A systematic review on drugs for synaptic plasticity in the treatment of dementia.

The aim of the present systematic review (SR) was to provide an overview of all published and unpublished clinical trials investigating the safety and efficacy of disease-modifying drugs targeting synaptic plasticity in dementia. Searches on CT.gov and EuCT identified 27 trials (4 phase-1, 1 phase-1/2, 18 phase-2, 1 phase-2/3, 1 phase-3, 1 phase-4, and 1 not reported). Twenty of them completed, and seven are currently active or enrolling. The structured bibliographic searches yielded 3585 records. A total of 12 studies were selected on Levetiracetam, Masitinib, Saracatinib, BI 40930, Bryostatin 1, PF-04447943 and Edonerpic drugs. We used RoB tool for quality analysis of randomized studies. Efficacy was assessed as a primary outcome in all studies except one and the main scale used was ADAS-Cog (7 studies), MMSE and CDR (4 studies). Safety and tolerability were reported in eleven studies. The incidence of SAEs was similar between treatment and placebo. At the moment, only one molecule reached phase-3. This could suggest that research on these drugs is still preliminary. Of all, three studies reported promising results on Levetiracetam, Bryostatin 1 and Masitinib.

The HIV-1 vpr protein acts as a negative regulator of apoptosis in a human lymphoblastoid T cell line: possible implications for the pathogenesis of AIDS.

Although apoptosis is considered one of the major mechanisms of CD4(+) T cell depletion in HIV-infected patients, the virus-infected cells somehow appear to be protected from apoptosis, which generally occurs in bystander cells. Vpr is an auxiliary HIV-1 protein, which, unlike the other regulatory gene products, is present at high copy number in virus particles. We established stable transfectants of CD4+ T Jurkat cells constitutively expressing low levels of vpr. These clones exhibited cell cycle characteristics similar to those of control-transfected cells. Treatment of control clones with apoptotic stimuli (i.e., cycloheximide/tumor necrosis factor alpha (TNF-alpha), anti-Fas antibody, or serum starvation) resulted in a massive cell death by apoptosis. In contrast, all the vpr-expressing clones showed an impressive protection from apoptosis independently of the inducer. Notably, vpr antisense phosphorothioate oligodeoxynucleotides render vpr-expressing cells as susceptible to apoptosis induced by cycloheximide and TNF-alpha as the control clones. Moreover, the constitutive expression of HIV-1 vpr resulted in the upregulation of bcl-2, an oncogene endowed with antiapoptotic activities, and in the downmodulation of bax, a proapoptotic factor of the bcl-2 family. Altogether, these results suggest that low levels of the endogenous vpr protein can interfere with the physiological turnover of T lymphocytes at early stages of virus infection, thus facilitating HIV persistence and, subsequently, viral spread. This might explain why apoptosis mostly occurs in bystander uninfected cells in AIDS patients.

In vitro cytotoxic effect of wheat gliadin-derived peptides on the Caco-2 intestinal cell line is associated with intracellular oxidative imbalance: implications for coeliac disease.

Coeliac disease (CD) is an inflammatory disorder of the upper small intestine in which gluten acts as an essential factor in its pathogenesis. Although it is generally accepted that cereal protein activation of the immune system is involved in CD progression, a non-immunomediated cytotoxic activity of gliadin-derived peptides on the jejunal/duodenal tract cannot be excluded. In this work, considering that (a) little has been reported about the intracellular metabolic events associated with gliadin toxicity, and (b) an important role for free radicals in a number of gastrointestinal disease has been demonstrated, we investigated the in vitro effects of gliadin-derived peptides on redox metabolism of Caco-2 intestinal cells during a kinetic study in which cells were exposed to peptic-tryptic digest of bread wheat up to 48 h. We found that the antiproliferative effects displayed by gliadin exposure was associated with intracellular oxidative imbalance, characterised by an increased presence of lipid peroxides, an augmented oxidised (GSSG)/reduced (GSH) glutathione ratio and a loss in protein-bound sulfhydryl groups. Significant structural perturbations of the cell plasma membrane were also detected. Additional experiments performed by using the specific GSH-depleting agent buthionine sulfoximine provide evidence that the extent of gliadin-induced cell growth arrest critically depends upon the 'basal' redox profile of the enterocytes. On the whole, these findings seem to suggest that, besides the adoption of a strictly gluten-free diet, the possibility for an adjuvant therapy with antioxidants may be considered for CD patients.

