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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.

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.

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

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.

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.

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 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.

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.

Objectives: Metastatic recurrence is the most frequent cause of death after surgical resection of lung cancer. Manipulation during surgery has been advocated as one of the causes contributing to promotion of spreading. Methods: We investigated if the detection of plasma circulating free DNA (cfDNA) is influenced by surgical manipulation in 25 lung cancer patients (17 males and eight females) undergoing complete resection; 20 health subjects formed the control group. Bloodstream levels of cfDNA were detected before surgery, one week and one month after surgery. Results: CfDNA levels measured preoperatively and in the control group were 23 07 ± 7 4 ng/mL and 7 5 ± 3 4 ng/mL respectively (p=0 0002); levels at one week and one month were 68 2 ± 36 2 ng/mL and 9 6 ± 3 1 ng/mL respectively. The difference between the three time points were statistically significant (preop vs. one week p=0 0006; one week vs. one month p=0 0003) with an increase in the first week and a strong decrease after one month. CfDNA levels at one month were not statistically different from those recorded in the control group. There was no correlation between preoperative cfDNA levels, tumour stage, grading and histology and patient demographics. No correlation was found between postoperative cfDNA, type of surgical procedure, histology and stage. After a median follow-up of 16 months no recurrence was detected. Conclusions: Surgical manipulation determines increased cfDNA levels in the early postoperative period; however, after one month they decrease within the normal range, at levels that are statistically comparable with healthy subjects.

Osteosarcoma is the most common malignant tumor of the bone. The major cause of death in osteosarcoma is the increase in metastatic potential, and the ezrin expression has been correlated with the metastasis development. Ezrin interacts with RhoGDI by dissociating it from RhoGTPases, which allow GTPases to load with GTP, activate RhoA to increase cell migration, and invasion. RhoGTPases have been found to contribute to pathologic processes including cancer cell migration, invasion, and metastasis and overexpression of either the GTPase itself or some elements of Rho signaling that have been detected in many human tumors, including Rac1 and RhoA. We have analyzed Rac1 and RhoA expression in the osteosarcoma tissues to understand the role of the ezrin-Rho family pathway in osteosarcoma metastatic progression. Moreover, we have blocked the ezrin expression using siRNA assay to investigate a possible correlation with RAC1 and RHOA expression in the osteosarcoma cell lines. Our immunohistochemical data showed that many osteosarcomas presented cytoplasmatic positivity for both Rac1 and RhoA and cases, both ezrin positive than ezrin negative, revealed the protein expression of Rac1 and RhoA. The results obtained by ezrin siRNA transfection showed that ezrin expression in the osteosarcoma cell lines might modulate, mainly, the Rac1 expression. It is possible that the mechanism of cell motility mediated by Rac1 and RhoA is maintained in osteosarcomas, and since the expression of ezrin, Rac1 and RhoA do not correlate with metastatic progression in osteosarcoma. However, osteosarcomas without metastasis displayed a positivity for Rac1 and RhoA expression compared with metastatic osteosarcomas and this could be a protective factor.

Osteosarcoma is the most common primary malignant tumour of the bone. Although new therapies continue to be reported, osteosarcoma-related morbidity and mortality remain high. Modern medicine has greatly increased knowledge of the physiopathology of this neoplasm. Novel targets for drug development may be identified through an understanding of the normal molecular processes that are deeply modified in pathological conditions. The aim of the present study is to investigate, by immunohistochemistry, the localisation of different growth factors and of the proliferative marker Ki-67 in order to determine whether these factors are involved in the transformation of osteogenic cells and in the development of human osteosarcoma. We observed a general positivity for NGF — TrKA — NT3 — TrKC — VEGF in the cytoplasm of neoplastic cells and a strong expression for NT4 in the nuclear compartment. TGF-p was strongly expressed in the extracellular matrix and vascular endothelium. BDNF and TrKB showed a strong immunolabeling in the extracellular matrix. Ki-67/MIB-1 was moderately expressed in the nucleus of neoplastic cells. We believe that these growth factors may be considered potential therapeutic targets in the treatment of osteosarcoma, although proof of this hypothesis requires further investigation.

