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Background: SARS-CoV-2 is associated with an increased risk of venous and arterial thrombosis, but the underlying mechanism is still unclear. Methods: We performed a cross-sectional analysis of platelet function in 25 SARS-CoV-2 and 10 healthy subjects by measuring Nox2 (NADPH oxidase 2)-derived oxidative stress and thromboxane B 2 , and investigated if administration of monoclonal antibodies against the S protein (Spike protein) of SARS-CoV-2 affects platelet activation. Furthermore, we investigated in vitro if the S protein of SARS-CoV-2 or plasma from SARS-CoV-2 enhanced platelet activation. Results: Ex vivo studies showed enhanced platelet Nox2-derived oxidative stress and thromboxane B 2 biosynthesis and under laminar flow platelet-dependent thrombus growth in SARS-CoV-2 compared with controls; both effects were lowered by Nox2 and TLR4 (Toll-like receptor 4) inhibitors. Two hours after administration of monoclonal antibodies, a significant inhibition of platelet activation was observed in patients with SARS-CoV-2 compared with untreated ones. In vitro study showed that S protein per se did not elicit platelet activation but amplified the platelet response to subthreshold concentrations of agonists and functionally interacted with platelet TLR4. A docking simulation analysis suggested that TLR4 binds to S protein via three receptor-binding domains; furthermore, immunoprecipitation and immunofluorescence showed S protein-TLR4 colocalization in platelets from SARS-CoV-2. Plasma from patients with SARS-CoV-2 enhanced platelet activation and Nox2-related oxidative stress, an effect blunted by TNF (tumor necrosis factor) α inhibitor; this effect was recapitulated by an in vitro study documenting that TNFα alone promoted platelet activation and amplified the platelet response to S protein via p47phox (phagocyte oxidase) upregulation. Conclusions: The study identifies 2 TLR4-dependent and independent pathways promoting platelet-dependent thrombus growth and suggests inhibition of TLR4. or p47phox as a tool to counteract thrombosis in SARS-CoV-2.

BACKGROUND/AIM Although still controversial, the current treatment for locally advanced neuroendocrine carcinoma of the cervix (NECC) relies on chemoradiation (CRT). The aim of this study is to evaluate the alternative role of combined chemotherapy and surgery in treating NECC. PATIENTS AND METHODS This is a retrospective series of patients undergoing radical surgery after neoadjuvant chemotherapy (NACT) for locally advanced NECC (stages IIB-IVA). Histological examination and immunohistochemistry were performed on surgical specimens to confirm diagnosis. Systematic literature search was conducted to identify other cases treated with chemotherapy and surgery. RESULTS Seven patients with a mean age of 49 years were identified. The mean greatest diameter at diagnosis was 59.3±24.7 mm. FIGO stage was IIB in 14.3% of patients, IIIB in 28.6%, IIIC in 42.9%, and IVA in 14.3%. The response to NACT was partial, ranging from 50% to 80%. Neuroendocrine markers were expressed in all cases. The mean progression-free survival (PFS) and overall survival (OS) were 15.0±30.6 months and 26.3±36.4 months, respectively. Eleven studies encompassing a total of 27 patients met eligibility criteria for the systematic review. CONCLUSION Surgery after NACT for locally advanced NECC may yield similar outcomes compared to CRT. The benefit of performing surgery as a primary approach could lie in the possibility of reserving CRT for recurrences. Since randomized clinical trials are difficult to be designed, an expert consensus is required to address the non-inferiority of radical surgery over CRT.

Background: The network of intermediate filament proteins underlying the inner nuclear membrane forms the nuclear lamina. A and B-type lamins are the major components of the nuclear lamina. Lamins have been associated with important cellular functions: DNA replication, chromatin organization, differentiation of the cell, apoptosis and in maintenance of nuclear structure. Little is known regarding the etiopathogenesis of adhesive capsulitis (AC); recently, a dysregulating fibrotic response starting from a subpopulation has been described within the fibroblast compartment that suddenly turns on an activated phenotype. Considering the key role of A-type lamin in the regulation of cellular stability and function we performed an immunohistochemical analysis to compare the lamin A/C expression between patients with AC and healthy controls. Materials and methods: Twenty-six patients [12M-14F, mean age (SD): 52.3 (6.08)] were enrolled. Tissue samples excised from the rotator interval were analysed for lamin A/C expression by immunohistochemistry. Controls were represented by samples obtained by normal rotator interval excised from patients submitted to shoulder surgery [6M-9F, mean age (SD): 57.1 (5.3)]. The intensity of staining was graded, and an H-score was assigned. Statistical analysis was performed. Results: The expression of lamin A/C was found to be significantly lower in the fibroblasts of patients with adhesive capsulitis when compared to controls (intensity of staining: p: 0.005; H-score: 0.034); no differences were found regarding the synoviocytes (p>0.05). Considering only patients with AC, lamin A/C intensity staining was found to be significantly higher in samples where acute inflammatory infiltrate was detected (p: 0.004). No significant changes in levels of lamin A/C expression were documented between the mild and severe adhesive capsulitis severity groups. Conclusions: Our study demonstrated that the activity of lamin A/C in maintaining nuclear structural integrity and cell viability is decreased in patients with adhesive capsulitis. The timing of the pathogenetic process is the key factor for cell functionality which is maintained in the early stages of the disease; on the contrary, the clinical severity of adhesive capsulitis plays a marginal role on nuclear stability. Level of Evidence: III

Background: Immunotherapy has revolutionized the approach to metastatic triple-negative breast cancers (mTNBC). Atezolizumab was approved for patients with mTNBC whose tumours express PD-L1, determined by SP 142 assay. To assess the availability and practice of SP142 test we administered a survey to all the 15 Pathology Departments of the Lazio Region during a 6-months period.Methods: The survey comprised 12 questions regarding the availability of SP142 in the Pathology Departments, the percentage of positive tests, the difficulties of pathologists in case close to cut-off value and the tested samples.Results: The SP142 assay was available only in 8 Centers. In case of positive result, most Centers (5/8, 62.5%) reported values of PD-L1 expression ranging from > 1 to Conclusion: Our results raise some important issues concerning the evaluation of PD-L1 in the “real-life” setting, providing strategies for its implementation.

Cancer immunotherapy has significantly improved the management of many malignancies in recent years. Although cervical cancer is the second most common women's cancer in the world, there are still few information about the role of checkpoint inhibitors in this neoplasm, especially in the neoadjuvant setting. In the present study, we retrieved 38 consecutive patients with squamous cell cervical cancer who underwent platinum-based neoadjuvant chemotherapy (NACT) followed by radical surgery. Pre-therapy biopsies were evaluated for the presence of tumor-infiltrating lymphocytes (TILs), including T (both cytotoxic CD8+ and helper CD4+) and B lymphocytes, macrophages, natural-killer cells, and eosinophils. Immunohistochemistry was performed to characterize the inflammatory cells and to evaluate programmed death-ligand 1 (PD-L1) expression on both neoplastic and inflammatory cells. We divided our study population in three groups using three cut-offs (10%, 10-40%,40%), for both TILs and PD-L1 evaluation. Pathological response to NACT was obtained from the histological reports of the post-therapy surgical specimens. We observed that all cases showed stromal TILs, with a predominance of CD3+/CD4+ T helper cells, thus supporting the strong immunogenic potential of cervical cancer. The vast majority of neoplasms expressed PD-L1: 100% on immune cells and 92% on tumor cells. Firstly, we noticed that the percentage of neoplastic cells PD-L1+ was positively associated with high TIL percentage (p = 0.0073) and with increased PD-L1 expression on inflammatory cells (p = 0.0297). Secondly, we observed a significant correlation between both the percentage (p = 0.0105) of TILs and the expression of PD-L1 (p = 0.01045) on inflammatory cells and pathological response to NACT. These results suggest that cervical cancer could be a good target for immunotherapy, also in the neoadjuvant setting. Furthermore, PD-L1 expression was significantly associated with stromal TILs that interestingly may predict pathological response to NACT.

