Targeting nicotinamide N-methyltransferase decreased aggressiveness of osteosarcoma cells.

BACKGROUND: Osteosarcoma (OS) is a primary bone malignancy that mostly affects young people, characterized by high metastatic potential, and a marked chemoresistance that is responsible for disease relapse in most patients. Therefore, it is necessary to identify novel molecules to setup targeted strategies to improve the clinical outcome. The enzyme nicotinamide N-methyltransferase (NNMT) catalyses the N-methylation of nicotinamide and other analogs, playing a crucial role in the biotransformation of drugs and xenobiotics. NNMT overexpression was reported in a wide variety of cancers, and several studies demonstrated that is able to promote cell proliferation, migration and resistance to chemotherapy. The aim of this study was to explore the potential involvement of NNMT in OS. METHODS: Immunohistochemical analyses have been performed to evaluate NNMT expression in selected OS and healthy bone tissue samples. Subsequently, OS cell lines have been transfected with vectors targeting NNMT mRNA (shRNAs) and the impact of this downregulation on migration, cell proliferation, and response to chemotherapeutic treatment was also analysed by wound healing, MTT, SRB and Trypan blue assays, respectively. RESULTS: Results showed that OS samples display a significantly higher NNMT expression compared with healthy tissue. Preliminary results suggest that NNMT silencing in OS cell lines is associated to a decrease of cell proliferation and migration, as well as to enhanced sensitivity to chemotherapy. Data obtained showed that NNMT may represent an interesting marker for OS detection and a promising target for effective anti-cancer therapy.

Alzheimer's Disease Risk and Progression: The Role of Nutritional Supplements and their Effect on Drug Therapy Outcome.

Alzheimer's disease (AD) is the most common neurodegenerative disease in the elderly population. Despite significant advancements in understanding the genetic and molecular basis of AD, the pathology still lacks treatments that can slow down or reverse the progression of cognitive deterioration. Recently, the relationship between nutrient deficiency and dementia onset has been highlighted. AD is in fact a multifactorial pathology, so that a multi-target approach using combinations of micronutrients and drugs could have beneficial effects on cognitive function in neurodegenerative brain disorders leading to synaptic degeneration. Primarily, this review examines the most recent literature regarding the effects of nutrition on the risk/progression of the disease, focusing attention mostly on antioxidants agents, polyunsaturated fatty acids and metals. Secondly, it aims to figure out if nutritional supplements might have beneficial effects on drug therapy outcome. Even if nutritional supplements showed contrasting evidence of a likely effect of decreasing the risk of AD onset that could be studied more deeply in other clinical trials, no convincing data are present about their usefulness in combination with drug therapies and their effectiveness in slowing down the disease progression.

In vitro effects of fermented papaya (Carica papaya, L.) on platelets obtained from patients with type 2 diabetes.

BACKGROUND AND AIM: Oxidative stress is associated with insulin resistance pathogenesis, insulin secretion deficiency, and complication onset. Fermented papaya preparation (FPP), a dietary supplement obtained by fermentation of the papaya fruit, may be used as an antioxidant in the prevention of diabetic complications. METHODS AND RESULTS: Platelets from 30 patients with type 2 diabetes mellitus (DM 2) and 15 healthy subjects were analyzed to evaluate the in vitro effects of FPP incubation. Na(+)/K(+)-adenosine triphosphatase (ATPase) activity, membrane fluidity, total antioxidant capacity (TAC), superoxide dismutase (SOD) activity, and conjugated diene levels were determined. In vitro FPP incubation improved platelet function, by enhancing Na(+)/K(+)-ATPase activity and membrane fluidity, and ameliorated the antioxidant system functionality, through an increase in TAC and SOD activity and a parallel decrease in conjugated diene levels in patients with DM 2. CONCLUSION: Our data suggest that the incubation with FPP may have a protective effect on platelets from patients with DM 2, by preventing the progression of oxidative damage associated with diabetes and its complications.