We verified if the nuclear factor-κB (NF-κB) was present on the margins of rotator cuff tears (RCTs). Because NF-κB regulates apoptosis and stimulates neoangiogenesis, we hypothesized that NF-κB has a role in the evolution of RCT and in possible mechanisms of RCT healing.Samples from tear margins, subacromial bursa, and healthy subscapular tendons were excised during arthroscopic treatment of patients with posterosuperior RCT. Sections were cut and stained with hematoxylin and eosin for morphologic evaluation and used for immunohistochemical analysis with NF-κB p65 antibody.The presence of NF-κB in the RCT margins and subacromial bursa increases with increasing tear size. NF-κB is also present in the subscapularis tendon of patients with large and massive RCT. Analogously, we observed that neoangiogenesis grows with increasing RCT size and is always present in the subscapularis tendon independently from RCT size. Statistical analysis indicates that NF-κB and neoangiogenesis are correlated, regardless of the dimension of the RCT.This is the first study that identifies the association between activated NF-κB and RCT. Activated NF-κB on the margins of RCT increases with increasing tear size. We hypothesized a series of possible causes responsible for NF-κB activation; however, we believe that activation is due to tissue hypoxia. Activated p65 directly stimulates neoangiogenesis, but the same factors that regulate NF-κB activation might also act as neoangiogenesis inductors.

Background and Aim: Diabetic nephropathy is the main cause of end-stage renal disease. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), a physiological tetrapeptide hydrolyzed by the angiotensin-converting enzyme (ACE), has antifibrotic effects in the cardiovascular system and in the kidney in experimental models of hypertension, heart failure and renal disease. The aim of the study was to evaluate the effect of Ac-SDKP in diabetic nephropathy and the potential additive effect of Ac-SDKP, when compared to ACE inhibitors alone, on the development of renal fibrosis. Method: Diabetes was induced in 28 Sprague-Dawley rats by a single intraperitoneal injection of streptozotocin. Control rats (n = 10) received only buffer solution. An ACE inhibitor (ramipril, 3 mg/kg/day) was administered to 11 diabetic rats. After 2 months, Ac-SDKP (1 mg/kg/day) was administered by osmotic minipumps for 8 weeks to 7 diabetic rats and to 6 diabetic rats treated with ramipril. Osmotic minipumps delivered saline solution in the corresponding sham-treated rats (diabetic rats, n = 10, and ramipril-treated diabetic rats, n = 5). Results: Diabetic rats showed a significant increase in blood glucose level, urinary albumin excretion and renal fibrosis, and a reduction of glomerular nephrin expression with respect to control rats. Ac-SDKP administration significantly reduced renal fibrosis in diabetic rats, without significantly reducing urinary albumin excretion. Ramipril treatment caused a significant decrease in albuminuria and renal fibrosis and restored glomerular nephrin expression. Administration of Ac-SDKP, in addition to ramipril, further reduced renal fibrosis with respect to ramipril alone, while it did not improve the antiproteinuric effect of ramipril. Conclusion: Ac-SDKP administration reduces renal fibrosis in diabetic nephropathy. Addition of Ac-SDKP to ACE inhibition therapy improves the reduction of renal fibrosis with respect to ACE inhibition alone, suggesting a beneficial effect of this pharmacological association in diabetic nephropathy.

Circulating free DNA (cfDNA) is present in higher concentration in non-small-cell lung cancer (NSCLC) patients than in controls. This study was designed to assess the sensitivity and specificity of Agilent 2100 Bioanalyzer to identify patients with NSCLC and to compare it with quantitative RealTime-PCR (RT-qPCR) assay. 30 NSCLC patients and 26 controls were analyzed. The amount of cfDNA was determined both through quantitative RT-PCR targeting the human β-actin gene and by Agilent 2100 Bioanalyzer. Performances of the assays were calculated by the receiver operating characteristic (ROC) curves. The mean cfDNA concentration, obtained through the use of Agilent 2100 Bioanalyzer, in NSCLC patients (94.5 ng/mL) was almost twice the concentration detected in controls (42.8 ng/mL) as well as found by RT-qPCR (22.5 ng/mL vs 7.1 ng/mL, p

