Longitudinal observation of antibody responses for 14 months after SARS-CoV-2 infection.

Better understanding of antibody responses against SARS-CoV-2 after natural infection might provide valuable insights into the future implementation of vaccination policies. Longitudinal analysis of IgG antibody titers was carried out in 32 recovered COVID-19 patients based in the Umbria region of Italy for 14 months after Mild and Moderately-Severe infection.Two FDA-approved immunoassays against SARS-CoV-2 Nucleocapsid protein (NCP) and anti-spike-receptor binding domain (S-RBD) were used for sequential serological tests at different time points. The demographics,clinical history and symptom profile associated with the magnitude and longevity of antibody responses were also analyzed. Anti-S-RBD IgG persisted in 96.8% (31 of 32) subjects at 14 months. Patients reporting loss of smell and taste during the clinical course of the disease developed significantly higher antibody titers. Anti-NCP IgG seronegative patients(n=7) at 10 months, tested positive for anti-S-RBD IgG at 12,13 and 14 months emphasizing on a higher false-negative rate for NCP protein-based antibody assays. This study also highlights the importance of adopting specific immunoassays for routine estimation of antibody titers and the decreased rate of re-infections in recovered patients.

Nuclear Lipid Microdomains Regulate Daunorubicin Resistance in Hepatoma Cells.

Daunorubicin is an anticancer drug, and cholesterol is involved in cancer progression, but their relationship has not been defined. In this study, we developed a novel experimental model that utilizes daunorubicin, cholesterol, and daunorubicin plus cholesterol in the same cells (H35) to search for the role of nuclear lipid microdomains, rich in cholesterol and sphingomyelin, in drug resistance. We find that the daunorubicin induces perturbation of nuclear lipid microdomains, localized in the inner nuclear membrane, where active chromatin is anchored. As changes of sphingomyelin species in nuclear lipid microdomains depend on neutral sphingomyelinase activity, we extended our studies to investigate whether the enzyme is modulated by daunorubicin. Indeed the drug stimulated the sphingomyelinase activity that induced reduction of saturated long chain fatty acid sphingomyelin species in nuclear lipid microdomains. Incubation of untreated-drug cells with high levels of cholesterol resulted in the inhibition of sphingomyelinase activity with increased saturated fatty acid sphingomyelin species. In daunodubicin-treated cells, incubation with cholesterol reversed the action of the drug by acting via neutral sphingomyelinase. In conclusion, we suggest that cholesterol and sphingomyelin-forming nuclear lipid microdomains are involved in the drug resistance.

Why high cholesterol levels help hematological malignancies: role of nuclear lipid microdomains.

BACKGROUND: Diet and obesity are recognized in the scientific literature as important risk factors for cancer development and progression. Hypercholesterolemia facilitates lymphoma lymphoblastic cell growth and in time turns in hypocholesterolemia that is a sign of tumour progression. The present study examined how and where the cholesterol acts in cancer cells when you reproduce in vitro an in vivo hypercholesterolemia condition. METHODS: We used non-Hodgkin's T cell human lymphoblastic lymphoma (SUP-T1 cell line) and we studied cell morphology, aggressiveness, gene expression for antioxidant proteins, polynucleotide kinase/phosphatase and actin, cholesterol and sphingomyelin content and finally sphingomyelinase activity in whole cells, nuclei and nuclear lipid microdomains. RESULTS: We found that cholesterol changes cancer cell morphology with the appearance of protrusions together to the down expression of ß-actin gene and reduction of ß-actin protein. The lipid influences SUP-T1 cell aggressiveness since stimulates DNA and RNA synthesis for cell proliferation and increases raf1 and E-cadherin, molecules involved in invasion and migration of cancer cells. Cholesterol does not change GRX2 expression but it overexpresses SOD1, SOD2, CCS, PRDX1, GSR, GSS, CAT and PNKP. We suggest that cholesterol reaches the nucleus and increases the nuclear lipid microdomains known to act as platform for chromatin anchoring and gene expression. CONCLUSION: The results imply that, in hypercholesterolemia conditions, cholesterol reaches the nuclear lipid microdomains where activates gene expression coding for antioxidant proteins. We propose the cholesterolemia as useful parameter to monitor in patients with cancer.