IL-1ß and TGF-ß weaken the placental barrier through destruction of tight junctions: an in vivo and in vitro study.

INTRODUCTION: Chorioamnionitis is a gestational pathological condition characterized by acute inflammation of the amniochorionic membranes and placentas leading to high concentrations of IL-1ß, Il-6, Il-8 and TGF-ß in the amniotic fluid. In normal conditions, the permeability of foeto-maternal barrier is due to the assembly and maintenance of different cellular junctional domains. METHODS: In the present study, first we aimed to evaluate the protein expression (by immunohistochemistry and western blotting) and mRNA (by real time PCR) levels of the molecular components of tight junctions (Zonula occludens-1 and occludin), and of adherent junctions (VE-cadherin and ß-catenin) in placentas from chorioamnionitis compared to that in normal pregnancies. RESULTS: Western blotting results showed a significant down-regulation of occludin in placentas affected with chorioamnionitis. No differences were detected for the other proteins analysed. We evaluated whether occludin expression was regulated by IL-1ß, IL-6, IL-8 and TGF-ß by means of in vitro studies using HUVEC cultures and demonstrated a key role of IL-1ß and TGF-ß in the disappearance of occludin at cellular border. CONCLUSIONS: We conclude by suggesting a pivotal role of these two cytokines in facilitating intra-placental infection via para-cellular way due to the disassembly of tight junctions at trophoblastic and endothelial cells in placental tissues.

In vitro evaluation of mesenchymal stem cell isolation possibility from different intra-oral tissues.

Mesenchymal stem cells (MSCs) are of great interest for the regeneration of tissues and organs. Bone marrow is the first sources of MSCs, but in the recent years there has been interest in other tissues for the isolation of these pluripotent cells. In this study, we investigated the features of MSCs isolated from different oral regions in order to evaluate their potential application in the regeneration of damaged maxillofacial tissues. Sampling from human periodontal ligament, dental pulp, maxillary periosteum as well as bone marrow were collected in order to obtain different stem cell populations. Cells were morphologically and immunophenotipically characterized. Their proliferation potential and their ability to differentiate in osteoblasts were also assessed. All tested cell population showed a similar fibroblast-like morphology and superimposable immunophenotype. Slight differences were observed in proliferation and differentiation potential. Cells isolated from human periodontal ligament, dental pulp, maxillary periosteum had the characteristics of stem cells. Considering their peculiar feature they may alternatively represent interesting cell sources in stem cell-based bone/periodontal tissue regeneration approaches.

The mesenchymal stem cell profile in psoriasis.

BACKGROUND: The expression of inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) and the level of total oxyradical scavenging capacity have been evaluated extensively in the cutaneous cells of patients with psoriasis. As yet, no indications are available about the undifferentiated cells, the mesenchymal stem cells (MSCs), isolated from skin. OBJECTIVES: To isolate MSCs in patients with psoriasis and to compare them with those obtained from atopic and healthy subjects, in order to analyse whether MSCs show some typical psoriatic profiles and to understand whether pathophysiological events leading to psoriasis start early at the stem cell level. METHODS: MSCs isolated from seven patients with psoriasis, seven patients with acute atopic dermatitis and seven healthy subjects were characterized by fluorescence-activated cell sorting analysis. VEGF and nitric oxide (NO) content was measured in conditioned medium, the expression of VEGF and iNOS was analysed by immunohistochemistry, and the total oxyradical scavenging capacity towards peroxynitrite was tested. RESULTS: VEGF content was highest in the medium conditioned by psoriatic perilesional MSCs, whereas NO concentration was maximally increased in medium conditioned by MSCs isolated from lesional psoriatic skin. The ability to neutralize the oxidizing effects of peroxynitrite was lower for MSCs isolated from lesional psoriatic skin compared with other MSCs, except for MSCs of lesional atopic skin. CONCLUSIONS: The microenvironment in psoriasis differs from those of atopic dermatitis and healthy skin; it could induce resident MSCs to produce angiogenic and proinflammatory mediators which lead to a reduction in the antioxidant capacity of these cells, contributing to the development of skin lesions in psoriasis.

