Circulating IL-17A Levels in Postmenopausal Women with Primary Hyperparathyroidism.

BACKGROUND: Primary hyperparathyroidism (PHPT) is a common cause of secondary osteoporosis in postmenopausal women. Th17 lymphocytes and the released cytokine IL-17A play an important role in bone metabolism. Th17 cells have been shown to be activated by PTH, and peripheral blood T cells from patients affected with PHPT express higher levels of IL-17A mRNA than controls. AIM: To investigate circulating levels of IL-17A and the ratio RANKL/OPG, as markers of osteoclastogenesis, in 50 postmenopausal PHPT women compared with postmenopausal osteoporotic non-PHPT women (n = 20). RESULTS: Circulating levels of IL-17A were similarly detectable in most PHPT and non-PHPT osteoporotic women (12.9 (8.4-23.1) vs. 11.3 (8.3-14.3) pg/ml, median (range interquartile), P = 0.759), at variance with premenopausal women where IL-17A was undetectable. In PHPT women, any significant correlations could be detected between circulating IL-17A levels and PTH levels. Nonetheless, significant negative correlations between circulating IL-17A and ionized calcium levels (r = -0.294, P = 0.047) and urine calcium excretions (r = -0.300, P = 0.045) were found. Moreover, PHPT women were characterized by positive correlations between IL-17A levels and femur neck (r = 0.364, P = 0.021) and total hip (r = 0.353, P = 0.015) T-scores. Circulating IL-17A levels did not show any significant correlation with sRANKL, OPG, and sRANKL/OPG ratio in PHPT women. CONCLUSIONS: In postmenopausal PHPT women, circulating IL-17A levels were similar to those detected in postmenopausal non-PHPT women, showing a disruption of the relationship observed in postmenopausal osteoporosis among circulating PTH, sRANKL, OPG, IL-17A, and bone demineralization in postmenopausal PHPT women. The data support an osteogenic effect of IL-17A in postmenopausal PHPT women.

Plasma fatty acid profile as biomarker of coronary artery disease: a pilot study using fourth generation artificial neural networks.

Many studies, focused on identifying new biomarkers for coronary artery disease (CAD) risk computation and monitoring, suggested a potential diagnostic role for fatty acids (FA). In the present study, we explored the potential diagnostic role of FA by using a data mining approach based on fourth generation artificial neural networks (ANN). Forty-one male subjects were enrolled. According to coronary angiography, 31 displayed CAD and 10 did not (non-CAD, control group). FA analysis was performed on plasma samples using a gas chromatography-mass spectrometry system and analyses were performed by an ANN method. The variables most closely related to CAD were low levels of alpha-linolenic acid, eicosapentaenoic acid, eicosatetraenoic and docosahexaenoic acids. High levels of 1,1-dimethoxyhexadecane, total dimethyl acetals and docosatetraenoic acid were related to non-CAD condition. This subset of variables, which were most closely correlated to the target diagnosis, achieved a consistent predictive rate. The average accuracy obtained was 76.5%, with 93% of sensitivity and 60% of specificity. The area under the ROC curve was equal to 0.79. In conclusion, our study highlighted the association between different plasma FA species, CAD and non-CAD conditions. The specific subset of variables could be of interest as a new diagnostic tool for CAD management.

A pilot observational study on magnesium and calcium imbalance in elderly patients with acute aortic dissection.