N-acetylcysteine inhibits apoptosis and decreases viral particles in HIV-chronically infected U937 cells.

Apoptosis or programmed cell death (PCD) is a type of death occurring in various physiological processes. Several data suggest that: (1) apoptosis may play a critical role in AIDS pathogenesis; (2) an increase of endocellular free radical levels can be associated with activation of previously latent HIV virus. Tumor necrosis factor (TNF), a cytokine capable of inducing oxygen free radicals and apoptosis, appears also to be involved in HIV activation. The present findings, which elucidate a relationship between the percentage of apoptotic cells, reduced glutathione (GSH) depletion and an increase of p24 antigenemia, suggest that pretreatment with N-acetylcysteine (NAC) is capable of decreasing the above-mentioned phenomena in HIV-infected U937 cells.

N-acetylcysteine protects epithelial cells against the oxidative imbalance due to Clostridium difficile toxins.

Toxins A and B from the anaerobic bacterium Clostridium difficile are the causative agents of the antibiotic-associated pseudomembraneous colitis. At the subcellular level, they inhibit the Rho family GTPases, thus causing alterations of the actin cytoskeleton. The cytoskeletal integrity is also controlled by the redox state of cells. Therefore, we have evaluated whether an oxidative imbalance could be involved in the toxin-induced cytopathic effects. Our results indicate that both toxins induce oxidative stress with a significant depletion of protein SH-groups. These responses and the cytoskeleton-dependent cell retraction and rounding are significantly counteracted by N-acetylcysteine but not by alpha-tocopherol. Our study provides the first evidence that the thiol supplier N-acetylcysteine impairs the cellular intoxication by acting on the cytoskeleton integrity. This also suggests a possible beneficial role for this drug during therapeutic intervention.

Down-modulation of CD4 antigen during programmed cell death in U937 cells.

It has been hypothesized that programmed cell death (PCD), an active cell suicide process occurring in place of necrosis, can be associated with the pathogenesis of acquired immunodeficiency syndrome (AIDS). The entry of human immunodeficiency virus (HIV) into competent cells is mediated by the CD4 molecule present on the surface of certain lymphocyte subpopulations as well as on some cultured cell lines, e.g. U937 myelomonocytic cells. The present paper focuses on some specific aspects of PCD induced by the cytokine tumor necrosis factor (TNF). The results obtained indicate that the exposure of U937 cells to cycloheximide facilitates TNF-mediated PCD via a short term cell death program and modifies the expression of CD4 surface molecules. This change in surface antigen expression, manifested by internalization of the CD4 molecule, occurs in cells in which apoptosis has been triggered, but not in cells undergoing necrosis. These results indicate that the progression of cell death could be associated with specific alterations of certain surface molecules and could have a role in the entry of HIV into cells.

Oxidation affects the regulation of hepatic lipid synthesis by chylomicron remnants.

The effects of native and oxidized chylomicron remnants on lipid synthesis in normal and oxidatively stressed liver cells were investigated using MET murine hepatocytes (MMH cells), a nontransformed mouse hepatocyte cell line that maintains a highly differentiated hepatic phenotype in culture. Lipid synthesis was determined by measuring the incorporation of [(3)H]oleate into cholesteryl ester, triacylglycerol, and phospholipid by the cells. The formation of cholesteryl ester and phospholipid was decreased by chylomicron remnants in a dose-dependent manner, while triacylglycerol synthesis was increased. Exposure of MMH cells to mild oxidative stress by incubation with CuSO(4) (2.5 microM) for 24 h led to significantly increased incorporation of [(3)H]oleate into triacylglycerol and phospholipid, but not cholesteryl ester, in the absence of chylomicron remnants. In the presence of the lipoproteins, however, similar effects to those found in untreated cells were observed. Oxidatively modified chylomicron remnants prepared by incubation with CuSO(4) (10 microM, 18 h, 37 degrees C) did not influence cholesteryl ester or phospholipid synthesis in MMH cells, but had a similar effect to that found with native remnants on triacylglycerol synthesis. These findings show that hepatic lipid metabolism is altered by exposure to mild oxidative stress and by lipids from the diet delivered to the liver in chylomicron remnants, and these effects may play a role in the development of atherosclerosis.

