The aim of the present study was to investigate the effects of a 3-week residential multidisciplinary non-pharmacological treatment program (including individually prescribed aerobic exercise and ...cognitive-behavioral therapy) on fibromyalgia symptoms and hypothalamic–pituitary–adrenal (HPA) axis function. Salivary and venous blood samples were collected from 12 female patients with fibromyalgia (age: 25–58) the day before and the day after the treatment period: saliva, eight times (every two hours from 0800 to 2200
h); venous blood, at 0800
h. Peripheral blood mononuclear cells (PBMC) were separated and analyzed for glucocorticoid receptor-
α (GR-
α) mRNA expression by semi-quantitative RT-PCR, while the salivary cortisol concentration was determined by RIA. At the same time, pain and aerobic capacity were evaluated. Aerobic capacity improved at the end of the treatment program. The slope of the regression of salivary cortisol values on sampling time was steeper in all patients after treatment, indicating that the cortisol decline was more rapid. Concomitantly, the area under the cortisol curve ‘with respect to increase’ (AUC
i) was higher and there was a significant increase in GR-
α mRNA expression in PBMC. The number of positive tender points, present pain, pain area and CES-D score were significantly reduced after the treatment, while the pressure pain threshold increased at most of the tender points. Our findings suggest that one of the active mechanisms underlying the effects of our treatment is an improvement of HPA axis function, consisting in increased resiliency and sensitivity of the stress system probably related to stimulation of GR-
α synthesis by the components of the treatment.
The proliferative capacity of immune cells is known to be sensitive to conditions of oxidative stress and lipid peroxidation. We tested the hypothesis that activated neutrophils can induce ...peroxidation in extracellular lipid substrates, and evaluated the effects of 4-hydroxy-2,3-
trans-nonenal (4-HNE) — the most reactive aldehydic product of lipid peroxidation — on mitogen-induced proliferation of human T lymphocytes. Neutrophils activated in the presence of extracellular lipid substrates (liposomes, cellular membranes) induced lipid peroxidation. By means of cytoimmunofluorescent labeling and confocal microscopy, the binding of 4-HNE to surface and cytoplasmic proteins of activated neutrophils was observed. Short (20 min) pre-treatment of cells with low concentrations of 4-HNE were able to dose-dependently decrease the proliferation of human peripheral blood lymphocytes challenged with PHA or anti-CD3 monoclonal antibody OKT3, as well as the proliferation of a tetanus specific human T-cell line challenged with tetanus toxoid. In these conditions, the binding of 4-HNE to surface and cytoplasmic proteins of lymphocytes was also observed. When the proliferative capacity of peripheral blood lymphocytes was monitored over several days after 4-HNE treatment and PHA challenge, a recovery and a rebound in cell proliferation was observed. Data reported indicate that the lipid peroxidation promoted by activated neutrophils can exert modulatory effects on the responsivity of human T cells, through the action of its most reactive product, 4-HNE.
We have used the enhanced uptake by FcR-bearing cells observed when Ag is administered as an immune complex to investigate the possible impact of specific antibodies on processing and presentation of ...antigen by accessory cells. The Ag Escherichia coli beta-galactosidase alone or bound to different mAb was incubated with peritoneal macrophages. These were subsequently exposed to a battery of Ag-specific T hybridoma clones. The resulting production of IL-2 was taken as a measure of effective presentation. The results of 43 mAb-T clone combinations showed a potentiation of presentation of Ag at substimulatory concentration in the majority of the cases, indicating that each mAb is conducive to FcR-mediated uptake by macrophages, and that each T clone can be stimulated by properly presented Ag. In contrast, nine combinations yielded a lower response, two of them falling to baseline values. We attribute these results, which corroborate our previous evidence of directional help in the beta-galactosidase system, to a modulation in enzymatic processing of Ag and its subsequent presentation imposed by the paratope of the mAb binding to the relevant epitope.
The measurement of single parameters of oxidative stress in biological fluids can often give results difficult to interpret as to the real involvement of oxidative processes in a given disease ...condition. In the present study we propose a novel integrated parameter, called “redox compensation index”, obtained by combining the results of two established and convenient procedures, i.e. the Fox-2 assay for plasma lipid hydroperoxides and the ferric reducing/antioxidant power (FRAP) assay for total antioxidant potential of plasma. These procedures were employed for the evaluation of oxidative stress in a group of patients with type 2 diabetes mellitus, a condition in which oxidative processes are implicated in the development of complications. In type 2 diabetic patients, plasma lipid hydroperoxides were directly correlated with levels of glycated hemoglobin. On the other hand, a significant inverse correlation was o b s e rved between levels of glycated hemoglobin and redox compensation values. The data reported suggest that the redox compensation index could represent a convenient parameter for the direct appraisal of oxidative status in clinical subjects, and are in support of the proposed role of protein glycation in production of oxidative alterations during type 2 diabetes mellitus.
