NADPH oxidase activation and 4-hydroxy-2-nonenal/aquaporin-4 adducts as possible new players in oxidative neuronal damage presents in drug-resistant epilepsy.

A correlation between epilepsy and cellular redox imbalance has been suggested, although the mechanism by which oxidative stress (OS) can be implicated in this disorder is not clear. In the present study several oxidative stress markers and enzymes involved in OS have been determined. In particular, we examined the levels of 4-hydroxy-2-nonenal protein adducts (HNE-PA), a by-product of lipid peroxidation, and the activation of NADPH oxidase 2 (NOX2), as cellular source of superoxide (O(2)(-)), in surgically resected epileptic tissue from drug-resistant patients (N=50). In addition, we investigated whether oxidative-mediated protein damage can affect aquaporin-4 (AQP4), a water channel implicated in brain excitability and epilepsy. Results showed high levels of HNE-PA in epileptic hippocampus, in both neurons and glial cells and cytoplasmic positivity for p47(phox) and p67(phox) suggesting NOX2 activation. Interestingly, in epileptic tissue immunohistochemical localization of AQP4 was identified not only in perivascular astrocytic endfeet, but also in neurons. Nevertheless, negativity for AQP4 was observed in neurons in degeneration. Of note, HNE-mediated post-translational modifications of AQP4 were increased in epileptic tissues and double immunofluorescence clearly demonstrated co-localization of AQP4 and HNE-PA in epileptic hippocampal structures. The idea is that sudden, disorderly, and excessive neuronal discharges activates NOX2 with O(2)(-) production, leading to lipid peroxidation. The resulting generation of HNE targets AQP4, affecting water and ion balance. Therefore, we suggest that seizure induces oxidative damage as well as neuronal loss, thereby promoting neuronal hyperexcitability, also affecting water and ion balance by AQP4 modulation, and thus generating a vicious cycle.

Betamethasone, progesterone and RU-486 (mifepristone) exert similar effects on connexin expression in trophoblast-derived HTR-8/SVneo cells.

Connexins (Cx) are membrane proteins able to influence cell trophoblast responses, such as proliferation, differentiation, migration and invasiveness. Likewise, glucocorticoids are also known to modulate many factors involved in implantation, including trophoblast gap-junction intercellular communication, although their influence on pregnancy is controversial. In order to investigate the effects of betamethasone, a synthetic glucocorticoid, on Cx and glucocorticoid receptor (GR) expression and localisation, as well as on cell proliferation, the extravillous trophoblast-derived HTR-8/SVneo cell line was used as a model. The results, confirmed by means of immunofluorescence, demonstrate that betamethasone selectively modifies GR and Cx expression, enhancing the GRα isoform without affecting GRß, and inhibiting Cx40 expression whilst increasing that of Cx43 and Cx45. Furthermore, betamethasone was shown to exert an inhibitory action on cell proliferation. In this model the abortion drug RU-486 (mifepristone), reported to be a GR antagonist, did not counteract this effect of betamethasone. On the contrary, it induced responses similar to those of the hormone. Knowing that RU-486 is also a potent progesterone-receptor antagonist, the effect of progesterone alone and in combination with the drug on Cx expression and cell proliferation was then tested. Progesterone showed the same effect as betamethasone on Cx expression, but it did not affect proliferation. Based on these results, neither the abortion effects of RU-486 nor the protective action of betamethasone and progesterone are exerted by modulation of Cx. RU-486 did not antagonise the progesterone effect, suggesting that its abortive action does not involve alteration of trophoblast Cx expression.

cAMP efflux from human trophoblast cell lines: a role for multidrug resistance protein (MRP)1 transporter.

