In vivo gamma-tocopherol supplementation decreases systemic oxidative stress and cytokine responses of human monocytes in normal and asthmatic subjects

We have recently reported that gamma-tocopherol (gammaT) reduces allergen- and zymosan-induced inflammation using rodent models. As an initial step in extending these observations to humans, we conducted an open-label, Phase I dosing study of two doses (one or two capsules daily for 1 week) of a gamma-tocopherol-rich preparation containing 623 mg of gamma-tocopherol, 61.1 mg of d-alpha-tocopherol, 11.1 mg of d-beta-tocopherol (11.1 mg), and 231 mg of d-sigma-tocopherol per capsule. Endpoints for this study include serum levels of 5-nitro-gamma-tocopherol, as a marker of oxidative stress, and changes in serum gamma-, alpha-, and delta-tocopherol and gamma-2'-carboxyethyl-6-hydroxychroman (CEHC) 6 and 24 h after the first dose and after 1 week of treatment. To assess the biological activity of this treatment, we obtained peripheral blood mononuclear cells at baseline and after 1 week of treatment with two capsules of a gamma-tocopherol-rich preparation/day and examined the inflammatory cytokine response of these cells in culture to ex vivo endotoxin/LPS (0.01 ng/ml) challenge. We also monitored a number of safety endpoints to examine how well this preparation is tolerated in eight normal volunteers (four allergic and four nonallergic) and eight allergic asthmatics. We further obtained human monocytes from a subset of these volunteers and treated them ex vivo with gammaT, alphaT, gamma-CEHC, and alpha-CEHC and assessed their actions on LPS-induced degradation of IkappaBalpha and JNK signaling and ROS generation. As detailed herein, this open-label study demonstrates that gamma-tocopherol-enriched supplementation decreased systemic oxidative stress, increased serum levels of gamma-tocopherol, and inhibited monocyte responses to LPS without any adverse health effects. Further, in vitro treatment of human monocytes with gamma-CEHC and alpha-CEHC inhibits ROS generation and LPS-induced degradation of IkappaB and JNK activation.     

Aged mice exhibit distinct peripheral B-cell phenotypes differing in apoptotic susceptibility: an ex vivo analysis.

BACKGROUND: Age-related changes in the antibody response have been classically associated with alterations in T-cell help, but increasing evidence shows that intrinsic B-cell defects exist. This article analyzes the apoptotic susceptibility of peripheral B-cells in aged and young control mice. MATERIALS AND METHODS: Freshly isolated lymphocytes from spleen and Peyer's patches (PPs) were labeled for B-cell lineage (B220(+) cells) and germinal center B subset (GCs, B220(+)/PNA(+) cells). Alternatively, splenic B-cells purified by MACS were used. Apoptosis was monitored by the Annexin V binding, incorporation of 3,3(')-dihexyloxacarbocyanine iodide (DiOC(6)(3)), propidium iodide (PI) staining, and morphological changes. Moreover, intracellular Bcl-2 expression and Bad phosphorylation status were also analyzed in B-cells. RESULTS: We showed in aging mice an enhanced Annexin V(+)/PI(-) cell percentage in splenic B-lymphocytes, which was correlated with a lower DeltaPsi(m). By contrast, no change in apoptosis was observed in compartments known to be enriched in activated B-cells (GCs and PPs). Analysis of Bcl-2 levels revealed no modification. When using B-cells purified by MACS, we strongly confirm data obtained on staining cells. Moreover, enhanced spontaneous apoptosis of splenic B-cells in aged mice was found to be correlated with a reduced phosphorylated Bad expression. CONCLUSION: Increased apoptosis of resting B-cells in old mice may be determined by an altered Bad phosphorylation, which in turn contributes to cell death by lowering the mitochondrial threshold for apoptosis.     