Acid sphingomyelinase as target of Lycium Chinense: promising new action for cell health.

BACKGROUND: Sphingomyelin plays very important roles in cell function under physiological and pathological conditions. Physical and chemical stimuli produce reactive oxygen species that stimulate acid sphingomyelinase to induce apoptosis. Antioxidant plants of the traditional Chinese Pharmacopoeia, such as Lycium Barbarum and Lycium Chinense, have become increasingly popular in Western countries. We investigated the effects of Lycium Chinense on acid sphingomyelinase and sphingomyelin species in relation to gene expression. METHODS: We prepared Lycium Chinense berry extracts and evaluated their antioxidant properties. Increasing amount of extracts was used to test cytotoxic and genotoxic effect on HepG2 cells. Gene expression, protein amount and enzyme activity of acid sphingomyelinase were tested by RT-PCR, immunoblotting and enzymatic activity assay, respectively. Sphingomyelin species were analyzed by UFLC MS/MS. A panel of 96 genes involved in oxidative stress, proliferation, apoptosis and cancer was used to test the effect of LC on gene expression. GLRX2, RNF7, and PTGS1 proteins were analyzed by immunoblotting. RESULTS: We showed that Lycium Chinense berries have high antioxidant properties, have an IC50value of 9.55 mg/mL, do not induce genotoxic effect and maintain high level of cell viability. The berry extracts inhibit acid sphingomyelinase activity and increase both very long fatty acid sphingomyelin species and unsaturated fatty acid sphingomyelin species. Among 96 genes, Lycium Chinense berries up-regulate Glutaredoxin 2 and Ring Finger Protein 7 genes and proteins, able to protect cells from apoptosis. Intrigantly, Lycium Chinense berries down-regulates Prostaglandin H synthase 1 gene but the protein is not expressed in HepG2 cells. CONCLUSION: The results identify acid sphingomyelinase as a novel target of Lycium Chinense berries to decrease saturated/unsaturated fatty acid sphingomyelin ratio, known to be useful for cell health. Consistent with these data, the berries regulate specifically gene expression to protect cells from apoptosis.

Gentamicin arrests cancer cell growth: the intriguing involvement of nuclear sphingomyelin metabolism.

The use of gentamicin for the treatment of bacterial infection has always been an interesting and highly speculated issue for the scientific community. Conversely, its effect on cancer cells has been very little investigated. We studied the effect of high doses of gentamicin on non-Hodgkin's T-cell human lymphoblastic lymphoma (SUP-T1). We showed that gentamicin delayed cell growth and induced cell death in lymphoma cells with a rather mild effect on lymphocytes. In SUP-T1 cells, GAPDH, B2M, CDKN1A and CDKN1B were down-expressed in comparison with lymphocytes. Gentamicin treatment in SUP-T1 cells restored the expression of GAPDH, B2M and CDKN1A to values similar to those of lymphocytes and caused overexpression of CDKN1B. The drug acted via sphingomyelin metabolism; in whole cells, sphingomyelinase activity was stimulated, whereas in purified nuclei, sphingomyelinase activity was inhibited and that of sphingomyelin-synthase was stimulated, with a consequent high level of nuclear sphingomyelin content. We suggest that the increase of nuclear sphingomyelin might enrich the nucleus of lipid microdomains that act as a platform for active chromatin and, thus, might be responsible for gene expression. It is possible that in lymphoblastic lymphoma, high doses of gentamicin induce a beneficial therapeutic outcome.

Critical role for the protons in FRTL-5 thyroid cells: nuclear sphingomyelinase induced-damage.

Proliferating thyroid cells are more sensitive to UV-C radiations than quiescent cells. The effect is mediated by nuclear phosphatidylcholine and sphingomyelin metabolism. It was demonstrated that proton beams arrest cell growth and stimulate apoptosis but until now there have been no indications in the literature about their possible mechanism of action. Here we studied the effect of protons on FRTL-5 cells in culture. We showed that proton beams stimulate slightly nuclear neutral sphingomyelinase activity and inhibit nuclear sphingomyelin-synthase activity in quiescent cells whereas stimulate strongly nuclear neutral sphingomyelinase activity and do not change nuclear sphingomyelin-synthase activity in proliferating cells. The study of neutral sphingomyelinase/sphingomyelin-synthase ratio, a marker of functional state of the cells, indicated that proton beams induce FRTL-5 cells in a proapoptotic state if the cells are quiescent and in an initial apoptotic state if the cells are proliferating. The changes of cell life are accompanied by a decrease of nuclear sphingomyelin and increase of bax protein.

Nuclear lipid microdomain as resting place of dexamethasone to impair cell proliferation.

The action of dexamethasone is initiated by, and strictly dependent upon, the interaction of the drug with its receptor followed by its translocation into the nucleus where modulates gene expression. Where the drug localizes at the intranuclear level is not yet known. We aimed to study the localization of the drug in nuclear lipid microdomains rich in sphingomyelin content that anchor active chromatin and act as platform for transcription modulation. The study was performed in non-Hodgkin's T cell human lymphoblastic lymphoma (SUP-T1 cell line). We found that when dexamethasone enters into the nucleus it localizes in nuclear lipid microdomains where influences sphingomyelin metabolism. This is followed after 24 h by a cell cycle block accompanied by the up-regulation of cyclin-dependent kinase inhibitor 1A (CDKN1A), cyclin-dependent kinase inhibitor 1B (CDKN1B), growth arrest and DNA-damage 45A (GADD45A), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes and by the reduction of signal transducer and activator of transcription 3 (STAT3) and phospho signal transducer and activator of transcription 3 (phoshoSTAT3) proteins. After 48 h some cells show morphological changes characteristic of apoptosis while the number of the cells that undergo cell division and express B-cell lymphoma-2 (Bcl-2) is very low. We suggest that the integrity of nuclear lipid microdomains is important for the response to glucocorticoids of cancer cells.

Nuclear lipid microdomain as place of interaction between sphingomyelin and DNA during liver regeneration.

Nuclear sphingomyelin is a key molecule for cell proliferation. This molecule is organized with cholesterol and proteins to form specific lipid microdomains bound to the inner nuclear membrane where RNA is synthesized. Here, we have reported the ability of the sphingomyelin present in the nuclear microdomain to bind DNA and regulate its synthesis, and to highlight its role in cell proliferation induced by partial hepatectomy. During G1/S transition of the cell cycle, sphingomyelin and DNA content is very high and it is strongly reduced after exogenous sphingomyelinase treatment. During the S-phase of the cell cycle, the stimulation of sphingomyelinase and inhibition of sphingomyelin-synthase are accompanied by the DNA synthesis start. To assess the specificity of the results, experiments were repeated with trifluoperazine, a drug known to affect the synthesis of lipids and DNA and to stimulate sphingomyelinase activity. The activity of sphingomyelinase is stimulated in the first hour after hepatectomy and sphingomyelin-DNA synthesis is strongly attenuated. It may be hypothesized that the nuclear microdomain represents a specific area of the inner nuclear membrane that acts as an active site of chromatin anchorage thanks to the stabilizing action of sphingomyelin. Thus, sphingomyelin metabolism in nuclear lipid microdomains is suggested to regulate cell proliferation.

Long-term persistence of IgG antibodies in recovered COVID-19 individuals at 18 months post-infection and the impact of two-dose BNT162b2 (Pfizer-BioNTech) mRNA vaccination on the antibody response: Analysis using fixed-effects linear regression model.

This era of emerging variants needs a thorough evaluation of data on the long-term efficacy of immune responses in vaccinated as well as recovered individuals, to understand the overall evolution of the pandemic. In this study, we aimed to assess the dynamics of IgG response over 18 months in n = 36 patients from the Umbria region in Italy, who had a documented history of COVID-19 infection in March 2020, and then compared the impact of two-dose BNT162b2 (Pfizer-BioNTech) vaccination on the antibody responses of these patients with the ones who did not receive any dose of vaccine. This is the longest observation (March 2020-September 2021) for the presence of antibodies against SARS-CoV-2 in recovered individuals along with the impact of 2 dose-BNT162b2 vaccination on these responses. Fixed-effect regression models were used for statistical analysis which could be also used to predict future titer trends. At 18 months, 97% participants tested positive for anti-NCP hinting towards the persistence of infection-induced immunity even for the vaccinated individuals. Our study findings demonstrate that while double dose vaccination boosted the IgG levels in recovered individuals 161 times, this "boost" was relatively short-lived. The unvaccinated recovered individuals, in contrast, continued to show a steady decline but detectable antibody levels. Further studies are required to re-evaluate the timing and dose regimen of vaccines for an adequate immune response in recovered individuals.

Effect of synthetic vitamin E-bonded membrane on responsiveness to erythropoiesis-stimulating agents in hemodialysis patients: a pilot study.

BACKGROUND: Oxidative stress, a recently identified factor related to the response to erythropoiesis-stimulating agents (ESAs), is increased in hemodialysis patients. The aim of this study was to verify whether ESA responsiveness improves if the anti-oxidant vitamin E (Vi-E) is placed on the blood-side layer of a synthetic polysulfone (PS) dialyzer. METHODS: This 8-month, controlled and open randomized study involved 2 groups of patients on stable ESA therapy undergoing hemodialysis using a PS dialyzer with or without Vi-E treatment. Hemoglobin, albumin, high-sensitivity C-reactive protein, interleukin-6, iron status, parathyroid hormone (PTH), Vi-E (alpha- and gamma-tocopherol levels) and the oxidative stress markers, advanced oxidation protein products, carbonyls and advanced glycation end products were evaluated every 2 months. The primary outcome measure was ESA resistance, the weekly ESA dose divided by the product between hemoglobin level and end-dialysis body weight. RESULTS: Nineteen of the 20 randomized patients completed the study. During the follow-up, the ESA resistance significantly decreased (p = 0.024), greater in the Vi-E group (37%) than in the PS group (20%), but this difference was not statistically significant (p = 0.596). Baseline PTH and Vi-E levels were associated with ESA resistance. In the secondary analysis, including these covariates in the model, the difference between groups in ESA resistance became significant (p = 0.042). CONCLUSIONS: Vi-E placed on the blood-side of a dialyzer may have a possible beneficial effect on ESA resistance in hemodialysis patients; baseline PTH levels seem to predict ESA resistance and were useful in showing the possible beneficial effect of Vi-E and should be considered in designing adequate-sized trials for testing this hypothesis.

Resveratrol Supported on Magnesium DiHydroxide (Resv@MDH) Represents an Oral Formulation of Resveratrol With Better Gastric Absorption and Bioavailability Respect to Pure Resveratrol.

Resveratrol attracts great interest because of the plethora of in vitro effects at the micromolar concentration range. Unfortunately, these effects are difficult to establish in vivo, due to the low concentration of resveratrol reached. This discrepancy is due to the molecules low solubility in water that favors the propensity for an intestinal absorption rather than in the gastric compartment. To address these challenges, we developed a Solid Dispersion of Resveratrol Supported by Magnesium Di Hydroxide formulation (Resv@MDH). This formulation displays increased water solubility and better bioavailability relative to pure resveratrol in the rabbit animal model. In our study, we evaluated the pharmacokinetics profile of resveratrol in six healthy human subjects following 180 mg of oral resveratrol administration, derived from Resv@MDH or pure resveratrol. Free resveratrol was evaluated in the whole blood sample by using HPLC - MS/MS. In comparison with pure resveratrol that displays an increase of the maximum plasma concentration, Cmax at about 90 min and 2 µM, Resv@MDH displays an earlier peak of resveratrol concentration with an increase of Cmax at about 30 min and 6 µM. The different kinetics suggest a main gastric route for resveratrol absorption from Resv@MDH, where, because of its improved dissolution rate, there seems to be a higher propensity for an acidic environment, as opposed to that with pure resveratrol. This conclusion is also supported by the numerical simulation analysis, which considers the principal steps during the oral route administration. Moreover, there is a 2-fold increase in the amount of free resveratrol with respect to pure resveratrol confirming a better bioavailability observed in the animal model. The characteristic feature of the pharmacokinetic profile of Resv@MDH implies that the beneficial properties of resveratrol in human health should be capitalized on it.