Cyclic adenosine 3'-5'-monophosphate (cAMP) is a second messenger, which exerts an important role in the control of human first-trimester trophoblast functions. In the present study we demonstrate the existence of a mechanism that is able to extrude cAMP from trophoblast-derived cell lines, and show evidence indicating the involvement of multidrug resistance protein (MRP) 1, a transporter belonging to the ATP-binding cassette family, in cAMP egress. MRP1 is expressed in trophoblast cell lines and cAMP efflux is highly reduced by the MRP1 inhibitor, MK-571. In addition, interleukin-1beta and estrone are able to enhance MRP1 gene expression and influence extracellular cAMP concentration. The occurrence of a MRP1-dependent cAMP efflux is also shown in human first-trimester placenta explants. Extracellular cAMP could represent a source for adenosine formation, which in turn could regulate cAMP-dependent responses in placental tissue. Evidence is provided that adenosine receptor subtypes are present and functional in human trophoblast-derived cells. A role for cAMP egress mechanism in the fine modulation of the nucleotide homeostasis is therefore suggested.

Somatostatin as a regulator of first-trimester human trophoblast functions.

We have tested the hypothesis that human early trophoblast is a target for somatostatin (SRIF) regulatory actions. We report for the first time that SSTR2A and 2B transcripts and proteins are present in first-trimester human chorionic villi and the trophoblast-derived HTR-8/SVneo and JAR cells. In both cell lines, SSTR are functional since SRIF inhibits cyclic AMP pathway, stimulates arachidonic acid release and enhances cell proliferation. Moreover, in HTR-8/SVneo cells, considered a good model of first-trimester EVT, SRIF also enhances migration. An involvement of the cyclic AMP pathway in mediating SRIF effects on proliferation and migration is suggested. Our data support the idea that SRIF regulates early trophoblast functions mainly through an interaction with SSTR2.

The evolution of the atheromatous plaque: ultrastructural evidence.

Our understanding of endothelial physiology is overdue compared with other areas of study. For too many years the complex mysteries of this thin single-layered cellular lamina covering the whole of the vascular network, from the large conduction vessel to the smallest resistance and diffusion vessel, have been hidden by an organ-based science more focused on organ pathology than on ultrastructural physiopathology. We tried to follow chronologically the alteration stages of this system of membranes in relation to the development of the atherosclerotic plaque in human biopsy.

Effect of nitric oxide on arachidonic acid release from human amnion-like WISH cells.

In order to clarify the possible interactions between nitric oxide (NO) and arachidonic acid (AA) pathways, human amnion-like WISH cells were perifused to measure the effects of the following substances on [(3)H]arachidonic acid release: (1) sodium nitroprusside (SNP), a nitric oxide donor; (2) 1,1,1-trifluoromethyl-6,9,12,15-heicosatetraen-2-one, a cytosolic phospholipase A(2) (cPLA(2)) inhibitor; (3)L -arginine, the substrate of nitric oxide synthase (NOS); (4) 3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole and 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, activator and inhibitor of soluble guanylyl cyclase, respectively; (5) a membrane-permeable non-hydrolyzable analogue of guanosine-3',5'-cyclic monophosphate (cGMP). Furthermore, the effect of SNP on prostaglandin E(2) (PGE(2)) release was tested. Exogenous and endogenous NO, as well as the guanylyl cyclase activator and cGMP analogue, significantly increased [(3)H]arachidonic acid release. Both soluble guanylyl cyclase and PLA(2) inhibitors counteracted SNP response. Exogenous NO increased PGE(2) release, although to a much lesser degree compared with arachidonic acid release. Our results indicate that NO stimulates AA release in WISH cells by activating PLA(2) through a cyclic GMP-dependent mechanism.

Sulmazole effects on PGE2 and D-Ala2-Met-enkephalinamide modulation of cyclic AMP synthesis and neurotransmitter release in a sympathetic ganglion.

In the guinea-pig superior cervical ganglion, the Gi blocking agent sulmazole enhanced the basal and prostaglandin E2-induced stimulation of cyclic AMP synthesis but had no effect on the prostaglandin-dependent inhibition of acetylcholine release. On the contrary sulmazole counteracted the inhibitory effect of D-Ala2-Met-enkephalinamide both on cyclic AMP formation and acetylcholine release. Moreover sulmazole eliminated the supra-additive effect of the combination of prostaglandin + opiate on cyclic AMP synthesis. The presence of a Gi-coupled opiate receptor at the pre-and postsynaptic levels is suggested.

Liposomes containing distamycins: preparation, characterization and antiproliferative activity.