Expression of CD38 in human neuroblastoma SH-SY5Y cells.

Human CD38 antigen is a 42-45 kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long C-terminal extracellular region. It is widely expressed in different cell types including thymocytes, activated T cells, and terminally differentiated B cells (plasma cells) and it is involved in cellular proliferation and adhesion. CD38 acts as an ectocyclase that converts NAD+ to the Ca2+ -releasing second messenger cyclic ADP-ribose (cADPR). It has been also demonstrated that increased extracellular levels of NAD+ and cADPR are involved in inflammatory diseases and in cellular damage, such as ischemia. In the present study, we have characterized the expression of CD38 in human neuroblastoma SH-SY5Y cell line. All-trans-retinoic acid (ATRA) treatment was used to induce cell differentiation. Our results indicate that: a) even if SH-SY5Y cells have a negative phenotype express CD38 at nuclear level, ATRA treatment does not influence this pattern; b) CD38 localizing to the nucleus may co-localize with p80-coilin positive nuclear-coiled bodies; c) purified nuclei, by Western blot determinations using anti-CD38 antibodies, display a band with a molecular mass of approximately 42 kDa; d) SH-SY5Y cells show nuclear ADP-ribosyl cyclase due to CD38 activity; e) the basal level of CD38 mRNA shows a time-dependent increase after treatment with ATRA. These results suggest that the presence of constitutive fully functional CD38 in the SH-SY5Y nucleus has some important implications for intracellular generation of cADP-ribose and subsequent nucleoplasmic calcium release.

Intravitreal micronized triamcinolone versus triamcinolone acetonide: a clinical and morphological comparative study.

Nowadays many authors suggest the use of intravitreal triamcinolone acetonide (TA) for the treatment of vitreoretinal diseases, although it can be associated with a high risk of local toxicity. In order to develop a safer injection for clinical use, the purpose of our study is to evaluate the in situ safety of two different triamcinolone preparations, a commercially available TA and a micronized triamcinolone. The experiments were performed on 18 adult male age-matched New Zealand rabbits. The clinical examination included funduscopy with an indirect ophthalmoscope and intraocular pressure (IOP) measurement. At the end of the clinical observations, the animals were sacrificed and the eyes enucleated and processed for the morphological evaluation. In our study the main side effect observed was the IOP elevation in the group injected with triamcinolone acetonide. In addition, in the TA-injected group, one eye was enucleated following an endophthalmitis. Our study highlights that doses as low as 4 mg of triamcinolone acetonide injected into the rabbit vitreous may have a local toxic effect in terms of IOP elevation, endophthalmitis occurrence and changes in the retinal morphology. In contrast, the micronized triamcinolone injection shows a less toxic effect in situ, thus suggesting the alternative use of this more reliable preparation which seems to be safer for a clinical use.

Changes of plasma membrane properties in a human pre-T cell line undergoing apoptosis.

A variety of cellular functions are modulated by the physical properties of the cell membrane, and the modification of intracellular transfer, resulting from loss of membrane integrity, may contribute toward setting the cell onto the pathway of apoptosis. Apoptosis in lymphoid cells can be induced in different ways and biochemical modifications occur at an early phase of cell death, while the morphological features of apoptosis are evident later. We previously reported that DMSO is an efficient apoptosis-inducing factor in the human RPMI-8402 pre-T cell line. The aim of the present study was to verify the effect of DMSO on the plasma membrane fluidity, the intracellular calcium concentration and the phosphodiesterase activity in DMSO-induced apoptosis. Our results show a modification of membrane fluidity associated with an increase of intracellular Ca(2+) concentration. Moreover, we demonstrate that these modifications are related to a decrease in the phosphodiesterase (PDE) activity. The correlation between the proceedings of added DMSO and the induction of apoptosis will provide significant information regarding the first part of the apoptotic process.

Sphingolipid microdomains mediate CD38 internalization: topography of the endocytosis.