Effects of dietary antioxidants on human DNA ex vivo

The protective effect of fruits and vegetables against cancer is well established. It is believed that this effect is mediated by antioxidants and decreased oxidative damage to DNA. However, the identity of the antioxidant(s) responsible is not clear. Moreover, a potentially damaging pro-oxidant effect of some antioxidants has been reported. In this study the ex vivo effects of several dietary antioxidants, including quercetin, various catechins, ascorbic acid and alpha-tocopherol, were investigated, at concentrations up to 200 microM, using the single cell gel electrophoresis (comet) assay for DNA damage. Lymphocytes from three healthy subjects were pre-incubated with these antioxidants, and the comet assay was performed on treated, untreated, challenged and unchallenged cells in parallel, oxidant challenge being induced by 5 min exposure to hydrogen peroxide (final concentrations H2O2: 30, 45, or 60 microM). Results using this ex vivo cellular assay showed protection by some antioxidants (quercetin, caffeic acid), no effect by some (catechin, epicatechin, catechin gallate, epicatechin gallate) and an apparently damaging effect by others (epigallocatechin, epigallocatechin gallate). Damage may have been caused by production of H2O2 from these polyphenolics. Neither ascorbic acid nor alpha-tocopherol protected or damaged DNA. Further study of the role of quercetin and caffeic acid in DNA protection is needed.     

Cyclic stretch downregulates arterial vascular connexin43 protein expression: an ex vivo study

Vascular cells may communicate through gap junctions that are formed by connexin (Cx) proteins. We investigated differential regulation of arterial gap junctions by steady and cyclic stretch and the underlying mechanotransduction pathways. Ex vivo culture of rabbit thoracic aortas was used to investigate regulation of Cx43 by cyclic stretch. After culturing for 6 or 24 h, Cx43 protein levels were quantified using Western blot. Cultures under a pulsatile pressure (mean 80 mmHg, pulse 30 mmHg) decreased Cx43 protein at both 6 and 24 h as compared with cultures under a steady pressure (80 mmHg). The regulation of Cx43 protein was mediated by pulsatile pressure-induced cyclic stretch, not by cyclic stress. Protein levels of active and total Src were also decreased by cyclic stretch at 24 h. The Src- specific inhibitor PP1 in steady culture only or in both steady and pulsatile culture conditions eliminated the difference in Cx43 protein levels between the two culture conditions. Addition of reactive oxygen species inhibitor apocynin to the pulsatile culture abolished the differences in Src and Cx43 protein levels between the two cultures. Thus, Src and reactive oxygen species appear to play a role in cyclic stretch-mediated regulation of Cx43 protein. These results are likely to have important implications in cardiovascular physiology and pathophysiology under conditions wherein significant alterations in the level of cyclic stretch are present.     

Effects of isoproterenol on IL-2 and cAMP production in peripheral T cells from asthmatic and non-asthmatic subjects sensitive to Candida

Immunity to Candida albicans (Candida) is characterized by a Th-1 type pattern of reactivity. Candida is rarely a cause antigen for bronchial asthma. Beta agonists have been found to inhibit secretion of IL-2 from T cells through intracellular cAMP elevation. We examined effects of isoproterenol (ISO) on Candida-stimulated T cells. Peripheral T cells obtained from six Candida-sensitive asthmatics, six Candida-sensitive non-asthmatic subjects, and six normal donors by Ficoll-Hypaque gradient centrifugation and nylon-wool column chromatography were incubated with Candida antigen or concanavalin A (Con A) in the absence or presence of ISO. Secretion of IL-2 and intracellular accumulation of cAMP were assayed by ELISA. Con A induced secretion of IL-2 in each of the three groups. Candida antigen induced IL-2 secretion in the normal and the non-asthmatic subjects, but not in the asthmatics. ISO, which reduced Con A-induced secretion of IL-2 in a dose-dependent manner, had no effect on Candida-induced secretion of IL-2. Although ISO increased the intracellular concentration of cAMP in untreated and Con A-treated T cells from all donors, cells from the normal and the non-asthmatic subjects, but not from the asthmatics, that were co-incubated with ISO and Candida had lower levels of cAMP than those treated with ISO alone. It is suggested that Candida antigen induces secretion of IL-2 and reduces ISO-inducible accumulation of cAMP in Candida-responsive IL-2 secreting cells, which may make Candida-induced secretion of IL-2 insensitive to ISO.     