The ultraviolet (UV) component of solar radiation is the driving force of life on earth, but it can cause photoaging and skin cancer. In this study, we investigated the effects of the glucosamine-derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA) on human primary fibroblasts (FBs) stimulated in vitro with environmental levels of UVB radiation. FBs were irradiated with 0.04 J cm-2 UVB dose, which resulted a mild dosage as shown by the cell viability and ROS production measurement. This environmental UVB dose induced activation of MAP kinase ERK 1/2, the stimulation of c-fos and at lower extent of c-jun, and in turn AP-1-dependent up-regulation of pro-inflammatory factors IL-6 and IL-8 and suppression of collagen type I expression. On the contrary, 0.04 J cm-2 UVB dose was not able to stimulate metalloprotease production. NAPA treatment was able to suppress the up-regulation of IL-6 and IL-8 via the inhibition of MAP kinase ERK phosphorylation and the following AP-1 activation, and was able to attenuate the collagen type I down-regulation induced by the UVBs. Taken together, our results show that NAPA, considering its dual action on suppression of inflammation and stimulation of collagen type I production, represents an interesting candidate as a new photoprotective and photorepairing agents.

Background. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare syndrome of unclear aetiology occurring after DNA-based vaccinations against COVID-19. The aim of this study was to investigate the DNA vaccine-encoded Sars-cov-2 soluble spike protein (SP). as a potential trigger of platelet activation in VITT. Methods. We studied three VITT patients and seven healthy controls (HCs) within 3 weeks from the first dose of ChAdOx1 nCoV-19, and one non vaccinated HC. Serum levels of SP and soluble angiotensin-converting enzyme 2 (sACE2), ACE2 expression in platelets and platelet response to VITT serum stimulation were studied. A thrombus retrieved during mechanical thrombectomy from one VITT patients, was analysed by immunohistochemistry for SP and ACE2. Neutrophil extracellular traps (NETs) markers and coagulation parameters were also measured. Results. We detected serum SP (up to 35 days post-vaccination) and sACE2 in all VITT patients, and respectively in two and three out of 7 vaccinated HCs. Only platelets from one non-vaccinated HC expressed ACE2. VITT sera markedly activated platelets and this activation was inhibited by both anti-SP and anti-FcγRIIA blocking antibodies. The thrombus showed positive immunohistochemical labelling of platelets using an anti-SP antibody with reduced ACE2 expression, compared to a thrombus from a pre-pandemic stroke patient. Markers of endothelial dysfunction, NETs and hypercoagulability state were present in all VITT sera. Conclusions. The present data provides first evidence that DNA vaccine-encoded Sars-cov-2 SP is detectable in VITT sera (several weeks post-vaccination) and in a platelet-rich thrombus, and that may contribute to the initial platelet stimulation in VITT patients.

High- and low-risk endometrial carcinoma (EC) differ in whether or not a lymphadenectomy is performed. We aimed to develop MRI-based radio-genomic models able to preoperatively assess lymph-vascular space invasion (LVSI) and discriminate between low- and high-risk EC according to the ESMO-ESGO-ESTRO 2020 guidelines, which include molecular risk classification proposed by “ProMisE”. This is a retrospective, multicentric study that included 64 women with EC who underwent 3T-MRI before a hysterectomy. Radiomics features were extracted from T2WI images and apparent diffusion coefficient maps (ADC) after manual segmentation of the gross tumor volume. We constructed a multiple logistic regression approach from the most relevant radiomic features to distinguish between low- and high-risk classes under the ESMO-ESGO-ESTRO 2020 guidelines. A similar approach was taken to assess LVSI. Model diagnostic performance was assessed via ROC curves, accuracy, sensitivity and specificity on training and test sets. The LVSI predictive model used a single feature from ADC as a predictor; the risk class model used two features as predictors from both ADC and T2WI. The low-risk predictive model showed an AUC of 0.74 with an accuracy, sensitivity, and specificity of 0.74, 0.76, 0.94; the LVSI model showed an AUC of 0.59 with an accuracy, sensitivity, and specificity of 0.60, 0.50, 0.61. MRI-based radio-genomic models are useful for preoperative EC risk stratification and may facilitate therapeutic management.

The development of a periostin-rich microenvironment in areas associated with insult, orchestrating pathways of repair and rebuilding, is documented. Literature lacks information regarding the presence of periostin in the context of rotator cuff tear (RCT). 55 consecutive patients with RCT were enrolled. Immunohistochemical periostin detection was performed on tissue samples excised from tear margins. Our study documented the presence of periostin in the margins of RCT. It is plausible that, when a tear occurs, multiple stimuli, both mechanical and inflammatory, lead to the development of a periostin-rich microenvironment as an attempt to tendon healing.

Background The network of intermediate filament proteins underlying the inner nuclear membrane forms the nuclear lamin. A- and B-type lamins are the major components of the nuclear lamina. Lamins function in many nuclear activities. The role of lamin A and transcription factors (NF-kB) as anti-apoptotic is well documented. Recently, lamin A has also been considered as a mechanosensor protein that is able to maintain nuclear integrity from mechanical insults. We aimed to verify how lamin A expression varies in healthy cuff cells and in those with different-sized tears where various mechanical stresses are present. Methods Forty-three patients with rotator cuff tear (RCT) [23M–20F, mean age (SD): 63.5 (6.1)] were enrolled. Tissue samples excised from the most medial point of tear margins were analyzed for lamin A expression by immunohistochemistry. Controls were represented by samples obtained by normal supraspinatus tendons excised from patients submitted to reverse shoulder prosthesis implant [8M–7F, mean age (SD): 67.9 (7.1)]. The intensity of staining was graded, and an H-score was assigned. Statistical analysis was performed. Results Our study revealed a moderate intensity of lamin A in the healthy cuff tendons, a higher expression of this protein in the small tears, and a significant decrease of lamin A with increasing tear size (p Conclusions Our study emphasizes the importance of early repair of small RCTs since nuclear stability is maintained, and the cellular function is protected by lamin A overexpression. High re-tear of massive cuff repair could be due to cellular apoptosis and nuclear modifications induced by lamin A lack. Level of evidence III

Harpagophytum procumbens (Burch.) DC. ex Meisn. is a traditional remedy for osteoarticular diseases, including osteoarthritis (OA), although the bioactive constituents and mechanisms involved are yet to be clarified. In the present study, an aqueous H. procumbens root extract (HPE, containing 1.2% harpagoside) was characterized for its effects on synoviocytes from OA patients and phytochemical composition in polyphenols, and volatile compounds were detected. HPE powder was dissolved in different solvents, including deionized water (HPEH2O), DMSO (HPEDMSO), 100% v/v ethanol (HPEEtOH100), and 50% v/v ethanol (HPEEtOH50). The highest polyphenol levels were found in HPEDMSO and HPEEtOH50, whereas different volatile compounds, mainly &beta, caryophyllene and eugenol, were detected in all the extracts except for HPEH2O. HPEH2O and HPEDMSO were able to enhance CB2 receptor expression and to downregulate PI-PLC &beta, 2 in synovial membranes, moreover, all the extracts inhibited FAAH activity. The present results highlight for the first time a multitarget modulation of the endocannabinoid system by HPE, likely ascribable to its hydrosoluble compounds, along with the presence of volatile compounds in H. procumbens root. Although hydrosoluble compounds seem to be mainly responsible for endocannabinoid modulation by HPE, a possible contribution of volatile compounds can be suggested, strengthening the hypothesis that the entire phytocomplex can contribute to the H. procumbens healing properties.

