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Polyunsaturated fatty acid damages lead to alterations in sperm function. This study aimed to investigate the involvement of F2-isoprostanes (F2-IsoPs), oxidized lipid products from arachidonic acid, in sperm quality impairment. For this purpose, F2-IsoP levels in semen and F2-IsoP localization in spermatozoa were explored in infertile subjects affected by idiopathic infertility or varicocele, as well as in fertile men. As compared to fertile men, in the idiopathic infertility and varicocele groups, sperm concentration, motility, morphology, viability, and fertility index were significantly lower and the mean scores concerning sperm apoptosis, necrosis, and immaturity were significantly higher. The idiopathic infertile group showed a reduction in sperm motility and fertility index, as well as an increase of apoptosis and necrosis percentages, in comparison to the varicocele group. The varicocele group showed the highest levels of F2-IsoPs, a significant increase of sperm immaturity, and a significant correlation between F2-IsoP levels and sperm immaturity. 8-Iso Prostaglandin F2α, biomarker of in vivo F2-IsoP, was clearly localized in sperm midpiece and cytoplasmic residues. Data show that F2-IsoP formation is relevant in semen and sperm from infertile patients with varicocele and high percentage of immaturity, suggesting that a correct fatty acid integrity is needed for sperm maturation.

F4-neuroprostanes (F4-NeuroPs), derived from the oxidative metabolization of docosahexaenoic acid (DHA), are considered biomarkers of oxidative stress in neurodegenerative diseases. Neurons and spermatozoa display a high DHA content. NeuroPs might possess biological activities. The aim of this in vitro study was to investigate the biological effects of chemically synthetized 4-F4t-NeuroP and 10-F4t-NeuroP in human sperm. Total progressive sperm motility (p &lt, 0.05) and linearity (p = 0.016), evaluated by a computer-assisted sperm analyzer, were significantly increased in samples incubated with 7 ng F4-NeuroPs compared to non-supplemented controls. Sperm capacitation was tested in rabbit and swim-up-selected human sperm by chlortetracycline fluorescence assay. A higher percentage of capacitated sperm (p &lt, 0.01) was observed in samples incubated in F4-NeuroPs than in the controls. However, the percentage of capacitated sperm was not different in F4-NeuroPs and calcium ionophore treatments at 2 h incubation. The phosphorylated form of AMPKα was detected by immunofluorescence analysis, after 2 h F4-NeuroP incubation, a dotted signal appeared in the entire sperm tail, and in controls, sperm were labeled in the mid-piece. A defined level of seminal F4-NeuroPs (7 ng) showed a biological activity in sperm function, its addition in sperm suspensions stimulated capacitation, increasing the number of sperm able to fertilize.

Summary The propolis extract was shown to possess the capacity to protect sperm membrane from the deleterious action of oxidative attack. Oxidative stress can induce propagation of a lipid peroxidation (LPO) chain reaction because spermatozoa contain high concentration of unsaturated fatty acids. This study aimed at evaluating in vitro the possible toxicity and/or the antioxidant properties of Propolfenol® in ejaculated human spermatozoa. A colorimetric assay determined the total flavonoid content by spectrophotometry and a high-performance liquid chromatography–diode array detection analysis the quantity of galangin, pinocembrin and caffeic acid phenylethilic ester (CAPE). Sperm parameters such as motility, vitality and DNA integrity were assessed utilising optical microscopy. The antioxidant properties Propolfenol® against LPO induced by tert-Butyl Hydroperoxide were evaluated using the C11-BODIPY581/591 probe. Chemical analysis of Propolfenol® revealed low quantities of galangin, pinocembrin and CAPE; cyclic voltammetry experiments showed that Propolfenol® may exert an antioxidant activity. A protective action of Propolfenol® (20 and 100 μg/ml) on induced LPO in human spermatozoa was detected. Propolfenol® may be proposed as the supplement in media for sperm preparation techniques or cryopreservation to counteract the increased presence of reactive oxygen species generated by these methods.

Background Osteoarthritis (OA) of the trapeziometacarpal joint (TMJ) is a disabling condition with a significant impact on quality of life. The optimal management of hand OA requires a combination of non-pharmacological and pharmacological treatments that include intra-articular (i.a.) therapy. EULAR experts recommend corticosteroid injections in TMJ OA and underline the usefulness of hyaluronic acid (HA). The aim of this study was the assessment of the efficacy and tolerability of i.a. injections of a hybrid formulation of HA (Sinovial H-L®) in comparison to triamcinolone in patients with TMJ OA. Methods This 6-months observational comparative study, retrospective analyzed the medical records of 100 patients with monolateral or bilateral TMJ OA, treated with two injections of Sinovial H-L® (Sinovial H-L Group) or of triamcinolone acetonide (Triamcinolone Group). Clinical assessments were recorded at the time of the first and second injection and after one, 3 and 6 months. The primary outcomes were the change in global pain on a Visual Analogue Scale (VAS) and in hand function evaluated by the Functional Index for Hand OA (FIHOA) from baseline to month 6. Secondary outcomes were the improvement of the duration of morning stiffness, Health Assessment Questionnaire (HAQ) and the Medical Outcomes Study 36-Item Short Form (SF-36). The comparison between the two groups of treatment were performed with the Wilcoxon rank-sum test for continuous variables and with chi-square or Fisher exact test for categorical variables. Statistical significance was set at p

