain what makes their in vitro counterparts – monocyte‐derived Langerhans’‐like cells (MoLCs) – unique compared with monocyte‐derived dendritic cells (MoDCs). Immature MoDCs were generated by incubating peripheral blood monocytes with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4. The addition of transforming growth factor‐β (TGF‐β) to this cytokine cocktail resulted in the generation of MoLCs. MoLCs showed a lower expression of CD83, CD86, HLA‐DR and CCR7 compared with MoDCs, regardless of their maturational status. Both immature and mature MoLCs secreted higher quantities of IL‐23 compared with MoDCs and this finding correlated with a higher secretion of IL‐17 in co‐culture of MoLCs with allogeneic CD4+ T cells. Mature MoLCs, which produced higher levels of IL‐12 and lower levels of IL‐10 compared with mature MoDCs, were more potent at inducing interferon‐γ (IFN‐γ) production by CD4+ T cells in the co‐culture system. In conclusion, the finding that mature MoLCs stimulate stronger T‐helper 1 and T‐helper 17 immune responses than mature MoDCs, makes them better candidates for use in the preparation of anti‐tumour DC vaccines.

Signaling through Toll-like receptor 3 and Dectin-1 potentiates the capability of human monocyte-derived dendritic cells to promote T-helper 1 and T-helper 17 immune responses

BACKGROUND AIMS Recent studies have shown that the ligation of Toll-like receptor 3 (TLR3) or Dectin-1 on human monocyte-derived dendritic cells (MoDC) elicits their maturation, but with a different outcome on immunomodulation. Therefore the aim of this work was to study the response of MoDC to the combined effect of polyinosinic:polycytydilic acid [Poly (I:C)] and curdlan, selective TLR3 and Dectin-1 agonists, respectively. METHODS Immature MoDC, generated from human monocytes, were treated with Poly (I:C), curdlan or their combination for 2 days. Phenotypic characteristics of MoDC were determined by flow cytometry, and cytokine production was measured by enzyme-linked immunosorbent assay (ELISA) and FlowCytomix, while the stimulatory capability of MoDC was tested using a mixed leukocyte reaction assay. RESULTS The combination of Poly (I:C) and curdlan induced phenotypic maturation of MoDC with the capability to stimulate an alloreactive response. Such treated MoDC up-regulated the production of interleukin (IL)-12, IL-23 and IL-10, compared with the effect of Poly (I:C) alone. Curdlan-treated MoDC stimulated the production of IL-17 by alloreactive CD4 (+) T cells more strongly than Poly (I:C)-treated MoDC. The opposite effect was observed for interferon(IFN)-γ production. When combined, these agonists primed MoDC to increase further the production of IFN-γ by CD4 (+) T cells in co-culture, especially those of naive (CD45RA (+)) phenotype, and IL-17 by memory (CD45RO (+)) CD4 (+) T cells. CONCLUSIONS Ligation of TLR3 and Dectin-1 receptor up-regulates T-helper (Th) 1 and Th17 immune responses compared with single agonists. These findings may have therapeutic implications for the use of MoDC in immunotherapy.

Androgen status in non-diabetic elderly men with heart failure

Abstract Purpose: We aimed at evaluating androgen status (serum testosterone [TT] and estimated free testosterone [eFT]) and its determinants in non-diabetic elderly men with heart failure (HF). Additionally, we investigated its associations with body composition and long-term survival. Methods: Seventy three non-diabetic men with HF and 20 healthy men aged over 55 years were studied. Echocardiography, 6-min walk test, grip strength, body composition measurement by DEXA method were performed. TT, sex hormone binding globulin, NT-proBNP, and adipokines (adiponectin and leptin) were measured. All-cause mortality was evaluated at six years of follow-up. Results: Androgen status (TT, eFT) was similar in elderly men with HF compared to healthy controls (4.79 ± 1.65 vs. 4.45 ± 1.68 ng/ml and 0.409 ± 0.277 vs. 0.350 ± 0.204 nmol/l, respectively). In HF patients, TT was positively associated with NT-proBNP (r= 0.371, p = 0.001) and adiponectin levels (r = 0.349, p = 0.002), while inverse association was noted with fat mass (r = −0.413, p < 0.001). TT and eFT were independently determined by age, total fat mass and adiponectin levels in elderly men with HF (p < 0.05 for all). Androgen status was not predictor for all-cause mortality at six years of follow-up. Conclusions: In non-diabetic men with HF, androgen status is not altered and is not predictive of long-term outcome.

Dose-dependent response of dendritic cells to 7-thia-8-oxo-guanosine and its modulation by polyinosinic:polycytidylic acid.

Targeting the endosomal Toll-like receptors (TLRs) by specific agonists seems to be a promising tool for stimulation of the immunogenicity of dendritic cells (DCs). Since the functional outcome upon the engagement of TLRs may be different, the aim of our study was to examine if and how different concentrations of 7-thia-8-oxo-guanosine (7-TOG), a selective TLR7 agonist, influence differentiation, maturation and functions of human monocyte-derived DCs (MoDCs) and if its effects on MoDCs could be modulated by co-ligation of TLR3. Immature MoDCs were treated with different concentrations of 7-TOG (25, 100 and 250 μmol/L) alone, or together with polyinosinic:polycytidylic acid, Poly (I:C) (10 ng/mL), a selective TLR3 agonist, for an additional 48 h. We showed that the highest concentration of 7-TOG stimulated the differentiation, maturation and allostimulatory capability of MoDCs. These changes were accompanied by an increased production of interleukin 12 (IL-12) and induction of T helper (Th)1 and Th17 immune responses. Both Th responses were significantly augmented by additional stimulation of MoDCs with Poly (I:C). The treatment of MoDCs with the intermediate concentration of 7-TOG resulted in the up-regulation of co-stimulatory molecule (CD86) and increased production of IL-1β and IL-6 by MoDCs, followed by the stimulation of the Th17 immune response. The lowest concentration of 7-TOG down-regulated the expression of CD40 on MoDCs and potentiated the Th2 immune response. The Th2 response was not significantly modulated by additional treatment of MoDCs with Poly (I:C), but this combination of TLR3/TLR7 agonists also stimulated both Th1 and Th17 responses. In conclusion, our results show that 7-TOG influences the phenotype and functions of MoDCs in a dose-dependent manner and suggests that fine-tuned signaling through TLR7 may be modified by the engagement of TLR3, resulting in a different outcome of immune response.