BACKGROUND: Magnesium (Mg) and calcium (Ca) are the principal essential elements involved in endothelial cell homeostasis. Extracellular changes in the levels of either alter endothelial contraction and dilatation. Consequently Mg and Ca imbalance is associated with a high risk of endothelial dysfunction, the main process observed during acute aortic dissection (AAD); in this clinical condition, which mainly affects elderly men, smooth muscle cell alterations lead to intimal tears, creating a false new lumen in the media of the aorta. AAD patients have a high risk of mortality as a result of late diagnosis because often it is not distinguished from other cardiovascular diseases. We investigated Mg and Ca total circulating levels and the associated pro-inflammatory mediators in elderly AAD patients, to gain further information on the pathophysiology of this disorder, with a view to suggesting newer and earlier potential biomarkers of AAD. RESULTS: Total circulating Mg and Ca levels were both lower in AAD patients than controls (p < 0.0001). Using Ca as cut-off, 90% of AAD patients with low Ca (<8.4 mg/dL) came into the type A classification of AAD. Stratifying AAD according to this cut-off, Mg was lower in patients with lower total Ca. Compared to controls, both type A and B AAD patients had higher levels of all the pro-coagulant and pro-inflammatory mediators analyzed, including sP-sel, D-dimer, TNF-α, IL-6, and CRP (p < 0.05). Dividing types A and B using the Stanford classification, no significant differences were found (p > 0.05) The levels of both ICAM-1 and EN-1 were lower in AAD than in a control group (p < 0.0001 and p < 0.05 respectively). CONCLUSIONS: These findings suggest that low Mg and Ca in AAD elderly patients may contribute to altering normal endothelial physiology and also concur in changing the normal concentrations of different mediators involved in vasodilatation and constriction, associated with AAD onset and severity.

Circulating sRAGE in the diagnosis of osteolytic bone metastasis.

Despite the clinical importance of metastasis to the skeleton, the diagnostic tools for early detection and monitoring of bone metastasis lack sensitivity and specificity. We evaluated a promising new serum biomarker, the soluble form of the Receptor of Advanced Glycosylated End-products (sRAGE). sRAGE is involved in the Wnt-signaling pathway, and has been reported to reduce the risk of cancer. We investigated the diagnostic potential of sRAGE to improve the detection and monitoring of bone metastasis. We measured sRAGE in the serum of control healthy subjects, patients with primary tumors and patients with bone metastasis. sRAGE was also correlated with the Wnt inhibitors DKK-1 and sclerostin, the bone resorption markers MMP-2, MMP-9 and TRAP5, and the metastatic marker survivin. sRAGE was significantly lower in primary tumor and metastatic patients than in healthy subjects. sRAGE also showed a strong negative correlation with DKK-1, sclerostin, MMP-2, MMP-9, TRAP5b and survivin. These results indicated that sRAGE might play a protective role in bone metastasis progression, and it may diagnostic significance for detecting and monitoring osteolytic metastases.

Epicardial adipocyte hypertrophy: Association with M1-polarization and toll-like receptor pathways in coronary artery disease patients.

BACKGROUND AND AIMS: In coronary artery disease (CAD) epicardial adipose tissue (EAT) shows an elevated inflammatory infiltrate. Toll-like receptors (TLRs) are important mediators of adipose tissue inflammation and they are able to recognize endogenous products released by damaged cells. Because adipocyte death may be driven by hypertrophy, our aim was to investigate in CAD and non-CAD patients the association between EAT adipocyte size, macrophage infiltration/polarization and TLR-2 and TLR-4 expression. METHODS AND RESULTS: EAT biopsies were collected from CAD and non-CAD patients. The adipocyte size was determined by morphometric analysis. Microarray technology was used for gene expression analysis; macrophage phenotype and TLRs expression were analyzed by immunofluorescence and immunohistochemical techniques. Inflammatory mediator levels were determined by immunoassays. EAT adipocytes were larger in CAD than non-CAD patients and do not express perilipin A, a marker of lipid droplet integrity. In CAD, EAT is more infiltrated by CD68-positive cells which are polarized toward an M1 state (CD11c positive) and presents an increased pro-inflammatory profile. Both TLR-2 and TLR-4 expression is higher in EAT from CAD and observed on all the CD68-positive cells. CONCLUSIONS: Our findings suggested that EAT hypertrophy in CAD promotes adipocyte degeneration and drives local inflammation through increased infiltration of macrophages which are mainly polarized towards an M1 state and express both TLR-2 and TLR-4.

Acute phase of aortic dissection: a pilot study on CD40L, MPO, and MMP-1, -2, 9 and TIMP-1 circulating levels in elderly patients.