Adipocytokines, including adiponectin, resistin, and visfatin may play an important role in the pathophysiology of osteoarthritis (OA). Spa therapy is one of the most commonly used non-pharmacological approaches for OA, but its mechanisms of action are not completely known. The aim of the present study was to assess whether a cycle of mud-bath therapy (MBT) influences the serum levels of adiponectin, resistin, and visfatin in patients with knee OA. As part of a prospective randomized, single blind-controlled trial evaluating the efficacy of MBT in knee OA, we included in this study 95 outpatients. One group (n = 49) received a cycle of MBT at the spa center of Chianciano Terme (Italy) in addition to the usual treatment, and one group (control group; n = 46) continued their regular care routine alone. Patients were assessed at basal time and at the end of the study (15 days) for clinical and biochemical parameters. Clinical assessments included spontaneous pain on a visual analog scale (VAS) score and the Western Ontario and McMaster Universities index (WOMAC) subscores for knee OA evaluated as total pain score (W-TPS), total stiffness score (W-TSS), and total physical function score (W-TPFS). Adiponectin, resistin and visfatin serum levels were assessed by enzyme immunoassay methods. At the end of the mud-bath therapy, serum adiponectin levels showed a significant decrease (p

Mechanical stimuli and hydrostatic pressure (HP) play an important role in the regulation of chondrocytes metabolism. Growing evidence demonstrated the ability of mechanical loading to modulate the expression of microRNA (miRNA) involved in chondrocytes homeostasis and in the pathogenesis of osteoarthritis (OA). The expression of miR-155, miR-181a and miR-223 in normal and OA chondrocyte cultures, and their potential modifications following exposure to three hours of a cyclic HP (1–5 MPa, frequency 0.25 Hz) were investigated. Also evaluated the expression of Chuk, regulator of the NF-kB pathway activation, which is a target gene of miR-223, was evaluated. Chondrocytes were collected immediately after pressurization (T0), and following 12, 24, and 48 h. Total RNA was extracted, reverse transcribed and used for real-time PCR. At basal condition, a significant increase of miR-155 and miR-181a was observed in OA in comparison to normal cells; on the contrary, no differences in miR-223 and Chuk expression levels were detected between normal and OA chondrocytes. miR-155 and miR-181a resulted significantly downregulated immediately after pressurization (T0) in OA cells. The pressure effect on miR-155 and miR-181a levels was maintained over time. No modifications of miR-223 were observed in response to HP, while Chuk levels resulted significantly reduced at T0 and after 12 h. Pressurization did not cause any modifications in normal cells. In conclusion, HP was able to modulate the expression of miRNA associated to OA pathogenesis. The preliminary results about Chuk response to pressure raised interest in its involvement in the possible HP induced NF-kB pathway modulation.

Background MicroRNAs (miRNAs) are a class of 19–23 nucleotides long non-coding RNAs that post-transcriptionally regulate the activity of target mRNAs. MicroRNAs are involved in cartilage homeostasis and play an important role in the pathogenesis of osteoarthritis (OA). They have been detected in human plasma and in synovial fluid and are considered as potential diagnostic biomarkers and therapeutic targets of OA. Balneotherapy is a common non-pharmacological treatment for OA patients. In a previous published prospective single-blind randomized clinical trial in patients with knee OA, we showed that a cycle of mud-bath therapy (MBT) in addition to conventional treatments induced an improvement on pain, functional capacity and quality of life in comparison to standard treatment alone. Objectives as part of this study we evaluated the whole blood levels of miR-155, 223, 181a, 146a and miR-let-7e, which are involved in the pathogenesis of OA. Methods Thirty-two patients aged between 50 and 75 years with knee OA defined by the ACR criteria were included for the current analysis, based on the availability of blood sample at basal time and after 2 weeks. Twenty-one patients (MBT group) were daily treated with a combination of daily local mud-packs at 42°C and baths in mineral water, at 37°C for 15 min, for a total of 12 applications over a period of 2 weeks, in addition to standard therapy; the other eleven patients (control group) continued their conventional treatment alone. Clinical parameters [global pain score by a 0–100 mm Visual Analog Scale (VAS); physical function, total pain score and total stiffness score (WOMAC)] and microRNAs expression were performed at basal time and after 2 weeks. Peripheral whole blood was collected into PAXgeneTM Blood RNA tubes and then stored a -80°C. Total RNA was extracted using the PAXgeneTM Blood miRNA kit and the relative expression of miR-146a, miR-155, miR-223, miR-181a and miR-let-7e were determined by qRT-PCR. Results At the end of MBT we observed a statistically significant improvement of clinical paramethers. Furthermore, we observed a significant decrease of miR-155, 181a and miR-146a (p Conclusions Our data showed that MBT can modify the expression levels of miR-155, 181a, 146a and 223 expression levels, that are up-regulated in OA. This MBT effect could be explained considering the role of the heat stress and of the hydrostatic pressure, since some miRNAs were found to be temperature and mechano-responsive. Further studies are needed to better explain the mechanism of action of MBT and the role of miRNAs in OA. References Cheleschi S et al. Int J Mol Sci. 2017. De Palma A et al. Clin Exp Rheumatol. 2017. Fioravanti A et al. Int J Biometeorol. 2015. Potla R. et al. RNA. 2015. Disclosure of Interest None declared