The genetic and epigenetic factors underlying the variable penetrance of homoplasmic mitochondrial DNA mutations are poorly understood. We investigated a 16-year-old patient with hypertrophic cardiomyopathy harboring a homoplasmic m.4277T>C mutation in the mt-tRNA Ile (MTTI) gene. Skeletal muscle showed multiple respiratory chain enzyme abnormalities and a decreased steady-state level of the mutated mt-tRNA Ile . Transmitochondrial cybrids grown on galactose medium demonstrated a functional effect of this mutation on cell viability, confirming pathogenicity. These findings were reproduced in transmitochondrial cybrids, harboring a previously described homoplasmic m.4300A>G MTTI mutation. The pathogenic role of the m.4277T>C mutation may be ascribed to misfolding of the mt-tRNA molecule, as demonstrated by the altered electrophoretic migration of the mutated mt-tRNA. Indeed, structure and sequence analyses suggest that thymidine at position 4277 of mt-tRNA Ile is involved in a conserved tertiary interaction with thymidine at position 4306. Interestingly, the mutation showed variable penetrance within family members, with skeletal muscle from the patient’s clinically unaffected mother demonstrating normal muscle respiratory chain activities and steady-state levels of mt-tRNA Ile , while homoplasmic for the m.4277T>C mutation. Analysis of mitochondrial isoleucyl-tRNA synthetase revealed significantly higher expression levels in skeletal muscle and fibroblasts of the unaffected mother when compared with the proband, while the transient over-expression of the IARS2 gene in patient transmitochondrial cybrids improved cell viability. This is the first observation that constitutively high levels of aminoacyl-tRNA synthetases (aaRSs) in human tissues prevent the phenotypic expression of a homoplasmic mt-tRNA point mutation. These findings extend previous observations on aaRSs therapeutic effects in yeast and human.

MicroRNAs play an important role in myocardial diseases. MiR-133a regulates cardiac hypertrophy, while miR-29b is involved in cardiac fibrosis. The aim of this study was to evaluate whether miR-133a and miR-29b play a role in myocardial fibrosis caused by Angiotensin II (Ang II)-dependent hypertension. Sprague–Dawley rats were treated for 4 weeks with Ang II (200 ng/kg/min) or Ang II + irbesartan (50 mg/kg/day in drinking water), or saline by osmotic minipumps. At the end of the experimental period, cardiac miR-133a and miR-29b expression was measured by real-time PCR, and myocardial fibrosis was evaluated by morphometric analysis. A computer-based prediction algorithm led to the identification of collagen 1a1 (Col1A1) as a putative target of miR-133a. A reporter plasmid bearing the 3′-untranslated regions (UTRs) of Col1A1 mRNA was constructed and luciferase assay was performed. MiR-133a suppressed the activity of luciferase when the reporter gene was linked to a 3′-UTR segment of Col1A1 (P

Inflammatory tenosynovitis is an inflammation that involves the tendons and synovial sheaths caused by minor trauma repeated for a long period of time. This inflammatory disease may be involved in the onset of tunnel carpal syndrome (CTS), because of the thickening of the tendon sheath that may produce an increase in the carpal canal pressure and damage of the median nerve in the wrist. Recent studies suggest that in patients with CTS pathological changes occur in the subsynovial connective tissue, such as vascular proliferation and non-inflammatory synovial fibrosis. However, little is known about the pathological mechanism of tenosynovial thickening. The aim of this study is to evaluate the potential role of vascular endothelial growth factor (VEGF), prostaglandin E2 (PGE2) and trasforming growth factor-beta (TGF-β) in the modifications of connective synovial tissue of CTS specimens in order to determine whether these factors play a role in the development of this disease. Ten specimens from patients with CTS and four control tissues (cadavers) were analyzed by immunohistochemistry using specific antibodies against these growth factors. A temporary increase in the production of these molecules was found in cells within the vessels and synovial lining during the intermediate phase of the syndrome, when the histology of the tenosynovium changes from oedematous to fibrotic. Our data confirm a close correlation between the expression of PGE2and VEGF. Recent histological examinations have shown a marked increase in vascular proliferation and reduction of fibroblast density in specimens from CTS patients during the intermediate phase. Our study indicates that the expression of TGF-β in fibroblasts and vascular endothelial cells of synovial connective tissues of CTS patients was significantly higher than in those of controls. These findings suggest that angiogenesis appears to take place as a part of a regenerative reaction that results in fibrosis. We believe that VEGF, TGF-β and PGE2may be potential therapeutic targets in the treatment of this disease although proof of this evidence requires further studies.