BACKGROUND: Acute aortic dissection (AAD) is an event which may be rapidly fatal without early diagnosis and treatment. Aging is one of the main risk factors that could leading to AAD. To date, no specific biomarkers are available to increase the speed of diagnosis. CD40 ligand (CD40L), myeloperoxidase (MPO), matrix metalloproteinase (MMP)-1, -2, -9 and metallopeptidase tissue inhibitor 1 (TIMP-1) are biologically related molecules which integrate inflammation, tissue injury and remodeling, all events associated to AAD. Our is a pilot study to evaluate whether circulating levels of these molecules may be used as potential biomarkers in timely diagnosis of AAD. RESULTS: Within 24 h of symptom onset, circulating CD40L, MPO, MMP-1,-2,-9 and TIMP-1 were quantified by enzyme-linked immunosorbent assays in 22 patients (40-86 years of age) with AAD of ascending aorta (type A according to Stanford classification) and 11 patients with AAD of descending aorta (type B). 30 healthy individuals age matched were used as control group compared to controls, both type A and B AAD patients had higher CD40L (p < 0.001) and MPO (p < 0.01) levels. MMP-1 was higher in the overall AAD group (p < 0.01). After Stanford classification, type A group had increased level compared to both control and type B (p < 0.01 and p < 0.05, respectively). TIMP-1 was higher in both A and B groups compared to controls (p < 0.001). No differences were observed in MMP-2 and MMP-9 levels. CONCLUSIONS: The simultaneous evaluation of CD40L, MPO and MMP-1 and TIMP-1, which may contribute to structural changes in aortic tissue in AAD patients, seems to be a novel promising diagnostic panel.

Epicardial adipose tissue inflammation is related to vitamin D deficiency in patients affected by coronary artery disease.

BACKGROUND AND AIMS: Alterations in epicardial adipose tissue (EAT) biology (i.e. increased fat thickness and inflammation) have been described in coronary artery disease (CAD) patients. In addition to its classic role in the regulation of calcium-phosphate homeostasis, vitamin D may exert immune-regulatory and anti-inflammatory effects. Whether EAT inflammation may be linked to vitamin D deficiency is still unknown. In the present study we evaluated plasma 25-hydroxycholecalciferol (25OHD) level in CAD patients and its relationship with EAT ability to locally metabolize vitamin D, EAT expression of inflammation-related molecules and EAT thickness. METHODS AND RESULTS: Plasma 25OHD level was quantified by an immunoluminometric assay. EAT expression of inflammation-related molecules (MCP-1, PTX3, TNFα, IL-6, adiponectin), vitamin D receptor (VDR), CYP27B1 (25OHD-activating enzyme) and CYP24A1 (1,25-dihydroxycholecalciferol-metabolizing enzyme) was performed by microarray. EAT thickness was quantified by echocardiography. Median plasma 25OHD level was 10.85 ng/mL and 83% of CAD patients displayed 25OHD level below 20 ng/mL. At decreasing plasma 25OHD concentration, we observed a down-regulation in CYP27B1 and CYP24A1 level and an increased expression of VDR and pro-inflammatory cytokines (MCP-1, PTX3, TNFα, IL-6) at EAT level. No correlation was observed between plasma 25OHD level and EAT thickness. CONCLUSION: Our data suggest an increased activation of inflammatory pathways at EAT level possibly related to systemic and local vitamin D deficiency in CAD patients. Whether maintaining an optimal vitamin D status may be helpful to reduce EAT inflammation and to prevent CAD and its progression needs further investigation.

Reciprocal regulation of calcium-/phosphate-regulating hormones in cyclists during the Giro d'Italia 3-week stage race.