The aim of this study was to evaluate the whole-blood levels of miR-155, miR-223, miR-181a, miR-146a, and miR-let-7e in patients with bilateral knee osteoarthritis (OA) after a cycle of mud-bath therapy (MBT). Thirty-two patients with knee OA defined by the ACR criteria were included. Twenty-one patients (MBT group) were daily treated with a combination of local mud-packs at 42 °C and baths in mineral water, at 37 °C for 15 min, for 12 applications over a period of 2 weeks, in addition to standard therapy; 11 patients (control group) continued their conventional treatment alone. Global pain score evaluated by visual analog scale (VAS), WOMAC subscores, and microRNA expression were evaluated at baseline and after 2 weeks. Peripheral whole blood was collected into PAXgene™ Blood RNA tubes, stored at − 80 °C, and total RNA was extracted. The expression of miR-155, miR-223, miR-181a, miR-146a, and miR-let-7e was determined by qRT-PCR. After MBT, we observed a statistically significant improvement of clinical parameters and a significant decrease of miR-155, miR-181a, miR-146a (p

The aim of our study was to evaluate the effects of gold (Au) and silver (Ag) nanoparticles (NPs) at different concentrations on cultured human osteoarthritic chondrocytes. Cell viability and inducible nitric oxide synthase expression were evaluated by light microscopy. Using transmission electron microscopy (TEM) and field emission gun-based scanning transmission electron microscopy/energy dispersive spectroscopy (FEG-STEM/EDS) allowed us to localize NPs. Gene expression of matrix metalloproteinases 1, 3 and 13 and A disintegrin and metalloproteinase with thrombospondin motifs -4 and -5 were carried out by real-time polymerase chain reaction. A cell viability test indicated a significant dose-dependent cytotoxic effect of both NPs. At concentrations of 160 and 250 μM NP light microscopy showed chondrocytes with signs of apoptosis and an increased presence of inducible nitric oxide synthase. Au-NPs were characterized by FEG-STEM/EDS and TEM analysis localized NPs in cytoplasm and in endocytotic vesicles. On the contrary, the Ag-NPs were undetectable by FEG-STEM/EDS and TEM. Increased gene expression, particularly in matrix metalloproteinase-3, was observed for both NPs (160 μM), but at a concentration of 250 μM the expression of the evaluated genes became lower. Our in vitro studies, although preliminary, suggest that engineered Au and Ag-NPs appear to be harmful for human osteoarthritic chondrocytes in high concentrations (160–250 μM). Copyright © 2013 John Wiley & Sons, Ltd.

Osteoarthritis (OA) is a multifaceted disorder defined by the alteration of homeostasis and degradation in articular cartilage. Recently mounting evidence suggests that OA should be conceived as an inflammatory disease rather than a simple wear-and-tear problem. Bradykinin (BK) and B2 receptors play a role in the pathogenesis of OA. The aim of this paper is to analyze preclinical and clinical studies examining the potential role of BK and of B2 receptor blockade in OA pathogenesis. We analyzed the data about the effects of BK in synoviocytes, endothelial cells and chondrocytes cultures and described the action of B2 receptor antagonists (Icatibant and Fasitibant). In conclusion, the BK is an endogenous proinflammatory molecule that is involved in the pathophysiology of OA, and B2 receptor antagonists are believed to be considered as a potential symptomatic therapy for this disease. There is a need for further preclinical and clinical trials to better explain the mechanisms of action and the efficacy and tolerability of the B2 receptor antagonists in OA.