Objective To delineate the panel of expression of phosphoinositide-specific phospholipase C (PI-PLC) signaling enzymes in normal endometrium and in endometriosis. Design Clinical/experimental study. Setting University. Patient(s) Healthy donor woman and endometriosis-affected woman. Intervention(s) Normal endometrium and endometriosis surgical biopsies were analyzed using gene expression analyses methodology (reverse transcriptase-polymerase chain reaction [PCR], bioanalyses). Main Outcome Measure(s) Gene expression (messenger RNA concentration) measures of 12 PI-PLC enzymes: PI-PLC β1, PI-PLC β2, PI-PLC β3, PI-PLC β4, PI-PLC γ1, PI-PLC γ2, PI-PLC δ1, PI-PLC δ3, PI-PLC δ4, PI-PLC e, PI-PLC η1, and PI-PLC η2. Result(s) PI-PLC β1, PI-PLC β3, PI-PLC δ1, and PI-PLC δ3 enzymes were detected, although differently expressed in normal and endometriosis tissues. Conclusion(s) The involvement of PI-PLC enzymes in inflammation and the consistency of susceptible endometriosis loci with PI-PLC genes mapping corroborate the hypothesis that PI signaling might be involved in the pathogenesis of endometriosis.

Introduction Primary gallbladder neuroendocrine tumors are extremely rare, representing 0.2% of all neuroendocrine tumors. The diagnosis is incidental in most cases. Case presentation We describe the case of a 57-year-old Caucasian man who underwent laparoscopic cholecystectomy for the evaluation of a gallbladder polyp that had been incidentally detected by ultasonography. Histologically, his lesion was composed of monomorphic cells that contained small round nuclei and that were organized in small nodular, trabecular, and acinar structures. His cells were positive for chromogranin A and synaptophysin, and a diagnosis of "typical" carcinoid of the gallbladder was made. His post-operative computerized axial tomography, 111In-pentetreotide scintigraphy, and hormone-specific marker results were negative. He is disease-free 45 months after surgical treatment. Conclusions Characteristic pathological findings of the gallbladder neuroendocrine tumors predict the prognosis. Whereas classical carcinoids of the gallbladder only rarely have a metastatic or invasive phenotype, the "atypical" variants are more aggressive and are associated with a poorer prognosis. Given the difficulty in distinguishing between benign and malignant lesions in the pre-surgical setting, we tend to consider each polypoid-like lesion of the gallbladder to be a high-risk lesion if it is larger than 1 cm and, as a result, to emphasize the need for cholecystectomy in all cases, relying on the pathological and immunohistochemistry analyses for the final diagnosis.

The controversy on whether or not inflammatory infiltrates in chronic lymphocytic thyroiditis predispose to cancer, has now merged into a debate over the role of the inflammatory infiltrates. The question is how and why some cells become transformed and what factors allow them to spread and in some cases become invasive. Here, we show that the amount of inflammatory mediators such as nitric oxide (NO) and high mobility group Box 1 protein (HMGB1) produced in thyroiditis microenvironment increases in tumors and could be involved in the cellular transformation process. NO and HMGB1 are known to attract macrophages that would promote angiogenesis, matrix remodelling and suppression of an efficient immune response. Inflammatory infiltrates could increase the risk of papillary cancer in patients with autoimmune lymphocytic thyroiditis. Cytokines and soluble inflammatory mediators involved in cancer-related inflammation are not only a target for innovative diagnostic and therapeutic strategies but they also represent a future challenge for oncologists.

Ac-SDKP (N-acetyl-seryl-aspartyl-lysyl-proline) is a physiological tetrapeptide hydrolysed by ACE (angiotensin-converting enzyme). In experimental models of hypertension, Ac-SDKP has antifibrotic effects in the heart; however, the role of Ac-SDKP in diabetic cardiomyopathy is currently unknown. The aim of the present study was to evaluate the effect of Ac-SDKP on cardiac systolic and diastolic function, and interstitial and perivascular fibrosis in the heart of diabetic rats. Diabetes was induced in 55 Sprague - Dawley rats by streptozotocin injection. Control rats (n=18) underwent only buffer injection. Out of the 55 diabetic rats, 19 were chronically treated with insulin and 13 with the ACEI (ACE inhibitor) ramipril (3 mg · kg-1 of body weight · day-1). At 2 months after the onset of diabetes, Ac-SDKP (1 mg · kg-1 of body weight · day-1) was administered by osmotic minipumps for 8 weeks to eight control rats, 13 diabetic rats, seven diabetic rats treated with ramipril and nine insulin-treated diabetic rats. Diabetic rats had a significant increase in blood glucose levels. Left ventricular interstitial and perivascular fibrosis, and TGF-β1 (transforming growth factor-β1) protein levels were increased in diabetic rats, but not in insulin-treated diabetic rats and ramipril-treated diabetic rats, compared with control rats. Ac-SDKP administration significantly reduced left ventricular interstitial and perivascular fibrosis in diabetic rats and in diabetic rats treated with ramipril. This was accompanied by a significant reduction in active TGF-β1 and phospho-Smad2/3 protein levels in myocardial tissue of diabetic rats. Echocardiography showed that diabetes was associated with increased end-systolic diameters, and depressed global systolic function and diastolic dysfunction, as assessed by transmitral Doppler velocity profile. These changes were completely reversed by insulin or ramipril treatment. Ac-SDKP treatment partially restored diastolic function in diabetic rats. In conclusion, Ac-SDKP administration in diabetic rats reduces left ventricular interstitial and perivascular fibrosis, active TGF-β1 and phospho-Smad2/3 levels, and improves diastolic function. Taken together, these findings suggest that, by inhibiting the TGF-β/Smad pathway, Ac-SDKP protects against the development of diabetic cardiomyopathy. © The Authors Journal compilation © 2010 Biochemical Society.

Matrix Gla protein (MGP) and fetuin-A are inhibitors of arterial calcifications. In blood of rats, calcium–phosphate–fetuin–MGP complexes, produced in bone, have been identified. Indeed, an association between bone resorption, release of such complexes, and arterial calcifications has been reported. We have investigated the synthesis and localization of fetuin-A and MGP in bone of hemodialysis patients and the possible contribution of bone cells in arterial calcifications. Bone biopsies from 11 hemodialysis patients were used for histology, in situ hybridization of fetuin-A and MGP messenger RNA (mRNA), immunohistochemistry of fetuin-A, and total, carboxylated, and non-carboxylated MGP proteins. Patients showed various types of renal osteodystrophy, or normal bone. MGP was synthesized and expressed (total and carboxylated) by osteoblasts, osteocytes, and most osteoclasts, while fetuin-A by osteoblasts and osteocytes. Fetuin-A and carboxylated MGP proteins were positive in the calcified matrix, while total MGP was negative. Osteoid seams were negative to fetuin-A, lightly positive to carboxylated MGP, and occasionally positive to total MGP. Undercarboxylated MGP was mostly undetectable. In adult humans, fetuin-A is produced also by osteoblasts, and not only by hepatocytes, as previously believed. MGP, essentially carboxylated, is synthesized by osteoblasts and most osteoclasts. Increased bone turnover can be an important contributor to arterial calcifications.

The known role of heat-shock proteins (HSPs) in the pathogenesis of atherosclerosis prompted us to investigate whether HSP90 is a target autoantigen of immune responses in patients with carotid atherosclerosis. Methods and results The presence of HSP90 on 26 cryostat and 6 paraffin embedded sections of carotid atherosclerotic plaques was determined by immunohistochemistry and immunofluorescence. Plaque-infiltrating T lymphocytes from 9 patients and circulating PBMC from 26 patients and 21 healthy subjects were tested by cell proliferation assay and by flow cytometry and ELISA for cytokine production in response to HSP90. ELISA was used to detect soluble HSP90 and anti-HSP90 antibodies in serum samples. Strong HSP90 immunoreactivity was detected in the muscle and endothelial cell layer and in the inflammatory infiltrate of carotid plaques. Plaque-derived and circulating T lymphocytes from patients proliferated in response to HSP90 whereas cells from healthy subjects did not. HSP90-specific T lymphocytes expressed IFN-γ and IL-4 suggesting concomitant Th1 and Th2 activation. ELISA detected soluble HSP90 in 42% and anti-HSP90 antibodies in 46% of patients' sera. Conclusions These new findings, showing that HSP90 is overexpressed in plaque and serum from patients with atherosclerosis and induces an immune response in these patients, implicate HSP90 as a possible target autoantigen in the pathogenesis of carotid atherosclerosis.