Calcium and phosphate are essential for cell functions, and their serum concentrations result from the balance between intestinal absorption, bony storage, and urinary excretion. Fibroblast growth factor 23 (FGF23), expressed by osteocytes and osteoblasts, acts in the kidney, leading to hypophosphatemia and low 1,25-dihydroxycholecalciferol synthesis, but suppresses parathyroid function. The aim of this study was to explore the effects of a high-energy demanding cycling race on this bone-kidney-parathyroid axis. We studied nine cyclists during the 2011 Giro d'Italia stage race. Pre-analytical and analytical phases followed academic and anti-doping recommendations. Serum parathyroid hormone (PTH), 25(OH)D, total calcium, inorganic phosphorus, and plasma FGF23 were measured on days -1, 12, and 22 and corrected for changes in plasma volume. Dietary calcium and phosphorus, anthropometric parameters (height, weight, and body mass index) and indexes of metabolic effort (net energy expenditure, power output) were recorded. Dietary calcium and phosphorus intakes were kept at the same levels throughout the race. Twenty-five (OH)D, PTH, and calcium concentrations remained stable. FGF23 increased 50% with a positive correlation with the indexes of metabolic effort and, consequently, phosphorous decreased, although only in the first half. The strong metabolic effort acts on the bone-kidney-parathyroid system, and the rise in FGF23 plasma concentration might be aimed at maintaining calcium and phosphorus homeostasis.

Plasma myeloperoxidase in patients with erectile dysfunction of arteriogenic- and non-arteriogenic origin: association with markers of endothelial dysfunction.

Endothelial dysfunction and the disruption of the nitric oxide-cyclic guanosine monophosphate (cGMP) pathway have been considered the early mechanisms for the development of erectile dysfunction (ED). Myeloperoxidase (MPO), a heme-containing enzyme mainly released by activated neutrophils and monocytes, may contribute to endothelial dysfunction by promoting oxidation of different substrates and thus may play a role in ED. MPO level and its correlation with different plasma biomarkers of endothelial dysfunction were studied in patient with ED of arteriogenic (A-ED) and non-arteriogenic (NA-ED) to assess potential differences between the two ED subgroups. Diagnosis of ED was based on the International Index of Erectile Function Score. Its etiology was classified with penile echo-color Doppler at baseline and after intracavernous injection of prostaglandin E1. MPO, soluble (s) cGMP, sICAM-1, sVCAM-1 and sP-Selectin were measured by enzyme-linked immunosorbent assay. MPO concentration in A-ED was significantly higher compared to control subjects and NA-ED patients. Plasmatic cGMP level resulted lower both in A-ED and in NA-ED patients, whereas no difference has been observed between the two ED groups. sICAM-1 concentration resulted higher in A-ED compared both to controls and NA-ED. sVCAM-1 level was the same in controls, A-ED and NA-ED patients. sP-Selectin concentration resulted higher both in A-ED and in NA-ED patients than in controls, whereas no difference has been observed between the two ED groups. Correlation analysis indicated a positive correlation between plasmatic MPO, sICAM-1 and sP-Selectin levels. MPO may represent an important link between oxidation, inflammation and cardiovascular diseases and may also represent a potential marker to distinguish between the two subgroups of ED patients. Moreover, in ED subjects circulating cGMP may reflect the local signaling dysfunction. The use cGMP as a potential marker for monitoring the disease needs further investigation.

IL-18 level in patients undergoing coronary artery bypass grafting surgery or valve replacement: which link with epicardial fat depot?

Interleukin-18 (IL-18) is a member of the interleukin-1 family of cytokines produced constitutively by different cell types and by adipose tissue. Due to the link between obesity, inflammation and cardiovascular diseases, we aimed to measure IL-18 circulating level in patients undergoing open-heart surgery both for elective coronary artery bypass grafting (CABG) or for valve replacement (VR), and we also evaluated whether epicardial adipose tissue (EAT) depot may be a potential source of IL-18. Circulating IL-18 protein was quantified by enzyme-linked immunosorbent assay. IL-18, IL-18 receptor 1 (IL-18 R1) and IL-18 receptor accessory protein (IL-18-RAP) gene expression in EAT depot were evaluated by one colour microarray platform. EAT thickness was measured by echocardiography. In this study we found that all cardiovascular patients (CABG and VR) have increased circulating IL-18 level compared to healthy control subjects (p < 0.0001), but no statistical significant difference was observed between CABG and VR groups (p = 0.35). A great increase in the gene expression of IL-18 (p < 0.05), IL-18 R1 (p < 0.01) and IL-18 RAP (p < 0.001) was observed in EAT samples obtained from CABG vs VR patients. In conclusion, CABG and VR patients had similar increased level of circulating IL-18 protein, but in EAT depots isolated from CABG gene expression of IL-18, IL-18 R1 and IL-18-RAP resulted higher than in VR patients. Future investigation on local IL-18 protein production, its autocrine-paracrine effect and its correlation with plasmatic IL-18 level could give more information on the relationship between IL-18 and coronary artery disease.