Probucol reduces hepatic cholesterol secretion in hyperlipidemic Yoshida rats.

In this study, perfused livers from Yoshida rats, either on a normal diet or on a diet with 0.3% probucol, were examined. The analysis of liver lipid content and of bile and lipoprotein secretion changes showed that probucol had a relevant effect on liver lipid biosynthesis. In particular, it reduced the production of triacylglycerols and, to a much greater extent that of cholesterol. In addition, probucol reduced plasma cholesterol concentration by decreasing esterified cholesterol in HDL1 and HDL2 fractions. Furthermore, HDL1 composition of both hepatic neosynthetized and circulating particles was strongly modified by probucol. Finally, probucol did not appear to induce significant differences in lipid bile secretion while phospholipid secretion from perfused livers was increased. These facts suggest that the hypolipidemic action of probucol is not mediated by an increase in bile steroid secretion, but rather by a direct reduction in hepatic lipoprotein cholesterol secretion. This secretion induces a modified plasma profile of HDL particles such that these variations are advantageous in terms of reverse cholesterol transport.

Thiol supplier N-acetylcysteine enhances conjugate formation between natural killer cells and K562 or U937 targets but increases the lytic function only against the latter.

In this in vitro study, an evaluation of the importance of intracellular oxidative balance on cell-mediated cytotoxicity was performed by analyzing the effects of the antioxidant N-acetylcysteine (NAC), a specific thiol supplier, on natural killer (NK) cell-mediated cytotoxicity. The results obtained indicate that an enhancement of target cell (TC) killing can be detected when a pre-exposure of effector cells (EC) to NAC was performed. However, this effect seems to depend upon the TC type used. In fact, the increase of EC activity was detected against the differentiated U937 TC while no changes were detected by the same effectors against K562 cells. The mechanism of this enhancement seems to be ascribable to an increased ability of NAC-exposed NK cells to form conjugates (binding) which, in turn, appears to be due to a specific effect of NAC on actin microfilaments. A role for NAC as a cytoskeleton thiol-modifier contributing to the activation of effector cells can thus be hypothesized.

The role of oxidative imbalance in progression to AIDS: effect of the thiol supplier N-acetylcysteine.

In this study we investigate the redox profile of HIV+ patients at different stages of disease with regard to immunological parameters, i.e., the number of circulating CD4+ and CD8+ lymphocytes. For this purpose, peripheral blood mononuclear cells (PBMCs) obtained from healthy donors, HIV+ patients in the asymptomatic phase, long-term nonProgressors (LTNPs), and AIDS patients have been considered. Cells have been exposed in vitro to the prooxidizing agent menadione, which is able to induce superoxide anion formation, and the susceptibility of the cells to the induced oxidative stress was estimated. Moreover, the possibility that the susceptibility of the cells to oxidative stress might be reduced by preexposing them to the antioxidizing agent N-acetylcysteine (NAC) has also been analyzed. The results obtained can be summarized as follows: (1) treatment with the prooxidant agent is capable of inducing massive morphological alterations in PBMCs. In particular, a significant correlation was found between the decrease in number of CD4+ lymphocytes in patients at different stages of disease and the susceptibility of their PBMCs to oxidative stress; (2) preincubation with NAC was able to preserve partially the ultrastructural characteristics of PBMCs isolated from HIV+ patients. In particular, a direct relationship was found between the efficacy of NAC protection and CD4 counts; (3) evaluation of the plasma index of peroxidation and the number of circulating CD4 lymphocytes indicates the existence of a positive correlation between "systemic" oxidative imbalance and stage of the disease; and (4) cells from LTNPs display either oxidative susceptibility or oxidative markers similar to those of healthy donor cells. Our study suggests that the redox profile of patients may be considered a predictive marker of AIDS progression and that the acute infection and the asymptomatic phase of the disease may represent a useful period in which the combined use of antiretroviral and antioxidant drugs may be beneficial.