This article describes the production and characterization of two liposome formulations containing antitumor drugs, namely distamycin A (Dist) and a new alkyl derivative of distamycin A (C16-Dist). Egg-PC/cholesterol liposomes (4:1 mol/mol) were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. The encapsulation efficiency was found to be almost complete for C16-Dist (99.8%), while native distamycin A showed a lower yield (19.0%). The in vitro antiproliferative activity of the distamycins-containing liposomes determined on human leukaemic K562 cells, was 11-fold and 8-fold higher for native and alkyl derivative distamycin A, respectively, compared with that of the corresponding free drugs. Liposomal formulations show an increase in the activity and specificity of distamycins in experimental antitumor therapy.

One case of immunotactoid glomerulopathy: morphological-ultrastructural aspects.

Starting from a group of 736 renal biopsy patients, evaluated by ultrastructural studies over a period of 22 years, the authors present a rare case of immunotactoid glomerulopathy, suggesting that these forms, until a few years ago considered in the same group as fibrillary glomerulonephritis, are in fact a separate entity; moreover, they may represent a very early manifestation of plasmacellular dyscrasia still at the initial stage.

Cholesterol vesicles in areas of gastric metaplasia of gallbladder epithelium.

Gastric metaplasia is a frequent epithelial change in gallbladder of lithiasic patients. Particles consistent with cholesterol-containing vesicles were searched in areas overlying gastric metaplastic cells, in gastric metaplastic cells, in areas overlying normal-looking epithelial cells and in normal-looking epithelial cells in 25 patients with radiolucent calculi and 10 control patients undergoing cholecystectomy for other reasons. Important trends were found confirming the peculiar role of mucous layer adherent to metaplastic gallbladder epithelium in beginning the nucleation process and interesting pictures concerning the formation of uni- and multilamellar vesicles in these "nucleating areas" were seen.

Birbeck granules: contribution to the comprehension of intracytoplasmic evolution.

We investigated the ultrastructure of Birbeck granules which are found in some malignant histiocytoses such as histiocytosis X, Letterer-Siwe disease, Hand-Schüller-Christian disease, eosinophilic granuloma of the bone and self-healing reticulohistiocytosis. The research is based on the systemic study of Birbeck granules, from their formation to intracytoplasmic development, examining with the electron microscope at regular intervals ultrathin sections derived from biopsies of two cases of Langerhans cell histiocytosis.

Treatment of cervical dentin hypersensitivity with resin adhesives: 4-week evaluation.

PURPOSE: To evaluate the effectiveness of a dentin bonding agent containing HEMA compared to a resin emulsion in the reduction of cervical dentin hypersensitivity (CDH). MATERIALS AND METHODS: The study was a randomized-within-subject, double blind design. Twenty patients who were suffering from CDH from at least two teeth (canines and/or premolars) were enrolled. The subjective perceptions of pain in response to tactile, air blasts and cold ethyl chloride stimuli were evaluated using a visual analog scale (VAS) at baseline and 10 minutes, 1 week and 4 weeks after the treatments. Scotchbond 1 (a dentin bonding agent similar to Single Bond in the USA) and MS Coat (a resin emulsion), both applied according to the manufacturers' directions, were selected for the study. RESULTS: Multiple regression ANOVA for repeated measures showed that both treatments significantly reduced the CDS with respect the baseline values after just 10 minutes. There were no significant differences between the ability of the two treatments to reduce CDH for each of the three evaluation stimuli or for subjective evaluation.

Hypoxia induces cell damage via oxidative stress in retinal epithelial cells.