Cancer cells are able to release high amounts of extracellular vesicles, thereby conditioning the normal cells in the surrounding tissue and/or in distant target organs. In the context of bone cancers, previous studies suggested that osteosarcoma cancer cells produce transforming extracellular vesicles able to induce a tumour-like phenotype in normal recipient cells. Indeed, phosphoinositide-specific phospholipase C (PI-PLC) enzymes are differentially expressed in osteosarcoma cell lines with increasing aggressiveness, thus providing helpful insights to better define their role and functions in this bone tumour. By confocal microscopy analysis, we demonstrated that osteosarcoma-derived extracellular vesicles convey all the assessed PI-PLC isoforms, and that they localize into cell membrane bubble-like structures, resembling extracellular vesicles about to be released, as conveyed and/or membrane protein. Cytofluorimetric analysis confirmed the presence of PI-PLC isoforms in the extracellular vesicles collected from conditioned media of osteosarcoma cells. These findings suggest the feasibility to use circulating extracellular vesicles as biomarkers of osteosarcoma progression and/or the monitoring of this distressing disease.

Mesenchymal cystic hamartoma (MCH) of the lung is a rare disease, with an indolent course in the majority of cases. It can be single or multifocal and it is composed of primitive mesenchymal cells admixed with cystic spaces. Only few cases have been reported in the literature, with variable clinical presentation. We describe the case of a huge MCH, presenting with spontaneous pneumothorax in a 65-year-old man. Further, we provide a brief overview of the literature and discuss the differential diagnosis with other entities, and the possible diagnostic pitfalls.

The nuclear lamina is essential for the maintenance of nuclear shape and mechanics. Mutations in lamin genes have been identified in a heterogeneous spectrum of human diseases known as &ldquo, laminopathies&rdquo, associated with nuclear envelope defects and deregulation of cellular functions. Interestingly, osteosarcoma is the only neoplasm described in the literature in association with laminopathies. This study aims characterized the expression of A-type and B-type lamins and emerin in osteosarcoma, revealing a higher percentage of dysmorphic nuclei in osteosarcoma cells in comparison to normal osteoblasts and all the hallmarks of laminopathic features. Both lamins and emerin were differentially expressed in osteosarcoma cell lines in comparison to normal osteoblasts and correlated with tumor aggressiveness. We analysed lamin A/C expression in a tissue-microarray including osteosarcoma samples with different prognosis, finding a positive correlation between lamin A/C expression and the overall survival of osteosarcoma patients. An inefficient MKL1 nuclear shuttling and actin depolymerization, as well as a reduced expression of pRb and a decreased YAP nuclear content were observed in A-type lamin deficient 143B cells. In conclusion, we described for the first time laminopathic nuclear phenotypes in osteosarcoma cells, providing evidence for an altered lamins and emerin expression and a deregulated nucleoskeleton architecture of this tumor.

Background: Bone hardness and strength depends on mineralization, which involves a complex process in which calcium phosphate, produced by bone-forming cells, was shed around the fibrous matrix. This process is strictly regulated, and a number of signal transduction systems were interested in calcium metabolism, such as the phosphoinositide (PI) pathway and related phospholipase C (PLC) enzymes. Objectives: Our aim was to search for common patterns of expression in osteoblasts, as well as in ES and SS. Methods: We analysed the PLC enzymes in human osteoblasts and osteosarcoma cell lines MG-63 and SaOS-2. We compared the obtained results to the expression of PLCs in samples of patients affected with Ewing sarcoma (ES) and synovial sarcoma (SS). Results: In osteoblasts, MG-63 cells and SaOS-2 significant differences were identified in the expression of PLC δ4 and PLC η subfamily isoforms. Differences were also identified regarding the expression of PLCs in ES and SS. Most ES and SS did not express PLCB1, which was expressed in most osteoblasts, MG-63 and SaOS-2 cells. Conversely, PLCB2, unexpressed in the cell lines, was expressed in some ES and SS. However, PLCH1 was expressed in SaOS-2 and inconstantly expressed in osteoblasts, while it was expressed in ES and unexpressed in SS. The most relevant difference observed in ES compared to SS regarded PLC ε and PLC η isoforms. Conclusion: MG-63 and SaOS-2 osteosarcoma cell lines might represent an inappropriate experimental model for studies about the analysis of signal transduction in osteoblasts

Endothelial cells (EC) act as leading actors in angiogenesis. Understanding the complex network of signal transduction pathways which regulate angiogenesis might offer insights in the regulation of normal and pathological events, including tumours, vascular, inflammatory and immune diseases. The effects of olive oil and of Blueberry extracts upon the phosphoinositide (PI)-specific phospholipase C (PLC) enzymes were evaluated both in quiescent and inflammatory stimulated human umbilical vein EC (HUVEC) using molecular biology (multiliquid bioanalysis) and immunofluorescence techniques. Oleuropein significantly increased the number of surviving HUVEC compared to untreated controls, suggesting that it favours the survival and proliferation of EC. Our results suggest that Oleuropein might be useful to induce EC proliferation, an important event during angiogenesis, with special regard to wound healing. Blueberry extracts increased the number of surviving HUVEC, although the comparison to untreated controls did not result statistically significant. Lipopolysaccharide (LPS) administration significantly reduced the number of live HUVEC. LPS can also modify the expression of selected PLC genes. Adding Blueberry extracts to LPS treated HUVEC cultures did not significantly modify the variations of PLC expression induced by LPS. Oleuropein increased or reduced the expression of PLC genes, and statistically significant results were identified for selected PLC isoforms. Oleuropein also modified the effects of LPS upon PLC genes’ expression. Thus, our results corroborate the hypothesis that Oleuropein owns anti-inflammatory activity. The intracellular localization of PLC enzymes was modified by the different treatments we used. Podosome-like structures were observed in differently LPS treated HUVEC.