Oxidative stress and the overproduction of reactive oxygen species (ROS) play an important role in the pathogenesis of osteoarthritis (OA). Accumulating evidence has demonstrated the involvement of microRNAs (miRNAs) dysregulation in disease development and progression. In this study, we evaluated the effect of oxidative stress on miR-146a and miR-34a expression levels in human OA chondrocytes cultures stimulated by H2O2. Mitochondrial ROS production and cell apoptosis were detected by flow cytometry. The antioxidant enzymes SOD-2, CAT, GPx, the transcriptional factor NRF2 and the selected miRNAs were analyzed by qRT-PCR. The H2O2-induced oxidative stress was confirmed by a significant increase in superoxide anion production and of the apoptotic ratio. Furthermore, H2O2 significantly up-regulated the expression levels of SOD-2, CAT, GPx and NRF2, and modulated miR-146a and miR-34a gene expression. The same analyses were carried out after pre-treatment with taurine, a known antioxidant substance, which, in our experience, counteracted the H2O2-induced effect. In conclusion, the induction of oxidative stress affected cell apoptosis and the expression of the enzymes involved in the oxidant/antioxidant balance. Moreover, we demonstrated for the first time the modification of miR-146a and miR-34a in OA chondrocytes subjected to H2O2 stimulus and we confirmed the antioxidant effect of taurine.

Tumour necrosis factor (TNF)-α is primarily involved in the regulation of cell proliferation and apoptosis; in addition it possesses pro-inflammatory properties. Anti-TNF-α strategies involve either administration of anti-TNF-α antibody or soluble TNF receptor to mop up circulating TNF-α. Etanercept, a recombinant human TNF-α receptor, was found to be effective in the treatment of rheumatoid arthritis. The impact of TNF-α inhibitors on human fertility is of notable interest. This in vitro study investigated the effect of different concentrations of TNF-α and etanercept used alone or in combination on sperm viability, motility, mitochondrial function, percentage of apoptosis and chromatin integrity in swim-up selected human spermatozoa. A negative effect of TNF-α (300 and 500 ng mL–1) and etanercept (from 800 µg mL–1 to 2000 µg mL–1) individually on sperm viability, motility, mitochondrial function, percentage of apoptotic spermatozoa and sperm DNA integrity was demonstrated. However, at concentrations of 100 and 200 µg mL–1, etanercept can block, in a significant way, the toxic effects of TNF-α (500 ng mL–1) on studied sperm characteristics. Our results confirm that TNF-α has a detrimental effect on sperm function and suggest, for the first time, that etanercept may counteract the in vitro toxic action of TNF-α. This data appears to be quite promising, although further studies, both in vivo and in vitro, are needed to understand the exact mechanism of action of TNF-α and TNF-α antagonists on sperm function.

VA694, a promising cyclooxigenase-2 (COX-2)-inhibiting hybrid drug endowed with nitric oxide (NO) releasing properties (NO-COXIB), showed COX-2-selective inhibitory effects, associated with interesting anti-inflammatory and anti-nociceptive activities. Therefore, we studied the effects of VA694 on cartilage metabolism, in comparison with Naproxcinod, a COX inhibitor and NO donor (CINOD), and Naproxen, a traditional non-steroidal-anti-inflammatory drug (NSAID) on human osteoarthritic chondrocyte cultures. IL-1β-stimulated chondrocytes showed a significant decrease in cell viability (P

Canakinumab is a human IgGκ monoclonal antibody that neutralizes the activity of interleukin (IL)-1β blocking interaction with IL-1β receptors. Our study aimed to evaluate the in vitro effect of canakinumab on human osteoarthritic (OA) chondrocytes cultivated in the presence or absence of tumor necrosis factor (TNF)-α. Articular cartilage was obtained from the femoral heads of patients with osteoarthritis (OA). Chondrocytes were incubated with two concentrations (1μg/ml and 10μg/ml) of canakinumab alone or with TNF-α (10ng/ml) for 48h. We evaluated cell viability, release of proteoglycans (PG) and nitric oxide (NO) in culture medium, inducible nitric oxide synthase (iNOS) and metalloproteinanes (MMP)-1,3,13 gene expression, apoptosis, necrosis and morphological feature by transmission electron microscopy (TEM). Canakinumab alone did not have cytotoxic effect. Cell viability was reduced significantly (p

Obesity is a major risk factor for arterial hypertension, coronary artery disease, dyslipidemias, and type 2 diabetes. Spa therapy has long been used for treating obesity and its comorbidities. Enlargement of adipose tissue has been linked to a dysregulation of adipokine secretion and adipose tissue inflammation. Adipokines are currently investigated as potential drug targets in these conditions. Our primary aim was to assess the clinical efficacy of a 3-week program of diet combined with spa therapy in obese patients with and without type 2 diabetes. The secondary aim was to examine whether this combined program influences the response of serum levels of leptin, adiponectin, visfatin, and high-sensitivity C-reactive protein. Fifty obese males were enrolled and 21 of these featured a type 2 diabetes. During the 3-week period of the study, the patients were on a 1,000-kcal diet and were involved in mineral bath and total body's mud-pack applications (15 procedures). Patients were assessed at baseline and at the end of the therapy for clinical and biochemical parameters (total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, glycemia, and adipokines). We showed that a 3-week program of spa therapy in obese patients induced significant decrease of body weight, body mass index, triglycerides, total cholesterol, low-density lipoprotein (LDL) cholesterol, glycemia, and serum levels of leptin and high-sensitivity C-reactive protein. So, a cycle of mud-bath therapy associated with a controlled diet may be a promising treatment for obesity and type 2 diabetes decreasing body weight and many risk factors for atherosclerosis and metabolic syndrome.