Plasma membranes of several cell types contain specialized microdomains (or lipid rafts) enriched in sphingolipids, cholesterol, sphingomyelin, and glycosyl-phosphatidylinositol-anchored proteins. These membrane domains are characterized by detergent insolubility at low temperatures and low buoyant density. Human CD38 is the prototype of a gene family encoding surface molecules endowed with multiple functional activities. The endocytosis of the human CD38 molecule has been investigated in normal lymphocytes and in a number of leukemia- and lymphoma-derived cell lines demonstrating that internalization after CD38 ligation is a reproducible event involving only a fraction of the whole amount of the surface molecule. This study reports the results obtained by conventional, confocal, and electron microscopy on the effects induced by the engagement of the molecule with agonistic mAb, reproducing the signals mediated by its natural ligand. The results demonstrate that the endocytosis induced as consequence of CD38 ligation is preceded by a thorough rearrangement of the cell surface with formation of glycosphingolipid- and cholesterol-rich plasma membrane microdomains. These data suggest that specialized raft microdomains might be the plasma membrane structure through which CD38 translocates at intracellular level. The CD38/lipid interactions during the coated pit formation trigger a process that generate membrane curvature, considered as the first step of CD38 endocytosis. Moreover, ultrastructural studies show that early CD38(+) endosomes are pleiomorphic and contain cisternal and vesicular regions. Late endosomes exhibit a complex organisation, containing uncoupled CD38-ligand multivesicular- or multilamellar-regions.

DMSO modifies structural and functional properties of RPMI-8402 cells by promoting programmed cell death.

Apoptosis in lymphoid cells can be induced in different ways depending on cell type and acquired signal. Biochemical modifications occur at an early phase of cell death while at late times the typical morphological features of apoptosis can be visualized. The aim of this study is to verify by multiparametric analyses the plasma membrane fluidity, the intracellular Ca2+ concentration and the nitric oxide synthase (NOS) activity during cell death progression induced by DMSO treatment. The RPMI-8402 human pre-T lymphoblastoid cell line was induced to cell death by DMSO. Analyses rescued at early times of treatment prove a substantial modification of plasma membrane fluidity associated with an increase of intracellular Ca2+. Moreover, these modifications are associated with an up regulation of NOS activity. Our results are consistent with the hypothesis that programmed cell death can be induced by up regulation of the intracellular Ca2+ associated with an increase of cell membrane fluidity. The apoptotic mechanisms seem to involve not only membrane damage and increased intracellular calcium levels but also production of nitric oxide.

Magnetic Resonance applications in dental implantology: technical notes and preliminary results.

PURPOSE: To evaluate the possible use of Magnetic Resonance Imaging (MRI) in the field of dental implantology, for identifying the mandibular nerve, as proposed by several authors. MATERIALS AND METHODS: MRI was used for the study of the mandible in ten subjects (five healthy volunteers and five subjects awaiting dental implants). Imaging was performed on a 1.0-T MR scanner with a brain coil. T2 TSE, T1 spin-echo and T2 gradient-echo sequences were performed, both parallel and perpendicular to the horizontal portion of the mandible, with a thickness of 3 mm. RESULTS: In all the subjects MRI clearly identified the intraosseous course of the inferior alveolar neurovascular bundle within the mandibular canal. CONCLUSIONS: MRI appears useful for the depiction of the mandibular canal before dental implantation. Further studies are required to compare the accuracy of MRI and CT based on a statistically significant sample.

Glycated low density lipoproteins modify platelet properties: a compositional and functional study.

The interaction between low density lipoproteins (LDL) and platelets might play a central role in the development of atherosclerosis in diabetes. The aim of the present study was to investigate whether the glycation of LDL is associated with modifications of their physico-chemical and functional properties and to study the action of glycated LDL (glycLDL) on platelets. LDL and platelets were isolated from 15 healthy subjects. The content of thiobarbituric acid-reactive substances and the generalized polarization of the fluorescent probe Laurdan were determined in LDL glycated in vitro. Platelets were incubated with native LDL, GlycLDL, and minimally oxidized LDL, and the following parameters were evaluated: platelet aggregation, nitric oxide production, intracellular Ca(2+) concentrations, Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase), and Ca(2+)-ATPase activities. GlycLDL showed increased thiobarbituric acid-reactive substance levels, a red shift of the Laurdan emission maximum, and a decrease in generalized polarization, indicating a higher polarity and a reduced molecular order compared with native LDL. GlycLDL caused a significant increase in platelet nitric oxide production, intracellular Ca(2+) concentration, and aggregating response to ADP; an inhibition of the platelet membrane Na(+)/K(+)-ATPase activity; and a stimulation of Ca(2+)-ATPase activity. Minimally oxidized LDL did not cause statistically significant changes in the parameters studied. The present work demonstrates that glycation induces compositional and structural changes in LDL and suggests that an altered interaction between glycLDL and platelets might play a role in the vascular complications of diabetes.

Reduced susceptibility to peroxidation of erythrocyte plasma membranes from centenarians.

The plasma membrane composition affects intracellular processes and the cellular susceptibility to free radical attack, which has been associated with the impairment of cellular functions occurring during senescence. The study of the modifications of the plasma membrane in centenarians might elucidate the biological mechanisms at the basis of longevity and successful aging. The work was performed in 190 subjects, divided into five groups according to the age range: (1) 21-40 years (n=25); (2) 41-60 years (n=30); (3) 61-80 years (n=30); (4) 81-99 years (n=50); and (5) centenarians (> or = 100 years) (n=55). The following determinations were performed on erythrocyte membranes: (i) the lipid peroxide level (Lp) evaluated as malondialdehyde content; (ii) susceptibility to in vitro oxidation evaluated as difference in the content of thiobarbituric acid-reactive substances before and after phenylhydrazine addition; (iii) unsaturated/saturated fatty acid ratio and individual polyunsaturated fatty acid composition measured by gas chromatography; and (iv) fluidity studied by means of the anisotropy of the probe 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Erythrocyte membranes from centenarians showed: (i) decreased basal lipid peroxide levels and reduced susceptibility to peroxidation in comparison with elderly subjects; (ii) increased unsaturated/saturated fatty acid ratio in comparison with every other age group; (iii) higher levels of eicosapentaenoic and docosahexaenoic acid and reduced content of linoleic and arachidonic acid in comparison with elderly subjects; and (iv) decreased anisotropy of TMA-DPH, i.e. higher fluidity compared with all the other age groups. In conclusion, the present work demonstrates that erythrocyte membranes from centenarians show some distinct features in comparison with elderly subjects that might act in a protective way against injuries.

Age-related modifications in human unstimulated whole saliva: a biochemical study.

Human whole saliva contains a number of antimicrobial agents, and lysozyme, lactoferrin, secretory IgA and peroxidase are among the best known. Peroxidase catalyzes a reaction involved in the inhibition of bacterial growth and metabolism, and the prevention of hydrogen peroxide accumulation, thus protecting proteins from the action of oxygen and reactive oxygen species (ROS). To better understand the role played by the oxidative stress in the aging process, we studied the relationship between total protein content, peroxidase activity, malondialdehyde (MDA) and nitric oxide (NO) content of human unstimulated whole saliva in 169 healthy subjects subdivided into groups according to age. Our results show a significant decrease in peroxidase activity with age. Moreover, the increase in saliva lipid peroxide levels indicates an enhanced free radical production that may contribute to tissue damage. On the other hand, findings concerning human unstimulated whole saliva NO content showed a significant increase in elderly subjects, suggesting that an enhanced NO production might depend on a stimulation of leukocyte-inducible NO synthase (i-NOS) activity. Our results suggest that during aging the oral tissues may become more susceptible to environmental factors due to a modification in the balance between different antimicrobial agents.

Modifications induced by LDL from type 1 diabetic patients on endothelial cells obtained from human umbilical vein.

The aim of the present work was to analyze the effect of LDL obtained from type 1 diabetic patients in good metabolic control on human umbilical vein endothelial cells (HUVECs) after a short incubation period to detect possible atherogenic modifications of endothelial properties. Cultured HUVECs were incubated for 3 h with culture medium alone (control HUVEC), with native LDL from 12 healthy men (control LDL), or with native LDL from 12 type 1 diabetic men (type 1 LDL) (100 pg/ml). After the incubation, the following parameters were evaluated: nitric oxide synthase (NOS) activity, cytoplasmic Ca2+ levels, Na+-K+-ATPase activity, plasma membrane fluidity determined by means of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), and plasma membrane conjugated diene (CD) content. The same experiments were repeated after bradykinin stimulation or in the presence of the antioxidant butylated hydroxytoluene (BHT), and nitric oxide (NO) production in intact HUVECs was also evaluated. HUVECs incubated with control LDL in comparison with control HUVECs showed a decreased fluidity of the membrane surface evaluated by TMA-DPH and a higher CD content. These alterations were prevented by the presence of BHT. HUVECs incubated with type 1 LDL in comparison with both control HUVECs and cells incubated with control LDL showed 1) increased NOS and Na+-K+-ATPase activity, cytoplasmic Ca2+ levels, and CD content, and 2) decreased fluidity of the membrane surface evaluated by TMA-DPH. These modifications were blunted--but not abolished--by the presence of BHT. After bradykinin stimulation either in the absence or in the presence of BHT, both cytoplasmic Ca2+ levels and NO production were increased in control HUVECs and in HUVECs incubated with control LDL, while a reduced response was observed in HUVECs incubated with type 1 LDL. The alterations observed in the endothelial function after the cell-LDL interaction might play a central role in the atherogenic process in diabetes.

Changes in the composition of human unstimulated whole saliva with age.

The aim of this study was to evaluate changes in the concentration of certain components of human unstimulated whole saliva during aging, in order to better understand the role played by aging in oral health. In particular, we studied total protein concentration, alpha-amylase activity, sialic acid content and calcium and phosphorus concentrations in 100 healthy subjects of both genders, aged between 10 and 80 years, who were subdivided into four groups according to their age: 10-25 years, 26-40 years, 41-65 years, and 66-80 years. Other than sialic acid, the concentrations of the components studied were not affected by age. There was a significant negative correlation between sialic acid content and age. Our data indicate the presence of a decreased submandibular/sublingual function with aging, thus suggesting the possibility of a concomitant reduction in the modulating action of unstimulated whole saliva on the oral flora.

[Dentinogenesis imperfecta. Scanning electron microscopic study and microanalysis]. / Dentinogenesi imperfetta. Studio al microscopio elettronico a scansione e microanalisi.

BACKGROUND: Dentinogenesis imperfecta (DI) is an inherited dentine defect which affects both the primary and secondary dentitions. Shields et al. in 1973 suggested a classification of DI within three types: type I, associated with osteogenesis imperfecta (OI), type II and type III. Although the varying clinical, radiographic and histological findings in DI have been described in detail, an available method for closer examination of the abnormal dentine matrix, electron microscopy, has seldom been used. Scanning and transmission electron microscopy studies can help to understand the pathogenesis of the different types of heritable dentine defects and the diagnosis and classification of these diseases. The aim of the present study was to evaluate a case of DI using scanning electron microscopy and microanalysis. METHODS: Dentine was obtained from tooth samples from a fourteen-year-old boy affected by DI and from third molars extracted for therapeutic reasons used as controls. Samples were observed with a scanning electron microscope, scanning electron micrographs were evaluated with an image analysis program and specimens were finally observed with a scanning electron microscope equipped for micro-analysis. RESULTS AND CONCLUSIONS: The results obtained showed that the total number of dentinal tubules was significantly reduced and the presence of a dentine mineralization defect in the patient affected by DI, in comparison to the controls.