Background Post-translational-modifications (PTMs) are implicated in the pathogenesis of autoimmune diseases, leading to the break of self-tolerance, generation of new autoantigens and production of autoantibodies. Anti-citrullinated-proteins antibodies (ACPA) play a key role in the pathogenesis of rheumatoid arthritis (RA) and many evidences showed that they are related to bone loss in RA. Osteoclasts are the primary targets of ACPA in the joint site, displaying citrullinated antigens on their surface. Carbamylation is another PTM generating the production of anti-carbamylated proteins antibodies (anti-CarP), recently proposed as a new biomarker for RA. Anti-CarP have been also studied in patients with systemic lupus erythematosus (SLE) with predominant joint involvement, showing a prevalence of 46.1%. Moreover, in SLE patients these autoantibodies have been also associated to bone erosive damage. The pathogenic mechanism of these autoantibodies is not completely clarified so far. Objectives The aim of the present study was to investigate the possible pathogenic role of anti-CarP in the induction of osteoclastogenesis. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from a buffy coat from healthy donors using a Ficoll Hypaque gradient. Cells were cultured for 14 days in α-MEM complete medium, supplemented with 25 ng/ml of macrophage-colony-stimulating-factor (MCSF) and 30 ng/ml of receptor activator of nuclear-factor-kappa-β-ligand (RANKL), for the differentiation of monocytes in osteoclasts. Simultaneously, PBMCs were co-cultured in presence of anti-carbamilated-vimentin autoantibodies (anti-Vim CarP) purified from sera of 3 patients with RA and 3 patients with SLE with a predominant joint involvement at 100 ng/ml and 1 μg/ml. As control, intravenous immunoglobulin (IVIg) were used at the same concentrations. After 14 days, osteoclast differentiation was evaluated by staining cells for tartrate-resistant acid phosphatase (TRAP), using leukocyte acid phosphatase kit. Results Anti-VimCarP purified from RA patients at 100 ng/ml were able to increase the median number of osteoclasts per well significantly more than untreated and IVIg at 100 ng/ml and 1 μg/ml (P=0.0008; P=0.0009 and P=0.0009, respectively). The same results were observed treating cells with 1 μg/ml of anti-VimCarP from RA patients (P=0.0008, P=0.002 and P=0.002, respectively). Similarly, anti-VimCarP purified from SLE patients at 100 ng/ml significantly increased the median number of osteoclasts than untreated and IVIg at 100 ng/ml and 1 μg/ml (P=0.002, P=0.004 and P=0.003, respectively). Analogously, we observed similar results co-culturing cells with 1 μg/ml of anti-VimCarP from SLE patients (P=0.002, P=0.02 and P=0.006, respectively). Conclusion For the first time, we demonstrated that anti-Vim CarP purified from RA and SLE patients are able to induce osteoclastogenesis, suggesting their potential role as a biomarker of joint erosiveness in these autoimmune diseases. Disclosure of Interests Laura Massaro: None declared, Tania Colasanti: None declared, francesca spinelli: None declared, Fulvia Ceccarelli: None declared, Riccardo Mancini: None declared, Arbi Pecani: None declared, enrica cipriano: None declared, Carlo Perricone Speakers bureau: BMS; Lilly, Celgene, Sanofi, Francesca Miranda: None declared, Simona Truglia: None declared, Francesco Natalucci: None declared, Martina Leopizzi: None declared, Valeria Di Maio: None declared, Carlo Della Rocca: None declared, cristiano alessandri: None declared, Guido Valesini: None declared, fabrizio conti: None declared

Background: Patients with Rheumatoid Arthritis (RA) have an increased risk of cardiovascular diseases (CVD). Inflammation and autoantibodies indipendently promote endothelial dysfunction, which is the earliest, reversible stage of atherosclerosis. In vitro studies have demonstrated that antibodies against carbamylated proteins (anti-CarP), recently described in RA patients, can induce the production of proatherosclerotic molecules like Vascular Cell Adhesion Molecule (VCAM-1) and activate Interleukin-1 Receptor-Associated Kinase (IRAK-1), Nuclear Factor kB (NF-kB), inducible Nitric Oxide Synthase (iNOS) in endothelial cells (1,2). Moreover, anti-CarP are associated to endothelial dysfunction and subclinical atherosclerosis in RA patients (3). Objectives: Aims of the present study were: 1) to analyze the role of vimentin as a target of autoantibody response in the serum of patients with RA and 2) investigate the expression of vimentin and carbamylated proteins in endothelial cells. Methods: Consecutive RA patients were enrolled in this study. Vimentin was carbamylated and used as an antigen for the detection of anti-Vimentin Carbamylated antibodies (CarVim), through immunoenzymathic methods. Cells were incubated with anti-vimentin and carbamylated antibodies. The presence of vimentin and carbamylated proteins was investigated by immunofluorescence on the immortalized endothelial cell line EA.hy 926. Results: Eighty-eight (88) RA patients were enrolled in this study (F:M = 79:9, mean age = 56 ± 13 years). Anti-CarVim antibodies were present in 9% of the patients with a mean titre of di 442 aU/ML (IQR 303 aU/ml). Vimentin and carbamylated proteins were detected in the endothelial cells by immunofluorescence (Figure 1). Conclusion: The results of this study confirm that Vimentin is one of the antigenic targets of anti-CarP antibodies present in the serum of RA patients. The presence of carbamylated proteins and vimentin in endothelial cells suggests that anti-CarVim could bind the modified protein and determine endothelial activation and subsequent endothelial dysfunction. Reference [1] Pecani A & Alessandri C et al, Arthr Res Ther 2016 [2] Pecani A et al, Reumatismo 2017 [3] Spinelli FR & Pecani A et al, BMC Musculoskelet Disord 2017. Disclosure of Interests: None declared

Calciphylaxis is a rare disease characterized by ectopic calcification of skin arterioles resulting in ischemia, thrombosis and necrosis. Since end stage renal disease patients are those mainly affected, the term calcific uremic arteriolopathy (CUA) is also suggested. Early clinical manifestations are subtle, while overt necrotic ulcers may quickly spread and become infected so as to result in ominous outcome. Diagnosis might not be easy due to the number of other ischemic and non-ischemic skin lesions observed in uraemia. Skin biopsy, has been proposed as the diagnostic test and is often considered, but not systematically performed due to the hypothetical risk of further spreading of the lesions. Such ambiguity could be responsible for misdiagnosis or underdiagnosis. We review here five consecutive cases recorded in our Unit, all submitted to skin biopsy but with inconsistent results which generated some clinical frustration. Thus, we decided to carefully re-evaluate all of them together with pathologists and dermatologists. However, even after this ex-post discussion, we could not reach a complete agreement on the final diagnosis. In the meanwhile, papers were published in the literature that started to shed some light on the role of skin biopsy in the diagnosis of CUA.