The objective of this prospective parallel randomized single-blind study was to assess that a cycle of mud-bath therapy (MBT) provides any benefits over usual treatment in patients with bilateral knee osteoarthritis (OA). Patients with symptomatic primary bilateral knee OA, according to ACR criteria, were included in the study and randomized to one of two groups: one group received a cycle of MBT at spa center of Chianciano Terme (Italy) in addition to the usual treatment, and one group continued their regular care routine alone. Clinical assessments were performed 7�days before enrollment (screening visit), at the time of enrollment (basal time), after 2�weeks, and after 3, 6, 9, and 12�months after the beginning of the study. All assessments were conducted by two researchers blinded to treatment allocation. The primary efficacy outcomes were the global pain score evaluated by Visual Analog Scale (VAS) and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) subscore for physical function (W-TPFS). Of the 235 patients screened, 103 met the inclusion criteria: 53 patients were included in the MBT group and 50 in the control group. In the group of patients treated with MBT, we observed a statistically significant (p�

The cytotoxicity of Au or Ag nanoparticles (NPs) on rat germinal cells was investigated in vitro. Rat germ cells separated by the STAPUT method in two different populations, pachytene spermatocytes (F5) and round spermatids (F3), were incubated (37°C, 60'-120') with 60 μM, 125 μM, 250 μM and 500 μM of Au/Ag-NPs. Cell viability was assessed with the Eosin Y test. Au or Ag-NPs were investigated with FEG-STEM/EDS and TEM. A dose-dependent effect on the viability of F3 and F5 populations was observed after incubation with Au-NPs or Ag-NPs (P

Semen from 33 patients were evaluated by light microscopy (LM) obtaining sperm concentration, percent motility, percentage of sperm with normal morphology (PAP staining), and percentage of dead sperm (Eosin Y stained). The samples were observed by polarizing microscopy (PM), that evaluates sperm morphology and the viability by birefringence of organelles, and it provides a PM index (percentage of birefringent, viable, motile sperm) and a percentage of dead, non-birefringent sperm. Sperm were processed for transmission electron microscopy (TEM) and TEM data were elaborated with a mathematical formula able to provide a fertility index (FI, number of sperm free of structural defects) and percentages of sperm immaturity and necrosis (dead sperm). To test the reliability of these techniques, the values of normal acrosome, nucleus, midpiece, and tail and the presence of cytoplasmic residues obtained with the three methods were compared. With the exception of cytoplasmic residues (P = 0.40), significant differences in the evaluation of each organelle were observed and TEM analysis resulted as the most stringent screening. In addition, relationships among relevant sperm variables were investigated. Motility showed positive correlations with the percentage of normal tail, midpiece, and PM index (P < 0.01), but it exhibited negative correlations with indices of sperm death (non-birefringent sperm: P < 0.05; percentage of eosin Y stained sperm: P < 0.05; necrosis: P < 0.01), which were positively correlated with each other (P < 0.01). Positive correlations were found between indices expressing normal sperm morphology: FI with PM index (P < 0.01) and with the percentage of normal sperm (PAP staining) (P < 0.01), which in turn were correlated with the PM index (P < 0.001). Sperm immaturity showed positive correlations (P < 0.01) with the presence of cytoplasmic residues detected with the three methods. In conclusion, LM, PM, and TEM are reliable techniques in evaluating sperm quality. PM appears to offer several advantages ‘midway’ between LM and TEM and it should be considered in sperm analysis.

Spermatozoa with a rare combination of two monomorphic sperm defects, dysplasia of the fibrous sheath (DFS) and alterations in head-mid-piece junction were analysed. The main focus was to explore the status of the centriole, a key organisation during fertilisation, using the centrin 1, a calcium-binding protein linked to this structure. The sperm quality was examined by light, scanning and transmission electron microscopy (SEM, TEM); immunocytochemistry was performed for tubulin, A-kinase anchor protein 4 (AKAP4) and centrin 1. Spermatozoa showed DFS defect associated with anomalies in head-tail attachment detected by SEM and TEM. Immunolocalisation of tubulin, AKAP4 and centrin 1 confirmed these alterations. Centrin 1 was visible in 67% of spermatozoa (in only 13% centrin localised in a normal position); in the majority of sperm centrin 1's location was altered, sometimes bent; often four spots, indicating the presence of two implantation fossae, were detected. At the centriolar level, immunoreactive fragments, frequently invading the entire short and thick tail, were observed. Centrin 1 is an essential component of the spermatozoa connecting piece and plays a role in centrosome dynamics during sperm morphogenesis and in zygotes and early embryos during spindle assembly. It is important to shed light on these rare conditions in order to better manage the patients during assisted reproductive technology.