Antioxidant N-acetyl-cysteine increasing cell adhesion capability could facilitate the biocompatibility processes.

Cell adhesion plays an important role in several cell processes and functions, including differentiation, proliferation and death. An important role for cell attachment to medical devices in biocompatibility studies has also been hypothesized. In this paper we report that the use of the antioxidant drug N-acetyl-cysteine is capable of increasing the adhesion properties of epithelial cells in culture. This is associated with a modification of specific cytoskeletal element assembly, such as microfilament system molecules. In contrast, no quantitative alterations in the expression of certain surface receptors for extracellular matrix molecules, such as VLA2, VLA3 and VLA6, are found. These data seem to indicate that intracellular oxidative balance, in particular of thiol groups, could play a key role in the cell adhesion properties and that N-acetyl-cysteine treatment, acting as 'thiol supply', could be of importance in several circumstances, including biocompatibility of medical devices.

Age-related changes in lipid secretion of perfused livers from male Wistar rats donors.

Male Wistar rats show typical age-related variations in the distribution of high-density lipoprotein subfractions that include an increase in HDL1 and a decrease in HDL2 proportion. The role of liver in these variations was evaluated by studying the lipoprotein and bile secretions from perfused livers of 14 +/- 1 and 3.5 +/- 0.5 month old Wistar rats (adult and young animals, respectively). The lipid content of lipoproteins secreted from adult livers was higher in HDL2 fraction and lower in VLDL fraction. The lipid output did not show significant age-related variations in the case of HDL1 fraction. However, the lipoproteins secreted from adult livers contained a higher proportion of phospholipids, and a lower proportion of triacylglycerols in comparison with lipoproteins secreted by young livers. Therefore, the molar ratio of core to surface lipids was lower in lipoproteins secreted by adult livers. Adult livers showed a reduction in bile flow by about 37% with a significantly higher phospholipid secretion. These findings suggest that both the hepatic metabolism of glycerophospholipids and their repartition between plasma and bile compartments are affected by aging process. In conclusion, present data show that the age-related increase in plasma HDL1 proportion, previously observed in this rat strain in vivo, are not due to a higher liver secretion of these particles. Conversely, liver appears to have a major role in the age-related VLDL increase and in the variations of phospholipid lipoprotein secretion.

Role of pre-existing redox profile of human macrophages on lipid synthesis and cholesteryl ester cycle in presence of native, acetylated and oxidised low density lipoprotein.

The importance of the interactions of modified lipids and macrophages in foam cell generation is clear; however, little attention has been paid to the role of intra-macrophagic redox potential as a modulator of their lipid synthesis and metabolism. In this study, the effects of previously induced non-toxic manipulations of intracellular redox balance on lipid synthesis in human monocyte-derived macrophages (HMDM) was evaluated. Cells, pre-treated with 2.5 microM of the pro-oxidising agent CuSO(4) or with 5 mM of the antioxidant and thiol supplier N-acetylcysteine (NAC), were exposed to radiolabelled oleic acid alone or in combination with native low density lipoprotein (LDL) or modified LDL to evaluate the incorporation of radioactivity into cholesteryl ester, triacylglycerols and phospholipids. CuSO(4)-treated macrophages synthesised more lipids than NAC-treated cells in absence of exogenous lipid, and, generally, in the presence of native or acetylated, but oxidised LDL. In addition, the activities of the enzymes involved in cholesteryl ester storage were also influenced by the pro-oxidant condition. The ratio values between acyl-coenzyme A:cholesterol acyl transferase and cholesteryl ester hydrolase activity suggest that in CuSO(4)-treated macrophages the hydrolysis of cholesteryl ester is favoured with respect to esterification. The interaction of HMDM with oxidised LDL showed a significant different pattern in term of lipid synthesis with respect to those induced by native or acetylated LDL, disrespectful of the initial redox profile of the cells. On the whole, these results suggest that the pre-existing internal redox condition is a further parameter able to modulate the effects of native or acetylated LDL-cell interaction, influencing both HMDM lipid synthesis profile and cholesterol storage. Moreover, oxidised LDL represent a carrier of additional factor(s) able per se to introduce perturbation in the synthetic pathway of lipids, which is not influenced by the redox potential of the macrophage.