Effects of life events and stress on neutrophil functions in elderly men

Background

Psychological stress or life events might significantly decrease neutrophil function in elderly individuals and lead to infectious diseases. However, relationships between these factors have not been examined in detail. We investigated the relationships between neutrophil function measured using the nitroblue tetrazolium (NBT) reduction test and measurements of psychological stress and life events among 81 men aged over 60?years.

Results

The numbers and scores for life events were significantly higher (p?Conclusions Chronic psychological stress due to life events decreases neutrophil functions among elderly men.     

Effects of zinc ex vivo on taurine uptake in goldfish retinal cells

Background

Taurine and zinc exert neurotrophic effects in the central nervous system. Current studies demonstrate that Na⁺/Cl^- dependent neurotransmitter transporters, similar to that of taurine, are modulated by micromolar concentrations of zinc. This study examined the effect of zinc sulfate ex vivo on [³H]taurine transport in goldfish retina.

Methods

Isolated cells were incubated in Ringer with zinc (0.1–100 µM). Taurine transport was done with 50 nM [³H]taurine or by isotopic dilution with taurine (0.001–1 mM) and 50 nM [³H]taurine.

Results

Zinc reduced the capacity of taurine transport without changes in affinity, and caused a noncompetitive inhibition of high affinity taurine transport, with an EC₅₀= 0.072 µM. The mechanism by which zinc affects taurine transport is unknown at the present.

Conclusions

There may be a binding site of zinc in the transporter that affects union or translocation of taurine, or possibly the formation of taurine-zinc complexes, rather than free zinc, could affect the operation of the transporter.

     

Effect of acute exercise on some haematological parameters and neutrophil functions in active and inactive subjects

In this work we studied the possible effects of acute exercise on some haematological parameters and on some functions of neutrophils in seven active and six inactive subjects. Physical exercise (10 min on a cycle ergometer at a heart rate of 150 beats · min^–1) induced a significant increase in total leucocyte, lymphocyte and neutrophil concentrations in active subjects; serum iron and ferritin concentrations were lower in active compared to inactive subjects. Cellular adhesion, bactericidal activity and superoxide anion production did not change after exercise, while we also observed some differences between active and inactive subjects before exercise. In particular, the neutrophils from active subjects showed a significantly higher percentage of adhesion, higher bactericidal activity and lower superoxide anion production. In conclusion, the training induced changes in some neutrophil functions, while acute exercise influenced, overall, leucocyte concentrations.     

Augmentation cystoplasty and extracellular matrix scaffolds: an ex vivo comparative study with autogenous detubularised ileum

Background

Augmentation cystoplasty (AC) with autogenous ileum remains the current gold standard surgical treatment for many patients with end-stage bladder disease. However, the presence of mucus-secreting epithelium within the bladder is associated with debilitating long-term complications. Currently, decellularised biological materials derived from porcine extracellular matrix (ECM) are under investigation as potential augmentation scaffolds. Important biomechanical limitations of ECMs are decreased bladder capacity and poor compliance after implantation.

Methodology/Principal Findings

In the present ex vivo study a novel concept was investigated where a two-fold increase in ECM scaffold surface-area relative to the resected ileal segment was compared in ovine bladder models after AC. Results showed that bladder capacity increased by 40±4% and 37±11% at 10 mmHg and compliance by 40.4±4% and 39.7±6% (ΔP = 0–10 mmHg) after AC with ileum and porcine urinary bladder matrix (UBM) respectively (p<0.05). Comparative assessment between ileum and UBM demonstrated no significant differences in bladder capacity or compliance increases after AC (p>0.05).

Conclusions

These findings may have important clinical implications as metabolic, infective and malignant complications precipitated by mucus-secreting epithelium are potentially avoided after augmentation with ECM scaffolds.     