The aim of this in vitro study was to examine the possible effect of [2-methyl-5-(4-methylsulphonyl)phenyl-1-phenyl-3-(2-n-propyloxyethyl)]-1H-pyrrole (VA441), a new selective cyclooxygenase (COX)-2 inhibitor, on human osteoarthritic (OA) chondrocyte cultivated in the presence or absence of interleukin-1β (IL-1β). In particular, we assessed the effects of 1 and 10 μM of VA441, celecoxib, and indomethacin on cell viability, COX-2 and inducible nitric oxide synthase (iNOS) gene expression, prostaglandin E2 (PGE2) production, and nitric oxide (NO) and metalloproteinase-3 (MMP-3) release. Furthermore, we carried out morphological assessment by transmission electron microscopy (TEM). The presence of IL-1β led to a significant increase in PGE2, MMP-3, and NO production, as well as a significant increase in gene expression of COX-2 and iNOS. All the drugs tested had a statistically significant inhibitory effect on PGE2 production and gene expression of COX-2 stimulated by IL-1β. VA441 and celecoxib significantly suppressed IL-1β-stimulated MMP-3 and NO and iNOS gene expression in a dose-dependent manner, while indomethacin did not show any significant effect on MMP-3 and NO production or on iNOS gene expression. TEM demonstrated that IL-1β severely alters the structure of chondrocytes; after coincubation with VA441 or celecoxib, the cells recovered their ultrastructure. Our data suggest that VA441 and celecoxib may have a beneficial effect on chondrocyte metabolism. Keywords:: VA441, celecoxib, indomethacin, cyclooxygenase-2 inhibitor, chondrocyte

It is well known that estrogens are implicated in the pathogenesis of osteoarthritis. Raloxifene is a selective estrogen receptor modulator used in the treatment of osteoporosis, though little is known about the possible effects of raloxifene on cartilage metabolism. The aim of our study was to evaluate the possible in vitro effects of raloxifene in human osteoarthritis chondrocytes cultivated in the presence or absence of Interleukin-1 beta (IL-1β) (5 ng/ml). The effects of 0.1 μM and 1 μM of raloxifene in the culture medium were assessed using an immuno-enzymatic method for proteoglycans and metalloproteinase-3 (MMP-3), and the Griess method for nitrite. Gene expression of inducible Nitric Oxide Synthase (iNOS) was detected by real-time PCR. A morphological analysis was performed by transmission electron microscopy (TEM). Cell viability was significantly (P

Objective To investigate the occurrence of ghrelin and obestatin in human semen. Design Prospective study. Setting University, center for research and therapy of male infertility. Patient(s) 112 consecutively selected men. Intervention(s) Family history, clinical and physical examination, radioimmunoassay for ghrelin and obestatin determinations, semen analysis, annexinV/propidium iodide assay. Main Outcome Measure(s) Ghrelin and obestatin detected in the semen and relationships with semen parameters and conditions influencing semen quality (smoking, varicocele, ex varicocele, leukocytospermia). Result(s) The levels of both peptides in semen were higher versus serum. Linear correlations between ghrelin and obestatin levels in serum and in semen were observed. Serum ghrelin levels were negatively correlated with the men's ages. Semen obestatin levels were positively correlated with sperm concentration and motility. Obestatin levels were decreased in the semen of smokers and in the presence of leukocytospermia. Conclusion(s) This is the first study on the presence of obestatin in human semen and its relationship with sperm concentration and motility, suggesting a possible role of the peptide in controlling cell proliferation and survival. Further investigations are required to explore the exact role of obestatin and ghrelin in human semen.

Objective To characterize the association of two systematic sperm defects. Design Case report. Setting University, Interdepartmental Centre for Research and Therapy of Male Infertility. Patient(s) Patient 1, 42 years old, and patient 2, 38 years old, both with severe asthenozoospermia. Intervention(s) Family history, physical examination, hormonal analysis, microbial assays, semen analysis, transmission and scanning electron microscopy, immunocytochemistry for tubulin, and fluorescence in situ hybridization (FISH) for chromosomes 18, X, and Y. Main Outcome Measure(s) Admixture of dysplasia of the fibrous sheath (DFS) and head-tail misalignment up to acephalic sperm detected by microscopic methods. Result(s) In both patients, DFS was present in incomplete form and was associated with acephalic sperm and abnormal head-tail attachment. In patient 2, spermatozoa were also affected by necrosis that may cause fragmentation leading to short flagella; submicroscopic examination allowed defining only the origin of these "stumpy" tails. Immunofluorescence confirmed the sperm alterations. FISH revealed an altered frequency of diploidy and disomy in patient 2 and a slight increase in diploidy in patient 1. Conclusion(s) The importance of ultrastructural sperm evaluation for correct identification of sperm pathologies is evident, particularly regarding assisted reproduction technology and genetic risk assessment.