Background:Carbamylation is a post-translational modification occurring under several conditions such as uremia, smoking and chronic inflammation as in rheumatoid arthritis (RA). Low-density lipoproteins (LDL) represent a target of carbamylation. Carbamylated-LDL (cLDL) have an increased inflammatory and atherogenic potential. Growing evidence supports an influence of modified lipids on bone cells homeostasis. However, the role of cLDL on bone cells physiology is still unknown.Objectives:Considering the rate of carbamylation and the role of anti-carbamylated proteins antibodies as markers of erosive disease in RA, the purpose of this study is to investigate the effect of cLDL on bone homeostasis.Methods:In-vitrocarbamylation of LDL was performed as previously described by Ok et al. (Kidney Int. 2005). Briefly, native LDL (nLDL) were treated with potassium cyanate (KOCN) for 4 hours, followed by excessive dialysis for 36 hours to remove KOCN. Both osteoclasts (OCs) and osteoblasts (OBLs) were treated at baseline with 20 μg/ml, 100 μg/ml and 200 μg/ml of cLDL or nLDL. To induce osteoclast differentiation, CD14+ monocytes were isolated from peripheral blood of healthy donors by magnetic microbeads separation and then cultured on a 96-wells plate in DMEM media supplemented with RANKL and M-CSF. After 10 days cells were fixed, stained for tartrate-resistant acid phosphatase (TRAP), a marker of OC differentiation, and counted. OBLs were isolated from bone specimens of 3 patients who had undergone to knee or hip arthroplasty for osteoarthritis and treated for 5 days with different concentrations of cLDL and nLDL. OBLs were fixed and stained for alkaline phosphatase positive activity (ALP), a marker of osteogenic differentiation. Total RNA was extracted from cell lysates. Copies of single-stranded complementary DNA (cDNA) were synthesized and analyzed by real-time PCR to evaluate RANKL and Osteoprotegerin (OPG) mRNA expression levels.Results:In OCLs culture, cLDL significantly decreased the number of OC compared to untreated cells (200 μg/ml p=0,0015) and nLDL treated cells (200 μg/ml p= 0,011; 20 μg/ml p= 0,0014) (Fig 1). Moreover, treatment with cLDL induced an increase of not terminally differentiated OCs, reduced dimensions of OCs, less intense TRAP staining and vacuolization (Fig 2). In OBLs culture, cLDL (20, 100 μg/ml) significantly reduced the ALP activity of OBLs compared with untreated cells (pFig 1.Fig 2.Fig 3.Fig 4.Conclusion:cLDL induce a significant depression of OC and OBL differentiation. Moreover, cLDL increase RANKL expression in OBL, unbalancing bone tissue turnover towards bone resorption. Accordingly, cLDL could be implicated in the bone loss characterizing several conditions associated to an increased carbamylation, such as RADisclosure of Interests:Bruno Lucchino: None declared, Martina Leopizzi: None declared, Tania Colasanti: None declared, Valeria Di Maio: None declared, cristiano alessandri Grant/research support from: Pfizer, Guido Valesini: None declared, fabrizio conti Speakers bureau: BMS, Lilly, Abbvie, Pfizer, Sanofi, Manuela Di Franco: None declared, Francesca Romana Spinelli Grant/research support from: Pfizer, Consultant of: Novartis, Gilead, Lilly, Sanofi, Celgene, Speakers bureau: Lilly

BACKGROUND Sclerostin (SC) is a monomeric glycoprotein expressed by osteocytes that affects bone formation. Recent studies have suggested a potential role for this protein in the pathophysiology of vascular diseases. The aim of the present study was to investigate SC expression in atherosclerotic plaques of patients affected by severe atherosclerotic disease who underwent carotid endarterectomy. We also evaluated possible differences in SC expression between patients with and without type 2 diabetes (T2D). METHODS This was a cross-sectional study involving 46 patients aged 55 to 80 years (mean, 71.1 ± 6.7 years, 36 men, 15 patients with T2D) who underwent carotid endarterectomy. Immunohistochemical levels of SC were evaluated in the atherosclerotic plaques by double-staining immunochemistry, and serum SC levels were evaluated by enzyme-linked immunosorbent assay. RESULTS Sclerostin was present in the atherosclerotic plaques of all subjects investigated and increased significantly in the media compared with the intima (P

Burning bones Background Citrullinated peptide are one of the main target of immune response in Rheumatoid Arthritis (RA) and antibodies to citrullinated peptides (ACPA) are involved in bone resorption. One of the target antigens – citrullinated vimentin – is expressed on the surface of osteoclast precursors where it can bind the antibodies starting the differentiation in mature osteoclast. Moreover, recent data demonstrated that serum levels of receptor activator of nuclear factor-κB ligand (RANKL) are higher in ACPA-positive RA patients. RANKL is the main osteoblast-derived cytokine inducing osteoclastogenesis. Antibodies directed against carbamylated proteins (Anti-CarP) have been recently described in RA patients with Rheumatoid Arthritis. The effect of anti-CarP on bone resorption has not been yet addressed. Objectives The aim of the study was to investigate in vitro the effect of anti-CarP on Osteoprotegerin (OPG) and RANK ligand (RANKL) production in osteoblast cultures. Methods Anti-CarP were investigated by ELISA in the sera of 88 RA patients using carbamylated fetal calf serum (CarFCS) and non-modified FCS as antigens. Anti-CarFCS were purified from the sera of 3 RA patients who tested highly positive for anti-CarP. Osteoblasts were isolated from the femoral head of 3 patients undergoing total hip arthroplasty and cultured in three different conditions – 1 ng/ml of anti-CarFCS, 10 ng/ml of anti-CarFCS or control medium for 4–6 days, until confluence. RNA was extracted from cell lysates and OPG and RANKL mRNA expression was analysed by Real-time PCR. Moreover, OPG and RANK expression was investigated by immunofluorescence on treated and non-treated cells. differences were determined either with two-way repeated measures analysis of variance (ANOVA) with Bonferroni’s multiple comparison test, using Prism 5.0 software. A p value Results In osteoblast cultures, anti-CarFCS decreased the expression of OPG and increased the expression of RANKL in a dose-dependent manner, leading to an increase in RANKL/OPG ratio (figure 1A and 1B). The result was confirmed by the immunofluorescence analysis demonstrating the subcellular co-location of OPG and RANKL in osteoblast cultures (figure 1C). Conclusions The results of the study confirm that anti-CarFCS can be detected in nearly 40% of RA patients. The increase of RANKL/OPG ratio in the osteoblast cultures treated with anti-CarFCS suggests an effect of such autoantibodies on osteoclastogenesis and osteoclasts activity, supporting their possible involvement in the development of bone erosions. Disclosure of Interest None declared

The phosphoinositide (PI) signal transduction pathway participates in liver metabolism. Abnormal activity or expression of PI-specific phospholipase C (PLC) enzymes has been described in different liver diseases. We resume the role of the PI metabolism in liver and PLC abnormalities in different liver diseases. Moreover, we present the results of PLC analyses in a normal human liver and an alcohol-damaged liver. PLC enzymes and the expression of the corresponding genes in liver biopsies from individuals deceased for complications of the alcoholic liver disease (ALD) at different stages compared with normal controls (deceased individuals with histologically normal livers without alcohol addiction anamnesis) were analyzed by using immunohistochemistry and molecular biology techniques. The expression panel of PLCs was described in normal and alcohol abuse liver. Our observations suggest that the regulation of PLC expression might be due to posttranscriptional events and that alcohol affects the epigenetic control of PLC expression belonging to PI signaling. We also describe the alternate expression of PLCB1 and PLCH1 genes in liver. Our results corroborate literature data suggesting that PLC enzymes are differently expressed in normal versus pathological liver, playing a role in the histopathogenesis of liver tissue damage. The expression and/or localization of selected PLC isoforms is especially affected in alcohol-related liver tissue histopathology. Our present observations confirm that the modulation of protein synthesis plays a role in the regulation of PLC enzymes. We also suggest that this modulation might act at the transcription level. Further studies are required to investigate related epigenetic mechanisms.