Intermittent corticotrophin treatment: Ventilatory tests in asthmatic subjects

Ventilatory tests (F.E.V.₁/F.V.C.) have been studied in 16 asthmatic subjects during intermittent administration of corticotrophin, and the responses correlated with the plasma cortisol levels. The improvement in respiratory function was found to last for a longer period than the enhanced adrenocortical activity.     

Abiotic elicitors mediated elicitation of innate immunity in tomato: an ex vivo comparison

Improvement of the host resistance by using hazard free chemical elicitors is emerging as an alternative approach in the field of plant disease management. In our present work, we have screened the efficacy and possible mechanism of abiogenic elicitors like Dipotassium hydrogen orthophosphate (K₂HPO₄), Oxalic acid (OA), Isonicotinic acid (INA), Salicylic acid (SA), Acetylsalicylate (AS), Arachidonic acid (AA) and Calcium chloride (CaCl₂) to stimulate innate immune responses in Lycopersicum esculentum Mill. Excised tomato leaves, treated with elicitors at three different concentrations, were found to stimulate defense and antioxidative enzymes, total phenol and flavonoid content after 24 h of incubation. CaCl₂ (0.5 %) followed by INA (2.5 mM) were found most effective in activation of all such defense molecules in tomato leaves. Furthermore, nitric oxide (NO), a key gaseous mediator in plant defense signaling, was also measured after subsequent elicitor application. Higher doses of elicitors showed an elevated level of reactive oxygen species (ROS) generation, enhanced lipid peroxidation rate and proline content, which indicates the extent of abiotic stress generation on the leaves. However, ROS production, lipid peroxidation rate and proline concentration remain significantly reduced as a result of CaCl₂ (0.5 %) and INA (2.5 mM) application. A sharp increase of total chlorophyll content was also recorded due to treatment of CaCl₂ (0.5 %). These results demonstrate the effects of different abiogenic elicitors to regulate the production of defense molecules. Results also suggest that among all such chemicals, CaCl₂ (0.5 %) and INA (2.5 mM) can be used as a potential elicitor in organic farming of tomato.     

New in vivo and ex vivo models for the experimental study of sheep scrapie: development and perspectives

Sheep scrapie is a prototypical transmissible spongiform encephalopathy (TSE), and the most widespread of these diseases. Experimental study of TSE infectious agents from sheep and other species essentially depends on bioassays in rodents. Transmission of natural sheep scrapie to conventional mice commonly requires one or two years. In an effort to develop laboratory models in which investigations on the sheep TSE agent would be facilitated, we have established mice and cell lines that were genetically engineered to express ovine PrP protein and examined their susceptibility to the infection. A series of transgenic mice lines (tgOv) expressing the high susceptibility allele (VRQ) of the ovine PrP gene from different constructs was expanded. Following intracerebral inoculation with natural scrapie isolates, all animals developed typical TSE neurological signs and accumulated abnormal PrP in their brain. The survival time in the highest expressing tgOv lines ranged from 2 to 7 months, depending on the isolate. It was inversely related to the brain PrP content, and essentially unchanged on further passaging. Ovine PrP transgene expression thus enhanced scrapie disease transmission from sheep to mice. Such tgOv mice may bring new opportunities for analysing the natural variation of scrapie strains and measuring infectivity. As no relevant cell culture models for agents of naturally-occurring TSE exist, we have explored various strategies in order to obtain stable cell lines that would propagate the sheep agent ex vivo without prior adaptation to rodent. In one otherwise refractory rabbit epithelial cell line, a regulable expression of ovine PrP was achieved and found to enable an efficient replication of the scrapie agent in inoculated cultures. Cells derived from sheep embryos or from tgOv mice were also used in an attempt to establish permissive cell lines derived from the nervous system. Cells engineered to express PrP proteins of a specified sequence may thus represent a promising strategy to further explore, at the cellular level, various aspects of TSE diseases.     