Changes in plasma dolichol levels, transport, and hepatic delivery during rat liver regeneration.

During the proliferative process that follows partial hepatectomy in the rat, the dolichol content increases in both plasma and liver. Its transport in the blood by lipoproteins also changes. The difference in the distribution of dolichols of various chain lengths in plasma and in the liver is further enhanced during liver regeneration. The dolichol released by perfused liver shows a homologue distribution more similar to that observable in blood than in the liver, thus confirming the importance of the liver as a regulatory site for the blood dolichol supply.

Effects of cholesterol uptake from high-density lipoprotein on bile secretion and 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in perfused rat liver.

Small aliquots of rat high-density lipoproteins (HDL) (388 +/- 67 nmol lipoprotein cholesterol) were labeled with [14C]cholesterol and administered as a bolus to perfused rat livers. Bile and perfusate samples were collected for 2 hours at 30-minute intervals. After perfusion, both the microsomes and lipid extracts were prepared from the livers. Lipid composition was examined in both liver and microsomes, and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity was evaluated in microsomes. Basal values of bile flow, lipid composition, and enzyme activity were evaluated using livers in which perfusion was discontinued before injecting the lipoprotein. In some experiments, the effect of perfusion per se was assessed by infusing saline instead of lipoprotein. After 10 minutes of lipoprotein perfusion, 50% of cholesterol administered was taken up by the perfused liver. During infusion, transient but significant increases in both bile flow and bile steroid secretion were observed. Cholesterol administration, even if rapid, represented less than 0.4% of total liver cholesterol content. However, this was enough to significantly increase the cholesterol to phospholipid (CH/PL) molar ratio in liver microsomes and at the same time decrease HMG-CoA reductase activity. In conclusion, the main response of the perfused liver to HDL cholesterol infusion is a reduced activity of the rate-limiting enzyme in cholesterol biosynthesis, due to the shift in the microsomal CH/PL molar ratio. A small proportion of the infused cholesterol enters bile as cholesterol and bile salts.

Redox-dependent modulation of lipid synthesis induced by oleic acid in the human intestinal epithelial cell line Caco-2.

The absorption, remodeling, and delivery of dietary lipids by intestinal cells are part of a complex multi-step process, the dynamics of which is influenced by the lipid composition of the diet and the physiological state of enterocytes. Emerging data indicate that, among the parameters known to modulate the cell functionality, the internal oxidative balance plays a pivotal role. In this study, we analyzed the effects of varying redox equilibria on the way in which the intestinal Caco-2 cell line utilize an exogenous lipid source such as oleic acid. Firstly, we manipulated the intracellular levels of soluble thiols (glutathione), and the amount of cell-associated products of lipid peroxidation, commonly regarded as two critical parameters characterizing the redox profile of the cells. Two different perturbants having opposite effects on the cell's redox profile were used: the pro-oxidizing agent CuSO4 (2.5 and 10 microM) and the antioxidant and thiol supplier N-acetylcysteine (NAC, 2.5 and 5 mM). The influence of these mild but critical manipulations on the incorporation of oleate (50 and 500 microM) into cholesterol, triacylglycerol, and phospholipid was then evaluated. We found that the emerging pro-oxidant condition induced by CuSO4 pre-exposure was associated with a significant up-regulation of phospholipid synthesis, while minor modifications were detected in that of triacylglycerols. Conversely, when a more reducing state was induced by NAC pre-treatment, there was a significant down-regulation of triacylglycerol synthesis, with minor modifications in that of phospholipids. In addition, the incorporation of oleic acid in the cholesteryl ester fraction appeared to be unmodified under all the redox conditions reported. On the whole, these results indicate that the pre-existing internal redox potential of the enterocytes is a critical factor that is able to differentially modulate lipid synthesis at the intestinal level. Thus, the adoption of a strategy designed to control/buffer the antioxidant capacity of the gastrointestinal tract could have important consequences for the modulation of lipid balance in the body.

N-acetylcysteine prevents TNF-induced mitochondrial damage, apoptosis and viral particle production in HIV-infected U937 cells.