OBJECTIVE: Treatment of wounds difficult to heal concerns 50% of the elderly population in Italy and is therefore an extreme relevance social burden. The present study shows how the treatment with autologous leuco-platelets reduces the healing time of wounds improving the functional recovery. PATIENTS AND METHODS: One hundred patients with ulcers of the legs were divided in two groups: 1) 50 patients treated with conventional therapies; 2) 50 patients treated with autologous leuco-platelet concentrate (LPC) and HYAFF as a scaffold. RESULTS: After 2 months a 49% reduction of the wound area was observed in the second group and in a remarkable share (about 65%) wound reduction was obtained in 15 days (4 LPC dressings). On the contrary, patients treated only by simple medications, showed a longer healing time and a greater percentage of failures. Morphometric analysis of bioptic samples obtained from the edge as well as from the bottom of the lesions obtained from the LPC group, detected an abundant presence of neoformed capillaries, characterized by a cubic, "reactive endothelium", close to the site of leuco-platelet infiltration. CONCLUSIONS: These results suggest that healing was promoted not only by limiting bacterial infections but also by the release of chemiotatic and proangiogenetic factors from leukocytes and platelets, improving the neoformation of capillaries.

Background Symptoms and signs of myocardial ischemia in the absence of obstructive coronary disease are common in hypertensive patients. This can be explained by coronary microvascular dysfunction (CMD) due to adverse remodeling of coronary arterioles which have also been reported in the spontaneously hypertensive rat (SHR). Objective The aim of this study was to compare the effect of Ramipril, Perindopril, Candesartan, Atenolol, Amlodipine, Indapamide and HMR1766 on coronary microvascular dysfunction (CMD) in the spontaneously hypertensive rat (SHR). Methods Eight groups of 24 week old SHR were treated for 8 weeks. Blood pressure (BP) was measured invasively at the end of treatment. After sacrifice, hearts were mounted on a Langendorff apparatus for measurement of hyperemic coronary flow (CF). Hearts were then processed for histomorphometric analysis. Results All compounds, except HMR1766, induced a significant reduction of BP. Perindopril and Candesartan increased hyperemic CF whereas the other compounds had no significant effect. Perindopril, Ramipril, Atenolol, Indapamide and HMR1766 induced significant reverse arteriolar remodeling whereas Candesartan and Amlodipine did not. Conclusions The effect of anti-hypertensive treatment on CMD is not only dependent on BP reduction. Compounds with comparable anti-hypertensive efficacy may exert different effects on CF and induce different degrees of reverse arteriolar remodeling. This article is protected by copyright. All rights reserved.

Chronic inflammation has been associated to cancer development by the alteration of several inflammatory pathways, such as Nuclear Factor-κB pathway. In particular, IκB kinase α (IKKα), one of two catalytic subunit of IKK complex, has been described to be associated to cancer progression and metastasis in a number of cancers. The molecular mechanism by which IKKα affects cancer progression is not yet completely clarified, anyway an association between IKKα and the expression of Maspin (Mammary Serine Protease Inhibitor or SerpinB5), a tumor suppressor protein, has been described. IKKα shuttles between cytoplasm and nucleus, and when is localized into the nuclei, IKKα regulates the expression of several genes, among them Maspin gene, whose expression is repressed by high amount of nuclear IKKα. Considering that high levels of Maspin have been associated with reduced metastatic progression, it could be hypothesized that the repression of IKKα nuclear translocation could be associated with the repression of metastatic phenotype. The present study is aimed to explore the ability of a glucosamine derivative, 2-(N-Carbobenzyloxy)l-phenylalanylamido-2-deoxy-β-d-glucose (NCPA), synthesized in our laboratory, to stimulate the production of Maspin in an osteosarcoma cell line, 143B. Immunofluorescence and Western blotting experiments showed that NCPA is able to inhibit IKKα nuclear translocation, and to stimulate Maspin production. Moreover, in association with stimulation of Maspin production we found the decrease of β1 Integrin expression, the down-regulation of metalloproteases MMP-9 and MMP-13 production and cell migration inhibition. Taking in account that β1 Integrin and MMP-9 and -13 have been correlated with the invasiveness of osteosarcoma, considering that NCPA affects the invasiveness of 143B cell line, we suggest that this molecule could affect the osteosarcoma metastatic ability.

Ezrin, a protein belonging to the Ezrin, radixin and moesin (ERM) family, was engaged in the metastatic spread of osteosarcoma. The Protein 4.1, Ezrin, radixin, moesin (FERM) domain of Ezrin binds the membrane Phosphatydil inositol (4,5) bisphosphate (PIP2), a crucial molecule belonging to the Phosphoinositide (PI) signal transduction pathway. The cytoskeleton cross-linker function of Ezrin largely depends on membrane PIP2 levels, and thus upon the activity of related enzymes belonging to the PI-specific phospholipase C (PI-PLC) family. Based on the role of Ezrin in tumour progression and metastasis, we silenced the expression of Vil2 (OMIM *123900), the gene which codifies for Ezrin, in cultured human osteosarcoma 143B and Hs888 cell lines. After Ezrin silencing, the growth rate of both cell lines was significantly reduced and morphogical changes were observed. We also observed moderate variations both of selected PI-PLC enzymes within the cell and of expression of the corresponding PLC genes. In 143B cell line the transcription of PLCB1 decreased, of PLCG2 increased and of PLCE differed in a time-dependent manner. In Hs888, the expression of PLCB1 and of PLCD4 significantly increased, of PLCE moderately increased in a time dependent manner; the expression of PLCG2 was up-regulated. These observations indicate that Ezrin silencing affects the transcription of selected PLC genes, suggesting that Ezrin might influence the expression regulation of PI-PLC enzymes.

Endothelial cells (EC) are the first elements exposed to mediators circulating in the bloodstream, and react to stimulation with finely tuned responses mediated by different signal transduction pathways, leading the endothelium to adapt. Neuropeptide Y (NPY), the most abundant peptide in heart and brain, is mainly involved in the neuroendocrine regulation of the stress response. The regulatory roles of NPY depend on many factors, including its enzymatic processing, receptor subtypes and related signal transduction systems, including the phosphoinositide (PI) pathway and related phospholipase C (PI-PLC) family of enzymes. The panel of expression of PI-PLC enzymes differs comparing quiescent versus differently stimulated human EC. Growing evidences indicate that the regulation of the expression of PLC genes, which codify for PI-PLC enzymes, might act as an additional mechanism of control of the PI signal transduction pathway. NPY was described to potentiate the activation of PI-PLC enzymes in different cell types, including EC. In the present experiments, we stimulated human umbilical vein EC using different doses of NPY in order to investigate a possible role upon the expression PLC genes. NPY reduced the overall transcription of PLC genes, excepting for PLCE. The most significant effects were observed for PLCB2 and PLCD1, both isoforms recruited by means of G-proteins and G-protein-coupled receptors. NPY behavior was comparable with other PI-PLC interacting molecules that, beside the stimulation of phospholipase activity, also affect the upcoming enzymes' production acting upon gene expression. That might represent a mode to regulate the activity of PI-PLC enzymes after activation.