Increased levels of neurotrophins are not specific for chronic migraine: evidence from primary fibromyalgia syndrome.

UNLABELLED: All data obtained in experimental animal pain models support the role of nerve growth factor (NGF) as a putative candidate intervening in the pathogenesis of chronic pain, including chronic daily headache (CDH). Few studies have been carried out to establish its role in maintaining pain states in humans. The present study was aimed at investigating cerebrospinal fluid (CSF) levels of NGF and brain-derived neurotrophic factor (BDNF), both measured by sensitive immunoassay, in 20 chronic migraine (CM) patients and 20 patients affected by primary fibromyalgia syndrome (PFMS), compared with those of 20 age-matched control subjects. Significantly higher levels of both neurotrophins and glutamate were found. A significantly positive correlation emerged between CSF values of BDNF and those of NGF (r = .61, P < .001; r = .53, P < .01) and glutamate (r = .44, P < .02; r = .51, P < .01) in CM and PFMS patients, respectively. These findings suggest the possibility of a NGF-mediated up-regulation of BDNF involved in the pathophysiological events underlying long-term neuroplastic changes in persistent chronic painful conditions, such as CM and fibromyalgia. NGF might indirectly exert its effect through enhancing glutamatergic transmission via BDNF. The above mechanisms could account for sustained central sensitization in both chronic pain states. PERSPECTIVE: This article presents findings of higher NGF and BDNF levels correlated to increased glutamate levels in the CSF of both chronic migraine and fibromyalgia patients. This opens new insights into the pathogenic mechanisms of chronic pain and offers clinicians new therapeutic perspectives targeting the above mechanisms in both painful disorders.

Serum deprivation alters lipid profile in HN9.10e embryonic hippocampal cells.

The understanding of the mechanism of apoptosis is important to improve the use of stem cells for the treatment of neurodegenerative disorders. Sphingolipids are bioactive molecules involved in the regulation of cell fate. In HN9.10e embryonic hippocampal cells, serum deprivation induces apoptosis preceded by sphingomyelinase activation and raise of ceramide levels. Increasing evidence indicates that individual ceramide species regulated by specific pathways in distinct subcellular compartments might carry out distinct cellular functions, but the ceramides species involved in embryonic hippocampal cell death induced by growth factor deprivation are unknown. In the present paper, by using the UFLC-MS/MS methodology, we have investigated the effect of serum deprivation on the lipid profile in HN9.10e cells. At 48h of serum deprivation, we detected a decrease in cholesterol and increase in sphingosine-1-phoshate 18:1, phosphatidylcholine 18:1 18:0, sphingomyelin 18:1 16:0 and in ceramides 18:1 16:0; we also found an increase in saturated/unsaturated fatty acid ratio in sphingomyelin. We hypothesize that the rearrangement of sphingo- and glycerolipids with increase of saturated fatty acids in serum-deprivated, neural cells might represent a cellular response aimed at holding cholesterol inside the cells.

Very-long-chain fatty acid sphingomyelin in nuclear lipid microdomains of hepatocytes and hepatoma cells: can the exchange from C24:0 to C16:0 affect signal proteins and vitamin D receptor?

Lipid microdomains localized in the inner nuclear membrane are considered platforms for active chromatin anchoring. Stimuli such as surgery, vitamin D, or glucocorticoid drugs influence their gene expression, DNA duplication, and RNA synthesis. In this study, we used ultrafast liquid chromatography-tandem mass spectrometry to identify sphingomyelin (SM) species coupled with immunoblot analysis to comprehensively map differences in nuclear lipid microdomains (NLMs) purified from hepatocytes and hepatoma cells. We showed that NLMs lost saturated very-long-chain fatty acid (FA; C24:0) SM in cancer cells and became enriched in long-chain FA (C16:0) SM. We also found that signaling proteins, such as STAT3, Raf1, and PKCζ, were increased and vitamin D receptor was reduced in cancer cells. Because recent researches showed a shift in sphingolipid composition from C24:0 to C16:0 in relation to cell life, we performed a comparative analysis of properties among C16:0 SM, C24:0 SM, and cholesterol. Our results led us to hypothesize that the enrichment of C16:0 SM could determine enhanced dynamic properties of NLMs in cancer cells with an increased shuttling of protein signaling molecules.