Circulating cytokines and growth factors in professional soccer players: correlation with in vitro-induced motor neuron death.

BACKGROUND: professional soccer players are susceptible to amyotrophic lateral sclerosis. Strenuous physical activity has been associated with persistent inflammatory conditions and elevation of systemic cytokine levels, which could contribute to the vulnerability of these athletes. To investigate changes induced by playing soccer in the systemic profiles of growth factors and of the principal cytokines involved in the inflammatory response, we compared the serum concentrations of these factors in Italian professional soccer players and sedentary subjects. We also investigated the effects of the sera on primary cultured motor neurons in relation to their cytokine and growth factor content. METHODS: serum concentrations of cytokines and growth factors were measured by a biochip array analyzer. Neurotoxicity of sera was assessed by immunocytochemical assays in primary motor neuron cultures from mouse embryos. RESULTS: circulating levels of interleukin-8, tumor necrosis factor-alpha and interleukin-4 were lower in soccer players than controls. However, the viability of primary cultured mouse motor neurons treated with sera from the two groups did not differ significantly. Vascular endothelial growth factor (VEGF) independently emerged as a systemic protective factor for motor neurons. CONCLUSIONS: we found significant alterations in circulating pro-inflammatory cytokines in Italian professional soccer players, showing an unbalanced inflammatory condition in these subjects. VEGF was a protective serum factor affecting motor neuron survival.

Modulation of biofilm of strains isolated from patients with chronic obstructive pulmonary disease by levofloxacin, moxifloxacin, ciprofloxacin, amoxicillin/clavulanic acid and ceftriaxone.

The ability of levofloxacin, moxifloxacin, ciprofloxacin, amoxicillin/clavulanic acid and ceftriaxone to interfere on biofilm produced by Pseudomonas aeruginosa, Haemophilus influenzae and Streptococcus pneumoniae isolated from patients with chronic obstructive pulmonary disease was evaluated. The effects of antibiotics were evaluated on formation of biofilm (at 1/2, 1/4 and 1/8 X MIC) and on preformed biofilm (at epithelial lining fluid peak concentrations) by means of a spectrophotometric method. Levofloxacin was the most active compound followed by ciprofloxacin, moxifloxacin and amoxicillin/clavulanic acid and ceftriaxone. Levofloxacin may contribute to clear the reservoir of pathogens involved in chronic obstructive pulmonary disease, thus leading to decreased occurrence of acute exacerbations.

Biodegradable microcarriers as cell delivery vehicle for in vivo transplantation and magnetic resonance monitoring.

Microcarrier culture systems offer an attractive method for cell amplification and as delivery vehicle. At the same time, super paramagnetic iron oxide (SPIO) nanoparticles represent a unique in vivo tracking system, already approved for clinical use. In our study, we tested the combination of clinically approved microcarriers and SPIO nanoparticles for cell-construct delivery and subsequent tracking after implantation. In order to mimic better a clinical setting, biodegradable macroporous microcarriers were employed as an alternative approach to expand human primary chondrocytes in a dynamic culture system for subsequent direct transplantation. In addition, cellseeded microcarriers were labeled with SPIO nanoparticles to evaluate the benefits of cell-constructs tracking with magnetic resonance. In vivo subcutaneous implants were monitored for up to 3 weeks and orthotopic implantation was simulated and monitored in ex vivo osteochondral defects.

Experimental derivation of the fluence non-uniformity correction for air kerma near brachytherapy linear sources.