Dendritic-cell immunotherapy: from ex vivo loading to in vivo targeting

The realization that dendritic cells (DCs) orchestrate innate and adaptive immune responses has stimulated research on harnessing DCs to create more effective vaccines. Early clinical trials exploring autologous DCs that were loaded with antigens ex vivo to induce T-cell responses have provided proof of principle. Here, we discuss how direct targeting of antigens to DC surface receptors in vivo might replace laborious and expensive ex vivo culturing, and facilitate large-scale application of DC-based vaccination therapies.     

Impact of age and hyperglycemia on the mechanical behavior of intact human coronary arteries: an ex vivo intravascular ultrasound study

Despite their advantages, percutaneous coronary interventional procedures are less effective in diabetic patients. Changes in the mechanical properties of vascular walls secondary to long-term hyperglycemia as well as other factors such as age may influence coronary distensibility. This investigation is aimed at deciphering the extent of these effects on distensibility of postmortem human coronary arteries in a controlled manner. Excised human left anterior descending (LAD) coronary arteries were obtained within 24 h postmortem. With the use of intravascular ultrasound, vascular deformation was analyzed at midregions of 51 moderate lesions. Intraluminal pressure was systematically altered using a computerized pressure pump system and monitored by a pressure-sensing guidewire. Distensibility, a normalized compliance term, was defined as the change in lumen area normalized by the initial reference area over a given pressure interval. With the use of multivariate analysis and repeated-measures ANOVA, coronary distensibility was independently influenced by hyperglycemia and age (P < 0.05) through the entire pressure range. Within physiological pressure range, distensibility was significantly reduced with age in nonhyperglycemic coronary specimens (10.55 +/- 4.41 vs. 6.99 +/- 2.45, x10(3) kPa(-1), P = 0.01), whereas the hyperglycemic vessels were stiff even in the younger group (7.90 +/- 5.82 vs. 7.20 +/- 3.36, x10(3) kPa(-1), P = 0.79). Similar results were observed with stiffness index and elastic modulus of the arteries. Hyperglycemia and age independently influenced the distensibility of moderately atherosclerotic LAD coronary arteries. The stiffening with age was overshadowed in the hyperglycemic group by as-yet-undetermined factors.     

SC-41930: an inhibitor of leukotriene B4-stimulated human neutrophil functions

SC-41930 was evaluated for effects on human neutrophil chemotaxis and degranulation. At concentrations up to 100 microM, SC-41930 alone exhibited no effect on neutrophil migration, but dose-dependently inhibited neutrophil chemotaxis induced by leukotriene B4 (LTB4) in a modified Boyden chamber. Concentrations of SC-41930 from 0.3 microM to 3 microM competitively inhibited LTB4-induced chemotaxis with a pA2 value of 6.35. While inactive at 10 microM against C5a-induced chemotaxis, SC-41930 inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis, with 10 times less potency than against LTB4-induced chemotaxis. SC-41930 inhibited [3H]LTB4 and [3H]fMLP binding to their receptor sites on human neutrophils with KD values of 0.2 microM and 2 microM, respectively. SC-41930 also inhibited neutrophil chemotaxis induced by 20-OH LTB or 12(R)-HETE. At concentrations up to 10 microM, SC-41930 alone did not cause neutrophil degranulation, but inhibited LTB4-induced degranulation in a noncompetitive manner. SC-41930 also inhibited fMLP- or C5a-induced degranulation, but was about 8 and 10 times less effective for fMLP and C5a, respectively. The results indicate that SC-41930 is a human neutrophil LTB4 receptor antagonist with greater specificity for LTB4 than for fMLP or C5a receptors.     