Retinal diseases (RD), including diabetic retinopathy, are among the most important eye diseases in industrialized countries. RD is characterized by abnormal angiogenesis associated with an increase in cell proliferation and apoptosis. Hypoxia could be one of the triggers of the pathogenic mechanism of this disease. A key regulatory component of the cell's hypoxia response system is hypoxia-inducible factor 1 alpha (HIF-1α). It has been demonstrated that the induction of HIF-1α expression can be also achieved in vitro by exposure with cobalt chloride (CoCl2), leading to an intracellular hypoxia-like state. In this study we have investigated the effects of CoCl2 on human retinal epithelium cells (hRPE), which are an integral part of the blood-retinal barrier, with the aim to determine the possible role of oxidative stress in chemical hypoxia-induced damage in retinal epithelial cells. Our data showed that CoCl2 treatment is able to induce HIF-1α expression, that parallels with the formation of reactive oxygen species (ROS) and the increase of lipid 8-isoprostanes and 4-hydroxynonenal (4-HNE) protein adducts levels. In addition we observed the activation of the redox-sensitive transcription factor nuclear factor-kappaB (NFkB) by CoCl2 which can explain the increased levels of vascular endothelial growth factor (VEGF). The increased number of dead cells seems to be related to an apoptotic process. Taken together these evidences suggest that oxidative stress induced by hypoxia might be involved in RD development through the stimulation of two key-events of RD such as neo-angiogenesis and apoptosis.

Comparative effects between electronic and cigarette smoke in human keratinocytes and epithelial lung cells.

Information about the harmful effects of vaping is sparse and inconsistent, therefore, since the use of electronic cigarettes (e-CIGs) has become increasingly popular as a tool to limit tobacco smoking, it is urgent to establish the toxicity of the commercial e-CIGs. Skin (HaCaT) and lung (A549) cells, the main targets of cigarette smoke (CS), were exposed to e-CIG vapor and CS using an in vitro system. The cytotoxic effect of the exposure was analyzed in both cell types by ultrastructural morphology, Trypan Blue exclusion test and LDH assay. In addition, pro-inflammatory cytokines were measured by the Bio-Plex assay. The cytotoxic components of e-CIG were restrained to the flavoring compound and, to a lesser extent, to nicotine although their effects were less harmful to that of CS. Humectants alone exhibited no cytotoxicity but induced the release of cytokines and pro-inflammatory mediators. Based on our results, we can state that exposure to e-CIG vapors results in far less toxic than exposure to CS. In fact, besides the deleterious effect of flavor and nicotine, even the humectants alone are able to evocate cytokines release. This study will hopefully promote the development of safer e-CIGs to help people quit smoking.

Resveratrol protects SR-B1 levels in keratinocytes exposed to cigarette smoke.

Cigarette smoking (CS) has been strongly linked to several health conditions including heart disease, lung cancer, and other respiratory and circulatory ailments. Deleterious effects of cigarette smoking on skin have also been well documented, but unlike effects on other organs, damage does not depend upon inhalation. The upper layer of the skin, the stratum corneum (rich in cholesterol fatty acids and ceramide), is very susceptible to damage induced by exposure to environmental stressors that can modify its lipid composition and thereby affect its function of protecting skin from dehydration. Scavenger receptor B1 (SR-B1) is involved in the uptake of cholesterol in several tissues including skin. We previously demonstrated that CS exposure induces formation of aldehyde (HNE) adducts that decrease SR-B1 expression. As topical resveratrol, a well-known polyphenolic stilbene, has been demonstrated to show benefits against skin disorders, we investigated its possible role as a protective agent against CS-induced reduction of SR-B1 expression in cutaneous tissue. In this study, we demonstrate that resveratrol at doses ranging from 0.5 to 10 µM is not toxic and is able to increase SR-B1 protein levels in a dose-dependent manner in human keratinocytes. Moreover, when the cells that were pretreated with various doses of resveratrol were exposed to CS, the loss of SR-B1 was prevented in a dose-dependent manner. In addition, in keratinocytes, resveratrol was also able to prevent an increase in HNE-protein adducts induced by CS. In particular resveratrol was able to prevent HNE-SR-B1 adduct formation. Thus, resveratrol seems to be a natural compound that could provide skin with a defense against exogenous stressors by protecting the essential cholesterol receptor, SR-B1.

Glycogenic acanthosis presenting as leukoplakia on the tongue.

This report describes a rare case of glycogenic acanthosis (GA) located on the tongue of a 72-year-old man. The patient presented with a white plaque that had been on the right side of the tongue for 5 years. Histologically, the lesion was characterised by thickened squamous epithelium with abundant intracellular glycogen deposits. No epithelial dysplasia was noted. This is the first report of GA involving the tongue. This benign condition should be added to the large number of lesions responsible for leukoplakia in this anatomical location.