Excitatory amino acids and multiple sclerosis: evidence from cerebrospinal fluid.

BACKGROUND: Recent evidence suggests an altered glutamate homeostasis in the brain of patients with multiple sclerosis (MS), as seen in experimental models of MS. OBJECTIVE: To test whether the excitotoxic insult contributes to the pathological process in MS by measuring glutamate and aspartate levels in the cerebrospinal fluid of MS patients and control individuals. PARTICIPANTS: Twenty-five patients with the relapsing-remitting form of MS during a stable clinical phase, 30 patients with relapsing-remitting MS during relapse, and 25 patients with the secondary progressive form of MS were included in the study. Data were compared with those of 20 age-matched control subjects without diseases of the central and peripheral nervous systems. METHODS: Glutamate and aspartate levels in the cerebrospinal fluid were measured by high-performance liquid chromatography. RESULTS: Cerebrospinal fluid glutamate levels were significantly higher in patients assessed during relapse compared with those of the patients with relapsing-remitting MS examined during the stable clinical phase and the controls (P<.001). The levels of glutamate detected in patients with relapsing-remitting MS during the stable phase who had active lesions were significantly higher than in those without neuroradiological evidence of disease activity (P<.001). Significantly higher levels of glutamate were found in patients with secondary progressive MS with an increase of 1 or more points on the Expanded Disability Status Scale score compared with stable patients with secondary progressive MS and control subjects (P<.001). CONCLUSIONS: Neurotoxic events occur in MS patients, and they can be responsible for oligodendrocyte and neuronal cell death in patients with this demyelinating disease. The manipulation of glutamate-altered homeostasis or antagonizing glutamate receptor-mediated excitotoxicity may have therapeutic implications in MS patients.

Gamma-tocotrienol metabolism and antiproliferative effect in prostate cancer cells.

In this study, we evaluated the antiproliferative effect of tocotrienols (T3) and the presence of a specific vitamin E metabolism in PC3 and LNCaP prostate cancer cells. These cell lines are able to transform tocopherols (T) and T3 in the corresponding carboxyethyl-hydroxychromans metabolites (CEHCs). The extent of this metabolism and the inhibitory effect on cell growth followed the order of magnitude alpha-T

How microgravity changes galectin-3 in thyroid follicles.

After long-term exposure to real microgravity thyroid gland in vivo undergoes specific changes, follicles are made up of larger thyrocytes that produce more cAMP and express more thyrotropin-receptor, caveolin-1, and sphingomyelinase and sphingomyelin-synthase; parafollicular spaces lose C cells with consequent reduction of calcitonin production. Here we studied four immunohistochemical tumor markers (HBME-1, MIB-1, CK19, and Galectin-3) in thyroid of mice housed in the Mouse Drawer System and maintained for 90 days in the International Space Station. Results showed that MIB-1 proliferative index and CK19 are negative whereas HBME-1 and Galectin-3 are overexpressed. The positivity of Galectin-3 deserves attention not only for its expression but also and especially for its localization. Our results highlighted that, in microgravity conditions, Galectin-3 leaves thyrocytes and diffuses in colloid. It is possible that the gravity force contributes to the maintenance of the distribution of the molecules in both basal membrane side and apical membrane side and that the microgravity facilitates slippage of Galectin-3 in colloid probably due to membrane remodelling-microgravity induced.

A firmer understanding of the effect of hypergravity on thyroid tissue: cholesterol and thyrotropin receptor.