Dynamics of pro-inflammatory and anti-inflammatory cytokine release during acute inflammation in chronic obstructive pulmonary disease: an ex vivo study

Inspiratory muscle weakness in patients with COPD is of major clinical relevance. For instance, maximum inspiratory pressure generation is an independent determinant of survival in severe COPD. Traditionally, inspiratory muscle weakness has been ascribed to hyperinflation-induced diaphragm shortening. However, more recently, invasive evaluation of diaphragm contractile function, structure, and biochemistry demonstrated that cellular and molecular alterations occur, of which several can be considered pathologic of nature. Whereas the fiber type shift towards oxidative type I fibers in COPD diaphragm is regarded beneficial, rendering the overloaded diaphragm more resistant to fatigue, the reduction of diaphragm fiber force generation in vitro likely contributes to diaphragm weakness. The reduced diaphragm force generation at single fiber level is associated with loss of myosin content in these fibers. Moreover, the diaphragm in COPD is exposed to oxidative stress and sarcomeric injury. This review postulates that the oxidative stress and sarcomeric injury activate proteolytic machinery, leading to contractile protein wasting and, consequently, loss of force generating capacity of diaphragm fibers in patients with COPD. Interestingly, several of these presumed pathologic alterations are already present early in the course of the disease (GOLD I/II), although these patients appear not limited in their daily life activities. Treatment of diaphragm dysfunction in COPD is complex since its etiology is unclear, but recent findings indicate the ubiquitin-proteasome pathway as a prime target to attenuate diaphragm wasting in COPD.     

High-resolution ex vivo magnetic resonance angiography: a feasibility study on biological and medical tissues

Background  

In biomedical sciences, ex vivo angiography is a practical mean to elucidate vascular structures three-dimensionally with simultaneous estimation of intravascular volume. The objectives of this study were to develop a magnetic resonance (MR) method for ex vivo angiography and to compare the findings with computed tomography (CT). To demonstrate the usefulness of this method, examples are provided from four different tissues and species: the human placenta, a rice field eel, a porcine heart and a turtle.     

Preservation of the in vivo state of mitochondrial network for ex vivo physiological study of mitochondria

Over the course of many years our laboratory has been engaged in the study of physiological functions of mitochondria ex vivo.We showed that the unavoidable destruction of mitochondrial-reticular network during traditional isolation of the mitochondria diminishes the observable ex vivo changes of mitochondrial processes in vivo. Comparing preparations obtained from quiescent and stressed rats, we found that the great difference in size of assemblies of mitochondria preserved in homogenate disappears when it is diluted for the measurement of respiration. This also leads to a decrease in the difference between respiration of mitochondria from quiescent and stressed animals.We developed a new method that provides ex vivo stable preservation of the in vivo network using a cytochemical procedure on glass-adhered lymphocytes in blood smear. We radically changed the incubation medium for the measurement of dehydrogenase activity that excludes an artefact of succinate dehydrogenase hyperactivation ex vivo by non-physiological components of the traditionally used solution. Our method made it possible to observe ex vivo two- to eightfold increase in succinate dehydrogenase activity by adrenaline in vivo, while the activity of α-ketoglutarate dehydrogenase changed reciprocally.The data obtained show that the structure changes of the network play an important role in physiological regulation of mitochondrial functions. Thus, it may be possible to correct mitochondrial dysfunctions in the organism by substances supporting the stability of mitochondrial network. The developed method is non-invasive, informative and, therefore, is convenient for clinical investigations, particularly of mitochondrial diseases.     

Clearance of radiation-induced apoptotic lymphocytes: ex vivo studies and an in vitro co-culture model

Lymphocytes are very sensitive to radiation. Our aim was to test the possibility of detecting apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure. Our in vitro data confirmed the dose-time-effect relationships involved in radiation-induced apoptosis. The detection of in vivo induction of apoptosis in circulating lymphocytes after exposure of animals to radiation appears to depend critically on the technique used to measure apoptosis. Among the different techniques we investigated, mitochondrial modification was the most appropriate; they allowed establishment of dose-time-effect relationships when animals were observed for 72 h. A model of in vitro phagocytosis of apoptotic lymphocytes by macrophages was developed to mimic clearance of apoptotic cells occurring in vivo. Together, our data show that mitochondrial labeling may make it possible to detect ex vivo radiation-induced apoptosis of lymphocytes before macrophage ingestion occurs. We propose the measurement of apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure.