Atrial natriuretic Peptide expression in human articular cartilage.

This immunohistochemical study aims to investigate the Atrial natriuretic peptide (ANP)-presence and localization in human articular cartilage. Fragments of articular cartilage covering the femoral head were removed from patients submitted to surgical operation after femoral neck fracture without joint disease. The samples were immunostained with anti-ANP antibody. The results demonstrate that ANP is present in chondrocytes in all the three zones of the articular cartilage. Superficial chondrocytes show strong ANP-immunopositivty. The presence of ANP in the articular cartilage suggests that ANP may play a role in cartilage metabolism by regulating transport of molecules through the different zones of the articular cartilage and in maintenance of its homeostasis; probably ANP could be also involved in the regulation of the balance between synovial fluid and the other body fluids.

Atrial natriuretic peptide and vasopressin-presence in the ciliary body of eye in the pig (sus domesticus).

The aqueous humor is produced in the ciliary body, therefore in this study we investigated the Atrial natriuretic peptide (ANP) and vasopressin (VP)-presence in the ciliary body of the pig eye since these peptide are involved in the homeostasis of body fluids. The results show ANP-presence in the epithelial cells and in the endothelial cells of the blood vessels and VP-presence in the epithelial cells, in the endothelium of canal of Schelmm and in the muscle cells of the blood vessels. These peptides might regulate the synthesis and the composition of the aqueous humor and regulate the hydrodynamic flow and haemodynamic flow of the blood.

Expression of gelatinases (MMP-2, MMP-9) in human articular cartilage.

Osteoarthritis (OA) is a chronic degenerative joint disorder characterized by destruction of the articular cartilage, subchondral bone alterations and synovitis. Matrix metalloproteinases (MMPs) are expressed in joint tissues of patients with osteoarthritis (OA). The objective of this study was to define the steady state levels of two different MMPs to provide more insight into the role of MMPs in cartilage destruction in OA. We investigated the expression of gelatinases through immunohistochemistry Our results show that high levels of MMP-2 and MMP-9 are present in OA and suggest that once these MMPs are fully activated they may contribute to the cartilage destruction in OA.

Orthodontic stress Bcl-2 modulation and human odontoblast survival.

This study assessed the effect of orthodontic traction on Bcl-2 expression and apoptosis in human dental pulp. It also explored, in absence of noxious stimuli the regeneration of odontoblasts during the entire life of the tooth. Twenty young patients, with Class II malocclusion and severe to moderate crowding, were referred for orthodontic assessment. Whole pulps were removed. Half the pulps were fixed, paraffin-embedded and processed for histology and immunohistochemistry using anti Bcl-2, Caspase 9 cleaved and Caspase 9 not cleaved antibodies. The rest of the samples, both orthodontically treated and not treated dental pulps, were immediately frozen at -80ºC after the extraction and quantitative PCR was performed. Histology showed alterations in pulp microanatomy after 8 months of treatment. Immunohistochemistry depicted a decreasing expression of Bcl-2 in dental pulp over time in the non-treated while a very weak to absent Bcl-2 expression was detected in the orthodontically treated tissues. Active and non-active forms of Caspases, were expressed in both groups of dental pulp, however staining for the non active form was stronger than the corresponding cleaved form in all samples. The increased expression was detected mainly at nuclear level. Real time qPCR results correlated with those of immunohistochemistry and exhibited a decreasing expression of Bcl-2 in the treated samples. Orthodontic traction may inhibit the expression of Bcl-2, favoring the onset of apoptosis and leading us to conclude that the physical stress in the absence of noxious stimuli might make odontoblasts regeneration less likely.

Expression of gelatinases (MMP-2, MMP-9) and cyclooxygenases (COX-1, COX-2) in some benign salivary gland tumors.

Salivary gland tumors, most of which are rare benign tumors, represent a histologically heterogenous group with the greatest diversity of morphological and cellular features. The aim of this study is to analyse the expression and possible interactions between gelatinases (MMP-2, MMP-9) and cyclooxygenases (COX-1, COX-2) in some benign salivary gland tumors. We investigated the expression of gelatinases and cyclooxigenases in control salivary gland, Pleomorphic adenoma and Warthin's tumor through immunohistochemistry and Reverse Transcription - Polymerase Chain Reaction (PCR). We identified the expression of both classes of enzyme in normal samples and in the two types of pathological samples without any quantitative differences. From the present data no significant differences emerge in the expression of these enzymes among the different pathologies examined. Nevertheless, due to the small number of samples included in this study, general statements regarding correlation between the degree of severity of the tumoral pathology and the quantitative expression of these potential tumoral markers can not be made.

Immunohistochemical and transcriptional expression of the matrix metalloproteinases MMP-2 and MMP-9 in normal and pathological human oral mucosa.

The oral cavity is exposed to chronic or recurrent, physical and chemical trauma that could lead to mucosal reactions (e.g. hyperplasia, dysplasia and tumors). The objective of this study is to investigate the expression and the possible changes of the two matrix metalloproteinases MMP-2 and MMP-9 in normal and pathological human oral mucosa samples. Normal oral mucosa samples and three different types of pathological conditions (hyperplasia, dysplasia and carcinoma) were used for this study. Immunohistochemical analysis was used to evaluate protein expression for the two enzymes, while Reverse Transcription ? Polymerase Chain Reaction (RT-PCR) was used to investigate gene expression. Image analysis was used to give a quantitative evaluation of the immunohistochemical data. In control samples we identified a weak expression of both MMP-2 and MMP-9 in the epithelial layers. In hyperplasia samples MMPs expression is limited to epithelial layers but the immunoreactivity is more intense than in the control. In dysplasia and carcinoma samples the two matrix metalloproteases are expressed not only in epithelium but also in some cells of the connective tissue and in the vessel walls. Qualitative RT-PCR and image analysis confirmed the immunohistochemical data. The results obtained in this study suggest the existence of a possible relationship between the entity of morphological disorganization of the oral mucosa in different pathologies and the increase of MMP-2 and MMP-9 expression.

Sphingomyelin inhibition of Ciona intestinalis (Tunicata) cytotoxic hemocytes assayed against sheep erythrocytes.

Hemocytes from the ascidian, Ciona intestinalis, are capable of lysing erythrocytes in vitro following cell membrane contact. With the aim of examining the mechanism of cytotoxicity, we performed inhibition experiments with lipid components of erythrocyte membranes. Cholesterol is not an inhibitor, whereas, among the phospholipids tested, (sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine) sphingomyelin inhibits the hemolytic activity of hemocytes. However, thin layer chromatography showed that sphingomyelinase activity was not contained in the chloroform-methanol extracts from hemocyte debris. The inhibition capacity of the components ceramide and phosphorylcholine suggests that the entire sphingomyelin molecule is involved in binding cytolysins. The lysin-lipid interactions probably cause changes in erythrocyte membrane permeability, leading to lysis.

The level of soluble Granzyme A is elevated in the plasma and in the Vgamma9/Vdelta2 T cell culture supernatants of patients with active Behçet's disease.

OBJECTIVE: Gramzyme A (GrA) is a serine proteinase with trypsin-like activity that is released extracellularly during the degranulation of cytotoxic cells. Among the cytotoxic cells, gamma/delta T cells participate in the early phases of the immune response and are known to express perforin and granzymes constitutively in agreement with their cytolytic pontential. METHODS: GrA activity was detected using the synthetic substrate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester in the plasma and supernatants of peripheral blood mononuclear cell cultured in the presence of Dimethylallyl pyrophosphate to obtain Vgamma9/Vdelta2 T cell expansion. RESULTS: Significantly high levels of GrA were found in the serum and supernatants of lymphocytes from patients with active Behçet's disease cultured in the presence of DMAPP. Levels were found to be significantly lower after remission. A positive correlation was observed between GrA levels in the supernatants and the Vgamma9/Vdelta2 T cell expansion factor. CONCLUSION: These results strongly suggest that Vgamma9/Vdelta2 T cells are active participants in the pathogenesis of the disease through their degranulation and granzyme release.

Presence of oxytocin, vasopressin and atrial natriuretic peptide and their modification in rat hypothalamic paraventricular nucleus during resistance training.

Many studies have demonstrated the physiological effects of oxytocin (OT), atrial natriuretic peptide (ANP) and vasopressin (VP) in the homoeostasis of body fluids during physical exercise. However, a little information is available about the related immunohistochemical changes in hypothalamic magnocellular neurosecretory system during and after the training. The aim of the present work was to study the immunohistochemical changes in OT, ANP and VP levels in the hypothalamic paraventricular nucleus during and after resistance exercise protocol. Three groups of Wistar rats were trained by a rung ladder protocol for 15, 30 and 45 days, respectively; a fourth group was left to rest for 15 days after the training. Finally, four sedentary groups were used as controls. The results show that resistance training induces a significant reduction in the percentage of OT-positive neurons, compared with sedentary controls. In contrast, this protocol did not induce any change in VP levels, and ANP levels did not change significantly. However, VP increased after the resting period of 15 days. Our work shows that neurons of the paraventricular nucleus are involved in body fluid homoeostasis during and after resistance exercise. The functional significance of these changes in OT and VP levels, during and after the protocol, needs to be further investigated.

Salmonella infection in illegally imported spur-thighed tortoises (Testudo graeca).

The prevalence of Salmonella infection was determined in a group of spur-thighed tortoises (Testudo graeca) seized during two smuggling attempts and in a population of captive Hermann's tortoises (Testudo hermanni) sheltered in a wildlife rescue centre. Salmonella spp. was isolated in 81 of 220 (36.8%) and in 17 of 67 (25.4%) cloacal swabs collected from the T. graeca and T. hermanni tortoises respectively. Overall, a total of 21 different Salmonella serotypes were found. Some of these serotypes are common to terrestrial chelonians while others have never been reported. All cultured serotypes were non-typhoidal but nonetheless many of these have been previously reported as source of human outbreaks of reptile-related salmonellosis. Eighty-two per cent and 5.3% of the isolates were resistant to two and three anti-microbial agents respectively. However, the isolates were highly susceptible to the anti-microbials of choice for the treatment of salmonellosis such as cephalosporins and fluoroquinolones. Our findings confirm that tortoises can be considered a reservoir for Salmonella and that care should be employed when handling and breeding these animals. Tight surveillance should be enforced to avoid illegal importation and prevent the trading of live tortoises, carriers of zoonotic pathogens.