Polymorphonuclear neutrophils (PMNs) are only regarded as being involved in the cleavage of exogenous big endothelin-1 (ET-1) to the active peptide. The aims of the present study were to investigate ...whether PMNs may themselves express mRNA for prepro-ET-1 (pp-ET-1, a long precursor of 212 amino acids) and to determine the capacity of several PMN stimulants to modulate mRNA expression and the release of ET-1 in culture medium. PMNs, isolated from seven healthy adult volunteers, were stimulated with lipopolysaccharide (LPS, 0.25–10μg/ml), or LPS (1μg/ml)+phorbol myristate acetate (PMA, 10ng/ml) or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (f-MLP, 10−5M) or tumour necrosis factor-α (TNF-α, 50IU/ml). They were found to express pp-ET-1 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Enzyme immunoassay (EIA) revealed low levels of ET-1 in the culture supernatants of PMNs stimulated for 3h with LPS (10μg/ml) and with LPS+PMA. Control unstimulated PMNs did not express pp-ET-1 mRNA. The local production of ET-1 by PMNs in vivo has significant implications in inflammatory diseases.
Because the precise immunopathological events occurring in appendicitis are not completely understood, possible local production of endothelin-1 (ET-1) in human appendix was investigated. We used ...immunohistochemistry and in situ hybridization to detect the presence, distribution, and phenotype of ET-1-positive cells and prepro-ET-1 (pp-ET-1) mRNA-expressing cells. ET-1-positive stromal cells and pp-ET-1 mRNA-expressing cells were detected with different distributions and relative frequencies in normal control appendix, histologically normal appendix, and inflamed appendix. Six of 20 histologically normal appendixes from patients with a clinical diagnosis of acute appendicitis had many ET-1-positive stromal cells and high pp-ET-1 mRNA expression, similar to inflamed appendix. Forty percent of the pp-ET-1 mRNA-expressing cells were neutrophils, and the other positive cells were mast cells and macrophages. We suggest that local production of ET-1 by neutrophils and other inflammatory cells could be a molecular sign of focal inflammation in histologically normal appendixes and that ET-1 could be implicated, with other cytokines, in the pathogenesis of appendicitis by inducing appendiceal ischemia through vasoconstriction.
We studied the humoral response of mice immunized with soluble CD4-rgp120 complex, testing polyclonal and monoclonal antibodies (mAbs) with the aim of identifying molecular changes that take place ...after the first interaction between human immunodeficiency virus and the cell surface. The antisera had a paradoxically high syncytia-blocking titer associated with anti-CD4 specificity, while their capacity to inhibit CD4-gp120 binding was relatively modest. One of the mAbs produced from these responders blocks syncytia formation but does not inhibit CD4 interaction with gp120. Apparently, this mAb interacts with the CD4 moiety of CD4-gp120 complex and prevents a post-binding event necessary for membrane fusion and viral infection.
Divicine is an aglycone derived from vicine, a glucosidic compound contained in fava beans (Vicia faba major or broad beans). In this study, we investigated the effect of divicine on cultured human ...myoblasts from normal subjects, in order to see if the drug may induce signs of oxidant stress in these cells. Myoblasts incubated 24 hours in the presence of 1 mM divicine, showed an increase of carbonyl groups and 4-hydroxynonenal (4-HNE) bound to cell proteins, as well as a significant release of iron and lactate dehydrogenase in the culture medium. Desferrioxamine (DFO), an iron chelator, significantly prevented protein oxidation and formation 4-HNE adducts. Our results can be interpreted as indicating that divicine autooxidizes both at extracellular level and into myoblasts thus inducing the release of free iron, which initiates oxidation of cellular proteins and lipids. DFO protects the cells by subtracting the free iron both at intracellular and extracellular level.
The proliferative capacity of immune cells is known to be sensitive to conditions of oxidative stress and lipid peroxidation. We tested the hypothesis that activated neutrophils can induce ...peroxidation in extracellular lipid substrates, and evaluated the effects of 4-hydroxy-2,3-trans-nonenal (4-HNE) - the most reactive aldehydic product of lipid peroxidation - on mitogen-induced proliferation of human T lymphocytes. Neutrophils activated in the presence of extracellular lipid substrates (liposomes, cellular membranes) induced lipid peroxidation. By means of cytoimmunofluorescent labeling and confocal microscopy, the binding of 4-HNE to surface and cytoplasmic proteins of activated neutrophils was observed. Short (20 min) pre-treatment of cells with low concentrations of 4-HNE were able to dose-dependently decrease the proliferation of human peripheral blood lymphocytes challenged with PHA or anti-CD3 monoclonal antibody OKT3, as well as the proliferation of a tetanus specific human T-cell line challenged with tetanus toxoid. In these conditions, the binding of 4-HNE to surface and cytoplasmic proteins of lymphocytes was also observed. When the proliferative capacity of peripheral blood lymphocytes was monitored over several days after 4-HNE treatment and PHA challenge, a recovery and a rebound in cell proliferation was observed. Data reported indicate that the lipid peroxidation promoted by activated neutrophils can exert modulatory effects on the responsivity of human T cells, through the action of its most reactive product, 4-HNE.
Confocal laser scanning fluorescence microscopy coupled to image analysis was employed in order to develop and evaluate procedures for the appraisal at the single-cell level of: (1) protein-bound ...4-hydroxynonenal, the specific product of membrane peroxidation (by means of immunocytochemistry with biotin-avidin revelation); (2) protein oxidation (by reaction of protein carbonyls with 2, 4-dinitrophenyl-hydrazine followed by immunocytochemistry of dinitrophenyl moieties); and (3) cellular protein thiols (by direct alkylation of sulfhydryl groups with thiol-specific fluorescent reagents possessing different cell permeabilities). The procedures proved able to reveal the subcellular distribution of cytochemical parameters useful as indices of oxidative stress conditions, and may allow "redox phenotyping" of isolated cells, which would provide an efficient tool in selected experimental models.
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