Objective To characterize a novel flagellar defect involving 98% of sperm tails. Design Case report. Setting Interdepartmental Centre for Research and Therapy of Male Infertility, Siena, Italy. Patient(s) A 45-year-old infertile man with severe asthenozoospermia. Intervention(s) Family history, physical examination, hormonal analysis, microbial assays, semen analysis, transmission and scanning electron microscopy, tubulin distribution investigated by immunocytochemistry, fluorescence in situ hybridization (FISH) for chromosomes 9, 16, 18, X, and Y. Main Outcome Measure(s) Flagellar abnormalities detected by microscopical methods. Result(s) An apparent heterogeneity was observed: extremely elongated tails prone to ruptures; coiled tails at different levels with a strongly rolled axoneme or with a curl in the final flagellar segment; and V-shaped, isolated, bent tails. Transmission electron microscopy revealed the presence of normal heads, disorganized flagellar structures, and dynein deficiency. The FISH analysis was normal. Conclusion(s) We report a new sperm defect, characterized by abnormal elongation of the tail, which was prone to ruptures at different levels, concomitant with coiled tails, which were impossible to measure in length. This defect remained constant in different examined ejaculates and applied to the entire sperm population of a sterile man, the son of first-degree cousins, indicating a potential genetic origin.

Semen parameters were evaluated by a variety of tests. A polarizing microscope (PM) which allows, in a single step, for the evaluation of the number, motility, viability of sperm using the phenomenon of birefringence was used. This approach avoids a Papanicolaou staining procedure modified for sperm (PAP) performed in fixed material. The aim of this study was to examine the birefringence of sperm structures, in live cells, for the direct analysis of morphology on fresh samples. Semen samples from 15 men of recently proven fertility and from 66 male patients who attended our center for semen analysis were examined by polarization microscope (PM) analysis in order to find an index representing a pool of sperm with normal morphology and progressive motility. The receiver operating characteristics (ROC) curves were used to determine the performance of the proposed diagnostic method. The difference between the two areas under the ROC curves (PAP=0.76 and PM=0.82) was quantitatively not significant (P=0.308); however, the curve of the PM method was always higher than the curve of PAP, revealing that, qualitatively, PM was more sensitive than PAP. The PM index can represent the percentage of motile sperm with normal morphology, which is the actual pool of sperm that can reach and fertilize the oocyte. We suggest a PM diagnostic cut-off value of 20%, since this value was the lowest found for individuals of proven fertility.

We analysed ejaculated spermatozoa from five infertile men with different balanced reciprocal translocations to contribute to the study of meiotic segregation of chromosomes 18, X and Y and also to evaluate sperm morphology by transmission electron microscopy (TEM) analysis. Conventional lymphocyte karyotype analyses highlighted different reciprocal balanced translocations: t(12;13), t(4;9), t(X;8), t(8;10) and t(3;16). Semen analysis was performed by light and TEM. Fluorescence in situ hybridization was performed directly on sperm nuclei using centromeric probes for chromosomes 18, X and Y. The carriers of the balanced reciprocal translocations considered in the present study showed a very similar pattern of sperm pathologies: diffused presence of apoptosis and immaturity. All patients showed meiotic segregation derangements, highlighted by the presence of sperm diploidies and sex chromosome disomies particularly related to the failure of the first meiotic division. However, an increased incidence of chromosome 18 aneuploidy was detected in spermatozoa from t(X;8) and t(8;10) carriers. We have also reported values from sex chromosomes such as t(X;8), although the X chromosome was involved in translocation. Since patients with reciprocal translocations and spermatogenetic impairment are candidates for intracytoplasmic sperm injection cycles, the study of sperm parameters, and particularly of the level of aneuploidy rates, would provide better information for couples at risk and would contribute to the data in the literature for a better understanding of the effects of chromosomal rearrangement on the whole meiotic process and, in particular, on chromosomes not involved in translocation.

Objective To characterize a flagellar defect involving 95% of the sperm population from an infertile man. Design Case report. Setting Interdepartmental Centre for Research and Therapy of Male Infertility, Siena, Italy. Patient(s) A 42-year-old infertile man with severe asthenozoospermia. Intervention(s) Family history, physical examination, hormonal analysis, microbial assays, semen analysis, transmission electron microscopy (TEM) and scanning electron microscopy (SEM), tubulin distribution investigated by immunocytochemistry, fluorescence in situ hybridization for chromosomes 18, X, and Y. Main Outcome Measure(s) Ultrastructural abnormalities of the flagellum detected by methods listed. Result(s) Ultrastructural analysis revealed, in 95% of sperm cells, the total absence of the axoneme and outer dense fibers at the principal piece level, whereas the midpiece appeared abnormally long. Tubulin localization showed a total disorganization of the axoneme with a network of microtubular structures emerging randomly at any level of the flagellum. Fluorescence in situ hybridization analysis was normal. Conclusion(s) We report a rare sperm tail defect, characterized by abnormal elongation of the midpiece and absence of the axoneme and the outer dense fibers at the principal piece level in 95% of flagella. This defect occurs in the vast majority of the sperm population from a sterile man, and therefore a genetic origin could be hypothesized.