It has been hypothesized that reactive oxygen intermediates (ROI) can activate human immunodeficiency virus (HIV) replication and that HIV can trigger programmed cell death (PCD). In this work, we studied PCD in U937 cultured cells chronically infected with HIV and exposed to tumor necrosis factor alpha (TNFα). This cytokine has been shown to induce apoptosis in some cell types and to produce intracellular free radical species including ROI. In addition, it was also demonstrated that HIV-induced PCD observable in U937 infected cells can be favored by TNF exposure. In one of our recent works, evidence was presented that the thiol supplier N-acetylcysteine (NAC) can 'protect', at least partially, HIV-infected cells from PCD and determine a significant decrease in viral progeny. In the present work, we demonstrate (a) that apoptosis can be easily induced by TNF only in infected U937 cells and not in control wild-type cells, (b) that daily treatment of TNF-exposed cells with low concentrations of NAC is able to impair viral progeny formation as early as 24 h, (c) that the mitochondrial damage induced by TNF is counteracted by preexposure to NAC, and (d) that NAC alone exerts changes in mitochondria which may be responsible for the protective effects exerted by this compound. Because of the radical producing capacity of TNF, these results seem to indicate that the protective effects of NAC may be due to the specific antioxidant nature of this substance which appears to be capable of impairing both the apoptotic machinery and viral replication by an intracellular mechanism involving mitochondrial integrity and function.

Combined effect of 3-aminobenzamide and N-acetylcysteine on HIV replication in chronically infected U937 cells.

The existence of a close relationship between apoptosis associated with oxidative stress and the increase of viral progeny in chronically HIV-infected cells has been previously reported. The possibility of modulating both phenomena by using an antioxidant such as N-acetylcysteine (NAC) has also been demonstrated. The present investigation was designed to study the role of the nuclear enzyme poly-(ADP-ribose)-polymerase (PARP) when HIV- infected cells are treated with tumour necrosis factor alpha (TNFα), a cytokine capable of inducing both apoptosis and intracellular oxygen free radical production. PARP overexpression may result in a rapid drop of intracellular NAD(+) and ATP concentration, thus contributing to cellular redox imbalance. We have used the specific PARP inhibitor 3- aminobenzamide (3-ABA), alone or in a combination with NAC. 3-ABA was only partially capable of inhibiting viral replication and apoptosis induced by TNFα. In contrast, the combination of NAC and 3-ABA led to an inhibition of apoptosis as well as to a marked decrease in viral particle production, with a parallel replenishment of intracellular reduced glutathione content. The results reported here confirm the potential role of antioxidant drug treatment in specific phases of HIV infection.

The antioxidant N-acetyl-cysteine protects cultured epithelial cells from menadione-induced cytopathology.

The effects of the antioxidant N-acetyl-cysteine (NAC) were assessed after short term exposure of A431 epithelial cells. The drug was able to protect, at least partially, the cells from the oxidative stress induced by the quinone menadione. In particular, the oxidizing agent-induced cell rounding and detachment from the substrate were strongly impaired by pre-exposure to the compound. The mechanism of such an effect seems to be ascribable to a target effect of the drug on the adhesion properties of the cells. In fact, a modification of morphological features of NAC-exposed cells and of their ability to adhere to different coated substrates was found. These changes resulted in a significant improvement of the A431 tumor cell adhesion pattern which was associated with a noticeable rearrangement of some cytoskeletal components, mainly of the microfilament system. These data add new importance to the subcellular activity of NAC and seem to indicate that the redox status of the cells, i.e. the intracellular balance between proxidants and antioxidants, could also play a role in their adhesive properties.

Effect of HDL1 infusion on biliary secretion in perfused rat liver.

The effects of HDL1 lipoprotein infusion on biliary lipid secretion were studied in the in vitro model of rat perfused liver. A strong increase in bile flow was observed during and after lipoprotein infusion. This caused a significant rise in cholesterol, phospholipid and bile salt secretions. However, only the percentage of cholesterol increased with respect to the other bile lipids. The changes observed in the cholesterol/phospholipid molar ratio values of liver membrane subfractions (i.e., liver plasma membrane, mitochondria plus lysosomes and microsomes) isolated from the perfused rat liver after HDL1 administration were not significant.