Objectives: The aim of the study was to determine whether the release by macrophages of matrix metalloproteinase (MMP)-12 and vascular endothelial growth factor (VEGF) - leading to inflammation, matrix degradation and neoangiogenesis - represents an effective pathway that underlies aortic wall remodeling in Stanford type A acute aortic dissection (AAD). Methods: Twenty-one consecutive patients with no genetic predisposition, with Stanford type A AAD were selected. In each patient, the levels of serum VEGF, MMP-12, serum interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1 were evaluated using enzyme-linked immunosorbent assay. Ascending aortic specimens were collected for immunohistochemical identification of any presence of inflammatory infiltrate, VEGF and CD31 expression. Results: A significant increase in serum VEGF (p = 0.044), MMP-12 (p = 0.007), IL-6 (p = 0.0001), IL-8 (p = 0.0001) and MCP-1 (p = 0.0001) levels was observed in the AAD group compared to the control group. Furthermore, all AAD samples were positive for VEGF in the tunica media and showed vessel growth and immune-inflammatory infiltrate. A large number of cases (62.79%) showed inflammation at the edge of the dissection and approximately half (51.42%) showed neovessels growing at the edge of the dissection. Conclusions: The results suggest that VEGF-mediated angiogenesis and matrix degradation play a role in AAD. Finally, we believe that MMP-12 should be considered a marker of AAD.

Background/Aims Gastrointestinal stromal tumors (GISTs) strongly express a receptor tyrosine kinase (RTK, c-KIT-CD117) harboring a KIT mutation that causes constitutive receptor activation leading to the development and growth of tumors; 35% of GISTs without KIT mutations have platelet-derived growth factor receptor alpha (PDGFRA) mutations, and the type of mutation plays an important role in the response to treatment. This study aimed to establish the frequency of stop codon mutations in the RTKs, KIT, and PDGFRA, in GISTs and correlate this molecular alteration with protein expression and treatment responsiveness. Methods Seventy-nine GISTs were analyzed for both KIT and PDGFRA mutations. Immunohistochemical expression was studied in tissue microarray blocks. Results We found three rare KIT mutations in exon 11 that induced a stop codon, two at position 563 and one at position 589, which have never been described before. All three tumors were CD117-, DOG1-, and CD34-positive. Two patients with a KIT stop codon mutation did not respond to imatinib therapy and died shortly after treatment. Conclusions The association between stop codon mutations in KIT and patient survival, if confirmed in a larger population, may be useful in choosing effective therapies.

Macrophages’ phenotypic and functional diversity depends on differentiating programs related to local environmental factors. Recent interest was deserved to the signal transduction pathways acting in macrophage polarization, including the phosphoinositide (PI) system and related phospholipase C (PLC) family of enzymes. The expression panel of PLCs and the subcellular localization differs in quiescent cells compared to the pathological counterpart. We analyzed the expression of PLC enzymes in unpolarized (M0), as well as in M1 and M2 macrophages to list the expressed isoforms and their subcellular localization. Furthermore, we investigated whether inflammatory stimulation modified the basal panel of PLCs’ expression and subcellular localization. All PLC enzymes were detected within both M1 and M2 cells, but not in M0 cells. M0, as well as M1 and M2 cells own a specific panel of expression, different for both genes’ mRNA expression and intracellular localization of PLC enzymes. The panel of PLC genes’ expression and PLC proteins’ presence slightly changes after inflammatory stimulation. PLC enzymes might play a complex role in macrophages during inflammation and probably also during polarization.

Background A large number of phospholipase C (PLC) enzymes, both mRNA transcripts and proteins, have been detected in osteoblasts, corroborating the importance of calcium regulation in bone tissue. MG-63 and SaOS-2 human osteosarcoma cell lines are actually considered osteoblast-like cells, and are therefore widely used as experimental models for osteoblasts. Objectives Our aim was to verify whether MG-63 or SaOS-2 cells might also represent appropriate experimental osteoblast models for signal transduction studies, with special regard to the phosphoinositide (PI) pathway. We analyzed the expression and the subcellular distribution of enzymes related to calcium signal transduction (the PI-specific PLC family), which are known to possess high cell/tissue specificity. Materials and Methods The expression of PLC genes was analyzed by performing RT-PCR experiments. The presence of PLC enzymes and their subcellular distribution within the cells was analyzed with immunofluorescence experiments. Results Osteoblasts, MG-63 cells, and SaOS-2 cells have expression panels similar to those of PLC enzymes. However, slight differences were found in the expression of enzymes belonging to the PLCη subfamily. Conclusions MG-63 and SaOS-2 osteosarcoma cell lines might not represent appropriate experimental models for studies that aim to analyze signal transduction in osteoblasts.

The definition of the number and nature of the signal transduction pathways involved in the pathogenesis and the identification of the molecules promoting metastasis spread might improve the knowledge of the natural history of osteosarcoma, also allowing refine the prognosis and opening the way to novel therapeutic strategies. Phosphatydil inositol (4,5) bisphosphate (PIP2), belonging to the Phosphoinositide (PI) signal transduction pathway, was related to the regulation of ezrin, an ezrin–radixin–moesin protein involved in metastatic osteosarcoma spread. The levels of PIP2 are regulated by means of the PI-specific Phospholipase C (PLC) enzymes. Recent literature data suggested that in osteosarcoma the panel of expression of PLC isoforms varies in a complex and unclear manner and is related to ezrin, probably networking with Ras GTPases, such as RhoA and Rac1. We analyzed the expression and the subcellular localization of PLC enzymes in cultured human osteosarcoma MG-63 cells, commonly used as an experimental model for human osteoblasts, using U-73122 PLC inhibitor, U-73343 inactive analogue, and by silencing ezrin. The treatment with U-73122 significantly reduces the number of MG-63 viable cells and contemporarily modifies the expression and the subcellular localization of selected PLC isoforms. U-73122 reduces the cell growth in cultured MG-63 ostesarcoma cell line involving PI-specific Phospholipases C.

The survival of osteosarcoma patients is connected to metastasis. The ezrin expression is associated with the development of metastasis and poor outcome in osteosarcoma. Ezrin is present in the cytoplasm and after phosphorylation assumes an active form and links F-actin to the cell membrane. This study evaluated ezrin and phosphorylated ezrin at site Tyr354 and Thr567 expression and its subcellular localization in osteosarcoma. We studied 50 osteosarcoma patients (mean follow-up 9.8 years). Ezrin expression was assessed using immunohistochemical and immunofluorescence analysis on tissue microarray and cultured cells of human osteosarcoma 143B. The western blot analysis was carried out on cultured cells. The majority of osteosarcomas, showing cytoplasmic positivity for ezrin, phosphorylated and unphosphorylated, were associated with membranous and nuclear positivity for phosphorylated ezrin Thr567 and phosphorylated ezrin Tyr354, respectively. Ezrin expression was associated with high-grade osteosarcoma (P=0.04), with metastasis (P=0.04) and with tumors that developed metastasis (P=0.04); phosphorylated ezrin Thr567 expression was present mostly in tumors with metastasis (P=0.01) and in osteosarcomas that did not develop metastasis (P=0.002). The osteosarcoma patients with ezrin expression have a short survival. The cytoplasmic ezrin expression in osteosarcoma matches its role of membrane-cytoskeleton linker protein. The subcellular trafficking of ezrin is not blocked and it is linked to ezrin phosphorylation, also in cancer. The phosphorylated ezrin Tyr354 nuclear localization suggests its possible role as a nuclear factor in osteosarcoma. The phosphorylated ezrin Thr567 phosphorylation may not be necessary in osteosarcoma metastatic progression but it was modulated. The ezrin expression is associated with more aggressive osteosarcomas and with metastasis.