Aim: To perform screening, related to A-kinase anchoring proteins 4 (AKAP4) and tubulin proteins, in spermatozoa with absent or severely reduced motility in order to detect the status of the fibrous sheath and the axonemal structure. Methods: An immunocytochemical study of tubulin, used as a positive control, and AKAP4 was carried out to detect the presence and the distribution of these proteins in different sperm samples. The morphological characteristics of sperm were studied by transmission electron microscope (TEM) and the results were elaborated using a formula reported in previous studies. PCR was carried out on DNA extracted from peripheral blood lymphocytes to analyse partial sequences of the Akap4 and Akap3 genes. Results: Immunolabelling of tubulin and AKAP4 showed different patterns, which led us to divide the patients into groups. In group I, the absence of AKAP4 and tubulin was revealed, although these patients did not show alterations in the Akap4/Akap3 binding site. TEM evaluation highlighted that a high presence of necrosis was associated with total sperm immotility. In group II, a regular AKAP4 and tubulin signal was present, although motility was reduced and TEM analysis revealed the presence of immaturity. In group III, in which a weak AKAP4 label associated with normal tubulin staining and reduced motility was observed, a severe disorganization of the fibrous sheath was highlighted by TEM. Conclusion: While the role of AKAP4 in sperm motility is unclear, absent or weak AKAP4-labelling seems to be associated with absent or weak sperm motility.

Background Canakinumab is a human IgGκ monoclonal antibody targeting Interleukin (IL)-1β; its action involves the neutralization of the signaling of IL-1β, the most important cytokine in the pathogenesis of Osteoartrhitis (OA). In response to IL-1β, chondrocytes secrete other proinflammatory cytokines, neutral metalloproteinases (MMPs), nitric oxide (NO); furthermore IL-1β inhibits chondrocytes proliferation and induces apoptosis. Objectives The aim of our study was to evaluate the possible in vitro effects of canakinumab on human OA chondrocytes cultures cultivated in the presence or absence of tumor necrosis factor (TNF)-α. Methods Chondrocytes, from femoral cartilage of five OA patients who underwent surgery for hip prostheses, were isolated using sequential enzymatic digestion. Primary cultures so obtained were incubated with canakinumab (1μg/ml and 10μg/ml) alone or with TNF-α (10ng/ml). We evaluated after 48h cell viability, release of proteoglycans (PG) and NO in culture medium, inducible nitric oxide synthase (iNOS) and MMP-1,3,13 expression, the percentage of apoptosis and necrosis. After 24h we performed IL-1β dosage (ELISA). Finally, we used a transmission electron microscope (TEM) for morphological assessment. Results Canakinumab alone at the two concentrations studied hadn9t cytotoxic effect and didn9t modify significantly IL-1β levels in the culture medium. TNF-α caused a significant decrease of the percentage of viable cell (P Conclusions It is generally accepted that IL-1β and TNF-α are the pivotal cytokines involved in OA physiopathology. Hence, the neutralization of these cytokines appears to be a logical development for OA therapy. In the present study we showed, for the first time, that canakinumab counteracts the negative effect of TNF-α on OA chondrocyte cultures, probably inhibiting IL-1β signaling. References Dhimolea E. Mabs 2010;2:3-13. Fioravanti et al. J Pharmacol Sci 2012;20:6-14. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5066

Spa represents a treatment widely used in many rheumatic diseases (RD). The mechanisms by which immersion in mineral or thermal water ameliorates RD are not fully understood. The net benefit is probably the result of a combination of factors, among which the mechanical, thermal and chemical effects are most prominent. Buoyancy, immersion, resistance and temperature play important roles. According to the gate theory, pain relief may be due to the pressure and temperature of the water on skin; heat may reduce muscle spasm and increase the pain threshold. Mud-bath therapy increases plasma β-endorphin levels and secretion of corticotrophin, cortisol, growth hormone and prolactin. It has recently been demonstrated that thermal mud-bath therapy induces a reduction in circulating levels of prostaglandin E2, leukotriene B4, interleukin-1β and tumour necrosis factor-α, important mediators of inflammation and pain. Furthermore, balneotherapy has been found to cause an increase in insulin-like growth factor-1, which stimulates cartilage metabolism, and transforming growth factor-β. Beneficial anti-inflammatory and anti-degenerative effects of mineral water were confirmed in chondrocytes cultures, too. Various studies in vitro and in humans have highlighted the positive action of mud-packs and thermal baths, especially sulphurous ones, on the oxidant/antioxidant system. Overall, thermal stress has an immunosuppressive effect. Many other non-specific factors may also contribute to the beneficial effects observed after spa therapy in some RD, including effects on cardiovascular risk factors (e.g. adipokines) and changes in the environment, pleasant surroundings and the absence of work duties.