Maintaining a good health requires the maintenance of a body homeostasis which largely depends on correct functioning of thyroid gland. The cells of the thyroid tissue are strongly sensitive to hypogravity, as already proven in mice after returning to the earth from long-term space missions. Here we studied whether hypergravity may be used to counteract the physiological deconditioning of long-duration spaceflight. We investigated the influence of hypergravity on key lipids and proteins involved in thyroid tissue function. We quantified cholesterol (CHO) and different species of sphingomyelin (SM) and ceramide, analysed thyrotropin (TSH) related molecules such as thyrotropin-receptor (TSHR), cAMP, Caveolin-1 and molecule signalling such as Signal transducer and activator of transcription-3 (STAT3). The hypergravity treatment resulted in the upregulation of the TSHR and Caveolin-1 and downregulation of STAT3 without changes of cAMP. TSHR lost its specific localization and spread throughout the cell membrane; TSH treatment facilitated the shedding of α subunit of TSHR and its releasing into the extracellular space. No specific variations were observed for each species of SM and ceramide. Importantly, the level of CHO was strongly reduced. In conclusion, hypergravity conditions induce change in CHO and TSHR of thyroid gland. The possibility that lipid rafts are strongly perturbed by hypergravity-induced CHO depletion by influencing TSH-TSHR interaction was discussed.

Maternal dietary loads of α-tocopherol depress protein kinase C signaling and synaptic plasticity in rat postnatal developing hippocampus and promote permanent deficits in adult offspring.

Vitamin E (α-tocopherol) supplementation has been tested as prophylaxis against gestational disorders associated with oxidative damage. However, recent evidence showing that high maternal α-tocopherol intake can adversely affect offspring development raises concerns on the safety of vitamin E extradosages during pregnancy. Besides acting as an antioxidant, α-tocopherol depresses cell proliferation and modulates cell signaling through inhibiting protein kinase C (PKC), a kinase that is deeply involved in neural maturation and plasticity. Possible effects of α-tocopherol loads in the maturing brain, where PKC dysregulation is associated to developmental dysfunctions, are poorly known. Here, supranutritional doses of α-tocopherol were fed to pregnant and lactating dams to evaluate the effects on PKC signaling and morphofunctional maturation in offspring hippocampus. Results showed that maternal supplementation potentiates hippocampal α-tocopherol incorporation in offspring and leads to marked decrease of PKC phosphorylation throughout postnatal maturation, accompanied by reduced phosphorylation of growth-associated protein-43 and myristoylated alanine-rich C kinase substrate, two PKC substrates involved in neural development and plasticity. Although processes of neuronal maturation, synapse formation and targeting appeared unaffected, offspring of supplemented mothers displayed a marked reduction of long-term synaptic plasticity in juvenile hippocampus. Interestingly, this impairment persisted in adulthood, when a deficit in hippocampus-dependent, long-lasting spatial memory was also revealed. In conclusion, maternal supplementation with elevated doses of α-tocopherol can influence cell signaling and synaptic plasticity in developing hippocampus and promotes permanent adverse effects in adult offspring. The present results emphasize the need to evaluate the safety of supranutritional maternal intake of α-tocopherol in humans.

Analysis method and characterization of the antioxidant capacity of vitamin E-interactive polysulfone hemodialyzers.

The lipophilic antioxidant vitamin E was used as a surface modifier (or coating agent) of hollow-fiber hemodialyzer membranes with the aim of increasing their biocompatibility and preventing oxidative stress, which are the main clinical drawbacks in hemodialysis (HD) therapy. At present, the redox chemistry of vitamin E-modified dialyzers is not well characterized and there is no standard method to assess the antioxidant capacity of these biomembranes under conditions that simulate those observed during HD therapy. With this study, we developed an original online method to determine the antioxidant capacity of vitamin E-modified dialyzer membranes during circulation experiments. This method is based on a spectrophotometric assay known as the ferric reducing/antioxidant power assay (FRAP). The principle of FRAP and its application to the qualitative and quantitative assessment of miniaturized polysulfone (PS)-based vitamin E-modified dialyzers (PS-VE) were verified by the accurate in vitro analysis of the iron-catalyzed oxidation of vitamin E. The antioxidant capacity of miniaturized PS-VE samples assessed in this study was of 14.5 microM Fe(2+), which corresponded to the transformation of nearly one-third of the vitamin E bound to the hollow-fiber membrane to its oxidation end product alpha-tocopherol quinone. This method shows good reproducibility and intra- and inter-assay precision, and can be easily adapted to determine the redox activity of every type of vitamin E-modified dialyzers during technological investigation, manufacturing control and clinical research.