Sclerostin, the secreted protein product of the SOST gene, which is mainly expressed by osteocytes, has recently been proposed as a negative regulator of bone osteoblastogenesis. Chronic elevation of PTH reduces SOST expression by osteocytes, while controversial results have been obtained by intermittent PTH administration. We have investigated the effects of intermittently administered PTH on SOST expression and sclerostin localization, comparing them with those of controls, as they appeared in three different bone segments of rat tibia: secondary trabecular metaphyseal and epiphyseal bone, and cortical diaphyseal bone. The histomorphometric results demonstrate that PTH enhances bone turnover through anabolic effects, as shown by the association of increased bone resorption variables with a significant rise in BV/TV, Tb.Th and Tb.N and a fall in Tb.Sp. PTH induces a SOST mRNA and protein fall in secondary metaphyseal trabeculae, diaphyseal bone and in epiphyseal trabeculae. Numbers of sclerostin immunopositive osteocytes/mm(2) show no change, compared with controls; there are fewer sclerostin-positive osteocytes in secondary metaphyseal trabeculae than in the other two bone areas, both in the control and PTH groups. The low numbers of sclerostin-positive osteocytes in the metaphyseal trabecular bone seem to be directly related to the fact that this area displays a high remodeling rate. The anabolic effects of PTH are in line with the fall of SOST mRNA and protein in all the three bone segments examined; the rise of bone turnover supports a negative role of SOST in bone formation.

A number of studies suggested that suicide may be associated with specific neurobiological abnormalities. Neurobiology studies focused upon abnormalities of signalling mechanisms with special regard to the serotonin system and the related Phosphoinositide (PI) signalling system. Previous data suggested the involvement of the PI-specific phospholipase C (PLC) family in neuropsychiatric disorders. By using PCR and morphological microscopy observation we examined the whole panel of expression of PLC isoforms in the brains of 28 individuals who committed suicide and in normal controls in order to evaluate the involvement of specific PLC isoforms. The overall PLC expression was reduced and a complex reorganization of the isoforms was observed. The knowledge of the complex network of neurobiological molecules and interconnected signal transduction pathways in the brain of suicide victims might be helpful to understand the natural history and the pathogenesis of the suicidal behavior. That might lead to obtain prognostic suggestions in order to prevent suicide and to new therapeutic agents targeting specific sites in this signalling cascade.

Selected Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes occupy the convergence point of the broad range of pathways that promote Rho and Ras GTPase mediated signalling, which also regulate the activation of ezrin, a member of the ezrin-radixin-moesin (ERM) proteins family involved in the metastatic osteosarcoma spread. Previous studies described that in distinct human osteosarcoma cell lines ezrin networks the PI-PLC with complex interplay controlling the expression of the PLC genes, which codify for PI-PLC enzymes. In the present study, we analyzed the expression and the sub-cellular distribution of RhoA and Rac1 respectively after ezrin silencing and after PI-PLC e silencing, in order to investigate whether ezrin-RhoGTPAses signalling might involve one or more specific PI-PLC isoforms in cultured 143B and Hs888 human osteosarcoma cell lines. In the present experiments, both ezrin and PLCE gene silencing had different effects upon RhoA and Rac1 expression and sub-cellular localization. Displacements of Ezrin and of RhoA localization were observed, probably playing functional roles.

Summary Objective Aim of this study is to investigate the effects of Glucosamine (GlcN) and its peptidyl-derivative, 2-(N-Acetyl)-l-phenylalanylamido-2-deoxy-β-d-glucose (NAPA), on extracellular matrix (ECM) synthesis in human primary chondrocytes (HPCs). Methods Dose-dependent effect of GlcN and NAPA on Glycosaminoglycan (GAG), Collagen type II (Col2) and Small Leucine-Rich Proteoglycans (SLRPs) was examined by incubating HPCs, cultured in micromasses (3D), with various amounts of two molecules, administered as either GlcN alone or NAPA alone or GlcN plus NAPA (G + N). Immunohystochemical and immunofluorescent staining and biochemical analysis were used to determine the impact of the two molecules on ECM production. Gene expression analysis was performed by TaqMan Real-Time Polymerase Chain Reaction (PCR) assays. Results The lowest concentration to which GlcN and NAPA were able to affect ECM synthesis was 1 mM. Both molecules administered alone and as G + N stimulated GAGs and SLRPs synthesis at different extent, NAPA and mainly G + N stimulated Col2 production, whereas GlcN was not effective. Both molecules were able to induce Insulin Growth Factor-I (IGF-I) and to stimulate SOX-9, whereas NAPA and G + N were able to up-regulate both Hyaluronic Acid Synthase-2 and Hyaluronic acid. Very interesting is the synergistic effect observed when chondrocyte micromasses were treated with G + N. Conclusions The observed anabolic effects and optimal concentrations of GlcN and NAPA, in addition to beneficial effects on other cellular pathways, previously reported, such as the inhibition of IKKα, could be useful to formulate new cartilage repair strategies.

Osteoprotegerin (OPG) and the receptor activator of nuclear factor (NF)-kB ligand (RANKL) are key regulators of osteoclastogenesis. The present study had the main aim of showing the localization of OPG and RANKL mRNA and protein in serial sections of the rat femurs and tibiae by immunohistochemistry (IHC) and in situ hybridization (ISH). The main results were: (1) OPG and RANKL mRNA and protein were co-localized in the same cell types, (2) maturative/hypertrophic chondrocytes, osteoblasts, lining cells, periosteal cells and early osteocytes were stained by both IHC and ISH, (3) OPG and RANKL proteins were mainly located in Golgi areas, and the ISH reaction was especially visible in active osteoblasts, (4) immunolabeling was often concentrated into cytoplasmic vacuoles of otherwise negative proliferative chondrocytes; IHC and ISH labeling increased from proliferative to maturative/hypertrophic chondrocytes, (5) the newly laid down bone matrix, cartilage-bone interfaces, cement lines, and trabecular borders showed light OPG and RANKL immunolabeling, (6) about 70% of secondary metaphyseal bone osteocytes showed OPG and RANKL protein expression; most of them were ISH-negative, (7) osteoclasts were mostly unstained by IHC and variably labeled by ISH. The co-expression of OPG and RANKL in the same bone cell types confirms their strictly coupled action in the regulation of bone metabolism.

Besides the control of calcium levels, the phosphoinositide-specific phospholipases C (PI-PLCs), the main players in the phosphoinositide signalling pathway, contribute to a number of cell activities. The expression of PI-PLCs is strictly tissue specific and evidence suggests that it varies under different conditions, such as tumour progression or cell activation. In previous studies, we obtained a complete panel of expression of PI-PLC isoforms in human umbilical vein endothelial cells (HUVEC), a widely used experimental model for endothelial cells (EC), and demonstrated that the expression of the PLC genes varies under inflammatory stimulation. The fibroblast growth factor (FGF) activates the PI-PLC γ1 isoform. In the present study, PI-PLC expression in FGF-treated HUVEC was performed using RT-PCR, observed 24 h after stimulation. The expression of selected genes after stimulation was perturbed, suggesting that FGF affects gene transcription in PI signalling as a possible mechanism of regulation of its activity upon the AkT-PLC pathway. The most efficient effects of FGF were recorded in the 3-6-h interval. To understand the complex events progressing in EC might provide useful insights for potential therapeutic strategies. The opportunity to manipulate the EC might offer a powerful tool of considerable practical and clinical importance.