Nicola Antonio Pascarelli

Granulomatosis with polyangiitis and intravenous immunoglobulins: A case series and review of the literature

Granulomatosis with polyangiitis, formerly known as Wegeners granulomatosis or disease, is a systemic, necrotizing small-vessel vasculitis, belonging to the group of anti-neutrophil cytoplasm antibody vasculitis. The therapeutic strategy includes, in most cases, corticosteroids associated, at least in severe forms of the disease, with immunosuppressive agents: cyclophosphamide and rituximab to induce remission, methotrexate, azathioprine and mycophenolate mofetil to prevent relapses. Intravenous immunoglobulins represent an alternative adjuvant therapy. We described 5 cases of patients with granulomatosis with polyangiitis treated with monthly high-dose intravenous immunoglobulins (500mg/kg/daily for 3 consecutive days for 9months). No patients experienced adverse reactions, and 4 patients (80%) achieved a complete remission after 9 courses of this therapy, which was maintained also 3months later, although we are unable to determine whether improvement in outcomes was a direct result of the IVIG. We also discussed the beneficial effects of intravenous immunoglobulins in patients suffering from granulomatosis with polyangiitis, reporting the previously published data.

Effects of gold and silver nanoparticles in cultured human osteoarthritic chondrocytes.

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

Raloxifene protects cultured human chondrocytes from IL-1β induced damage: a biochemical and morphological study.

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<0.01) reduced by the IL-1β, and restored to basal levels by raloxifene at both of the concentrations used. The presence of IL-1 β led to a significant decrease (P<0.01) in proteoglycan levels as well as a significant increase of MMP-3 and NO (P<0.01). When the cells were co-incubated with IL-1β and raloxifene, a significant and dose-dependent increase in proteoglycans and a reduction of MMP-3 and nitric oxide (NO) were detected. iNOS was noticeably expressed in IL-1β stimulated chondrocytes, while raloxifene decreased in a very significant manner the gene expression of iNOS at both of the concentrations used. The results of the biochemical evaluation were confirmed by TEM. Our data suggest that raloxifene may have a potential chondroprotective role in osteoarthritis.

Sperm with fibrous sheath dysplasia and anomalies in head-neck junction: focus on centriole and centrin 1.

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 1s 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.

18,X,Y aneuploidies and transmission electron microscopy studies in spermatozoa from five carriers of different reciprocal translocations

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.

Mud-bath therapy in addition to usual care in bilateral knee osteoarthritis: Economic evaluation alongside a randomized controlled trial.

To perform a cost‐effectiveness analysis of mud‐bath therapy (MBT) in addition to usual treatment compared to usual treatment alone in patients with bilateral knee osteoarthritis (OA).

Abnormal elongation of midpiece, absence of axoneme and outer dense fibers at principal piece level, supernumerary microtubules: a sperm defect of possible genetic origin?

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.

Hydrostatic Pressure Regulates MicroRNA Expression Levels in Osteoarthritic Chondrocyte Cultures via the Wnt/β-Catenin Pathway

Mechanical loading and hydrostatic pressure (HP) regulate chondrocytes’ metabolism; however, how mechanical stimulation acts remain unclear. MicroRNAs (miRNAs) play an important role in cartilage homeostasis, mechanotransduction, and in the pathogenesis of osteoarthritis (OA). This study investigated the effects of a cyclic HP (1–5 MPa), in both normal and OA human chondrocytes, on the expression of miR-27a/b, miR-140, miR-146a/b, and miR-365, and of their target genes (MMP-13, ADAMTS-5, IGFBP-5, and HDAC-4). Furthermore, we assessed the possible involvement of Wnt/β-catenin pathway in response to HP. Chondrocytes were exposed to HP for 3h and the evaluations were performed immediately after pressurization, and following 12, 24, and 48 h. Total RNA was extracted and used for real-time PCR. β-catenin was detected by Western blotting analysis and immunofluorescence. In OA chondrocytes, HP induced a significant increase (p < 0.01) of the expression levels of miR-27a/b, miR-140, and miR-146a, and a significant reduction (p < 0.01) of miR-365 at all analyzed time points. MMP-13, ADAMTS-5, and HDAC-4 were significantly downregulated following HP, while no significant modification was found for IGFBP-5. β-catenin levels were significantly increased (p < 0.001) in OA chondrocytes at basal conditions and significantly reduced (p < 0.01) by HP. Pressurization did not cause any significant modification in normal cells. In conclusion, in OA chondrocytes, HP restores the expression levels of some miRNAs, downregulates MMP-13, ADAMTS-5, and HDAC-4, and modulates the Wnt/β-catenin pathway activation.

A case of severe asthenozoospermia: a novel sperm tail defect of possible genetic origin identified by electron microscopy and immunocytochemistry

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.

Clinical and biochemical effects of a 3-week program of diet combined with spa therapy in obese and diabetic patients: a pilot open study

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.