The PI3K-Akt cascade is a key signaling pathway involved in cell proliferation, survival, and growth. Activating PIK3CA mutations have been reported in breast carcinoma (BC). The aim of this study was to characterize the PIK3CA mutations at exons 9 and 20 in a series of 176 sporadic and 22 hereditary BCs and to correlate the results with clinicopathologic parameters and survival. In sporadic BC, 68 missense mutations were detected. PIK3CA mutations were significantly associated with ER+ in HER2-negative cases. A higher frequency of PIK3CA mutations was present in lobular carcinoma compared with ductal carcinoma (50% vs. 35%). There was no association between the survival and PIK3CA mutational status. In hereditary BC, PIK3CA mutations were found only in the BRCA2 group. The PIK3CA mutation seems to characterize the luminal-type BC, in both sporadic and BRCA2 mutated forms, and is absent in the basal-type BC, in both the sporadic and BRCA1 mutated forms.

Sclerostin, encoded by the SOST gene, is a recently identified protein which seems to affect bone remodeling by inhibiting bone formation via Wnt pathways. A previous study on OPG and RANKL, two cytokines involved in the control of osteoclastogenesis, showed that the anabolic effect produced by intermittent treatment with parathyroid hormone was characterized by an increase in OPG/RANKL mRNA ratio in the primary spongiosa of metaphyseal bone of rat femur, and by its falling in the secondary spongiosa, in comparison to controls (Silvestrini et al. (2007a)). Considering that Wnt pathway components seem to regulate osteoclast formation and bone resorption by repression of RANKL transcription and by positive regulation of OPG gene in osteoblastic cells, we have evaluated, in the same rats, whether and how SOST mRNA and protein in the primary and secondary metaphyseal bone are affected by PTH. SOST mRNA and protein significantly fell in both primary and secondary spongiosa where only a few osteocytes were positive to sclerostin. These data show that in the two metaphyseal areas no relationship does exist between the trends of OPG and RANKL mRNA and that of SOST, suggesting that there are no direct links between the effects induced by PTH on these molecules, at least in terms of gene expression.

We have studied the effects of the treatment with corticosterone (CORT), parathyroid hormone (PTH), or both (CORT + PTH), and of their withdrawal (CORT-rec and CORT + PTH-rec), on the osteoprotegerin (OPG) and receptor activator of nuclear factor-kB ligand (RANKL) localization and expression and on histomorphometric parameters in primary and secondary spongiosa of rat femur and tibia metaphyses. In the secondary spongiosa of the CORT group, the bone remodeling and the OPG/RANKL mRNA ratio decreased. In the PTH group, the bone turnover and the structural and connectivity indices increased, and the OPG/RANKL mRNA ratio fell; this ratio rose, however, in the primary spongiosa. In the CORT + PTH group, remodeling values intermediate between those of the CORT and PTH groups, were detected in the secondary spongiosa, where OPG and RANKL mRNA rose. Return towards control values was found in the recovery groups. The Cartilage Growth Plate Width was reduced in the CORT and CORT + PTH groups and returned to normal values in the recovery groups, while it was not affected by PTH. Independently of treatments, both OPG and RANKL mRNA and proteins were co-localized in the same cartilage and bone cells and in several bone marrow cells. In conclusion, the catabolic effects induced by CORT treatment occur together with an OPG fall and a RANKL rise. In the PTH group in which the bone turnover increase, the OPG and RANKL mRNA expressions differ in the primary and secondary spongiosa, confirming that the bone tissue in these sites can have different metabolic trends.