Prognostic value of Ki67 from clinically and histologically 'normal' distant mucosa in patients surgically treated for oral squamous cell carcinoma: a prospective study.

The frequent spread of oral squamous cell carcinoma (OSCC) has been explained by the persistence of genetically altered mucosa after surgery. This study examined whether clinically and histologically 'normal' mucosa distant from the primary tumour (from the opposite cheek) has an abnormal proliferative status, and whether this is associated with poor prognosis in terms of local recurrence or lymph node metastasis. The prospective study included 47 consecutive patients with OSCC. Disease-free survival endpoints were defined as the duration between surgical resection and the diagnosis of recurrence, lymph node metastasis, death or last follow-up. Proliferative status was evaluated by Ki67 expression. The mean Ki67 value (16.5+/-8.9) in the distant mucosa was statistically higher than that in controls (8.8+/-2.9). Abnormally high Ki67 values (>20%) were detected in 11 OSCC patients (23%). Multivariate analysis showed that Ki67 value in distant mucosa was a powerful independent prognostic factor, greater than tumour differentiation or clinical stage for the disease-free survival rate; it was statistically negative regarding local recurrence. Some patients surgically treated for OSCC have an abnormal proliferative status in areas distant from the primary tumour. The Ki67 value in these areas is a promising prognostic factor.

Predictive Role of p53 Protein as a Single Marker or Associated to Ki67 Antigen in Oral Carcinogenesis.

p53 over-expression has been proposed as a reliable marker associated to oral carcinogenesis, although only about 50% of oral carcinomas (OSCC) are associated with p53 over-expression and even p53-negative lesions can progress to OSCC. The aim of the study was to determine whether the combination of p53 over-expression and p53 low-expression associated with Ki67 over-expression (high Ki67/p53 ratio) could lead to a more sensitive parameter. Immunohistochemical expression of Ki67 and p53 was measured in 54 specimens from OSCC; 27 specimens from moderate/severe epithelial dysplasia; 32 specimens from oral leukoplakias without epithelial dysplasia, and 13 specimens with normal epithelium. p53 over-expression was found in 31 (53%) samples from OSCC, in 10 (37%) samples from severe dysplasias, and in 5 (15%) samples from non-dysplastic lesions, while the combination of high p53 values with high Ki67/p53 ratio was observed in 93% of OSCC, in 81% of dysplastic lesions, and in 50% of non-dysplastic lesions. This parameter may have a clinical implication to detect early lesions with an impairment of p53 pathway, and probably at risk of progress to OSCC.

On the interaction between xanthine oxidase and actin.

Xanthine oxidase increases the rate of actin polymerization. This occurs at oxidase concentrations as low as 40 nM provided the concentration of the polymerizing agent is low (0.5 mM MgCl2). In the presence of 0.1 M KCl plus 1 mM MgCl2 as the polymerizing agents, xanthine oxidase does not affect the rate of the polymerization but increases significantly the rate of the conversion of F(ATP)actin into F(ADP.Pi)actin and probably also the rate of the orthophosphate release.

Biodegradable microparticles for sustained delivery of tetracycline to the periodontal pocket: formulatory and drug release studies.

This paper describes the production and characterization of biodegradable microparticles containing tetracycline, designed for periodontal disease therapy. The influence of production parameters on microparticle characteristics and antibiotic release modality was studied. Microparticles were made by using different preparation procedures and different polyesters, namely poly(L-lactide), [L-PLA] poly(DL-lactide), [DL-PLA] and poly(DL-lactide-co-glycolide) 50:50, [DL-PLG]. A double emulsion preparation method together with a concentrated salt solution as external phase gave the best results in terms of tetracycline incorporation efficacy. In vitro release experiments demonstrated that tetracycline is slowly and appropriately released from microparticles. Release kinetics were found to be influenced by the type of polymer utilized for microparticle production. In vitro experiments, simulating in vivo conditions were carried out for up to 30 days. Only DL-PLG microparticles showed significant changes in their morphology, whereas L-PLA and DL-PLA were found almost intact after the same period of time.