In brachytherapy, one of the elements to take into account for measurements free in air is the non-uniformity of the photon fluence due to the beam divergence that causes a steep dose gradient near the source. The correction factors for this phenomenon have been usually evaluated by two available theories by Kondo and Randolph [Radiat. Res. 13, 37-60 (1960)] and Bielajew [Phys. Med. Biol. 35, 517-538 (1990)], both conceived for point sources. This work presents the experimental validation of the Monte Carlo calculations made by Rodriguez and deAlmeida [Phys. Med. Biol. 49, 1705-1709 (2004)] for the non-uniformity correction specifically for a Cs-137 linear source measured using a Farmer type ionization chamber. The experimental values agree very well with the Monte Carlo calculations and differ from the results predicted by both theoretical models widely used. This result confirms that for linear sources there are some important differences at short distances from the source and emphasizes that those theories should not be used for linear sources. The data provided in this study confirm the limitations of the mentioned theories when linear sources are used. Considering the difficulties and uncertainties associated with the experimental measurements, it is recommended to use the Monte Carlo data to assess the non-uniformity factors for linear sources in situations that require this knowledge.

Breast-conserving treatment for ductal carcinoma in situ: Impact of boost and tamoxifen on local recurrences.

PURPOSE: Ductal carcinoma in situ represents 15 to 20% of all breast cancers. Breast-conserving surgery and whole breast irradiation was performed in about 60% of the cases. This study reports local recurrence rates in patients with ductal carcinoma in situ treated by breast-conserving surgery and whole breast irradiation with or without boost and/or tamoxifen and compares different therapeutic options in two European countries. PATIENTS AND METHODS: From 1998 to 2007, 819 patients with pure ductal carcinoma in situ were collected, both in France (266) and Italy (553). Median age was 56. All underwent breast-conserving surgery and whole breast irradiation; 391 (48%) received a boost (55% in France and 45% in Italy, P=0.017) and 173 (22.5%) tamoxifen (4.5% in France and 32% in Italy, P<0.0001). RESULTS: With a 90-month median follow-up, there were 51 local recurrences (6.2%), including 27 invasive (53%). The 5- and 10-year local recurrence rates were 4% and 8.6%. Two patients developed axillary recurrence and 12 (1.5%) metastases (seven after invasive local recurrence); 41 (5%) patients had contralateral breast cancer. In the multivariate analysis, high nuclear grade and lack of tamoxifen are the most powerful predictors of local recurrence, with 2.6 (95% confidence interval [95% CI]: 1.74-3.89, P=0.0012) and 2.85 (95% CI: 1.42-5.72, P=0.04) odds ratio (OR) estimates, respectively. Age, margin status and boost did not influence local recurrence rates. CONCLUSIONS: This study confirms the ductal carcinoma in situ treatment heterogeneity among countries and the unfavourable prognostic role of nuclear grade. Tamoxifen reduces local recurrence rates and might be considered for some subgroups of patients, but further confirmation is required. The boost usefulness still remains unclear.

Low frequency pulsed electromagnetic field affects proliferation, tissue-specific gene expression, and cytokines release of human tendon cells.

Low frequency pulsed electromagnetic field (PEMF) has proven to be effective in the modulation of bone and cartilage tissue functional responsiveness, but its effect on tendon tissue and tendon cells (TCs) is still underinvestigated. PEMF treatment (1.5 mT, 75 Hz) was assessed on primary TCs, harvested from semitendinosus and gracilis tendons of eight patients, under different experimental conditions (4, 8, 12 h). Quantitative PCR analyses were conducted to identify the possible effect of PEMF on tendon-specific gene transcription (scleraxis, SCX and type I collagen, COL1A1); the release of pro- and anti-inflammatory cytokines and of vascular endothelial growth factor (VEGF) was also assessed. Our findings show that PEMF exposure is not cytotoxic and is able to stimulate TCs' proliferation. The increase of SCX and COL1A1 in PEMF-treated cells was positively correlated to the treatment length. The release of anti-inflammatory cytokines in TCs treated with PEMF for 8 and 12 h was significantly higher in comparison with untreated cells, while the production of pro-inflammatory cytokines was not affected. A dramatically higher increase of VEGF-A mRNA transcription and of its related protein was observed after PEMF exposure. Our data demonstrated that PEMF positively influence, in a dose-dependent manner, the proliferation, tendon-specific marker expression, and release of anti-inflammatory cytokines and angiogenic factor in a healthy human TCs culture model.