Dietary conjugated linoleic acid downregulates the AlCl3-induced hyperactivation of compensatory and maladaptive signalling in the mouse brain cortex.

Oxidative stress, hyperactivation of compensatory mechanisms (unfolded protein response, UPR; nuclear factor erythroid 2-related factor 2, Nrf2) and the stimulation of maladaptive response (inflammation/apoptosis) are interconnected pathogenic processes occurring during Alzheimer's disease (AD) progression. The neuroprotective ability of dietary Conjugated linoleic acid (CLAmix) in a mouse model of AlCl3-induced AD was recently described but, the effects of AlCl3 or CLAmix intake on these pathogenic processes are still unknown. The effects of dietary AlCl3 or CLAmix - alone and in combination - were examined in the brain cortex of twenty-eight BalbC mice divided into 4 groups (n = 7 each). The neurotoxic effects of AlCl3 were investigated in animals treated for 5 weeks with 100 mg/kg/day (AL). CLAmix supplementation (600 mg/kg bw/day) for 7 weeks (CLA) was aimed at evaluating its modulatory effects on the Nrf2 pathway while its co-treatment with AlCl3 during the last 5 weeks of CLAmix intake (CLA + AL) was used to investigate its neuroprotective ability. Untreated mice were used as controls. In the CLA group, the NADPH oxidase (NOX) activation in the brain cortex was accompanied by the modulation of the Nrf2 pathway. By contrast, in the AL mice, the significant upregulation of oxidative stress markers, compensatory pathways (UPR/Nrf2), proinflammatory cytokines (IL-6, TNFα) and the proapoptotic protein Bax levels were found as compared with control. Notably, in CLA + AL mice, the marked decrease of oxidative stress, UPR/Nrf2 markers and proinflammatory cytokines levels were associated with the significant increase of the antiapoptotic protein Bcl2. The involvement of NOX in the adaptive response elicited by CLAmix along with its protective effects against the onset of several pathogenic processes triggered by AlCl3, broadens the knowledge of the mechanism underlying the pleiotropic activity of Nrf2 activators and sheds new light on their potential therapeutic use against neurodegenerative disorders.

Conjugated linoleic acid downregulates Alzheimer's hallmarks in aluminum mouse model through an Nrf2-mediated adaptive response and increases brain glucose transporter levels.

Mitochondrial dysfunction, oxidative stress, inflammation and glucose dysmetabolism are pathological signs of Alzheimer's disease (AD). Dietary aluminum (Al) overload is often used to induce AD in rodents and trigger the onset of oxidative-stress hallmarks resembling those of the human disease. The Nuclear factor erythroid 2-related factor 2 (Nrf2), owing to its key role in redox homeostasis, mitochondrial function and inflammation, is a promising drug target for neurological disorders, but only a few data are available on its modulatory effects on glucose transporter expression levels. While it has been found that the protective effect of Conjugated linoleic acid (CLA) occurs through the activation of an Nrf2-mediated adaptive response, its beneficial effect on the considered pathological signs in the Al-induced model has not been established yet. Thirty-five male BalbC mice were divided into 5 groups: two Al-intoxicated groups were treated for 5 weeks with low or high Al doses (8 or 100 mg/kg/day in drinking water, respectively; L or H). Two groups of animals, orally supplemented with CLA (600 mg/kg bw/day) for 7 weeks (2 preliminary weeks plus the 5-week treatment with Al; CLA + L, CLA + H) were used to investigate its protective effect, while untreated mice were used as control (Cntr). We provide evidence that mitochondrial dysfunction, Nrf2 alteration, inflammation and Acetylcholinesterase (AChE) hyperactivation can occur even from L exposure. Interestingly, animal pre-treatment with an allometric CLA dose led to significant downregulation of the toxic effects elicited by L or H, likely through the activation of an adaptive response. In conclusion, CLA ability to increase the level of glucose transporters - along with its antioxidant and anti-inflammatory effect - expands the therapeutic targets of these molecules and comes out as an intriguing suitable candidate for the treatment of multifactorial disease.

Protective effect of Rumenic acid rich cow's milk against colitis is associated with the activation of Nrf2 pathway in a murine model.

Dietary supplementation with pure cis9, trans11 isomer of Conjugated Linoleic Acid -known as Rumenic Acid (RA)- improves cytoprotective defenses downstream through the activation of nuclear factor-E2-related factor-2(Nrf2). This capability, when Rumenic Acid is consumed in the form of foods, is still unknown. The ability of standard (St) or cow milk naturally-enriched in RA (En) to activate Nrf2 pathway and its impact on dextran sodium sulfate (DSS)-induced colitis was comparatively evaluated. Activity of Nrf2 pathway was investigated in colonic tissue of BALB/c mice, receiving 4-week supplement with skimmed milk (SK), St or St reinforced with pure RA (RSt) providing increasing RA dose (0, 124 or 404mg RA/kg-1 b.w, respectively). Next, the anti-oxidant/ anti-inflammatory effect produced by St or En treatment (383mg RA/kg-1 b.w.) was explored. Finally, macroscopic and histomorphologic features of colitis were evaluated in animals challenged with 5% (w/v) DSS, at the end of St or En treatment. Significant activation of Nrf2 pathway is associated with RSt and En intake (P<0.05), but not with SK or En treatment. En pre-treatment offers better protection, in comparison with St, against pro-oxidant, pro-inflammatory signs (P<0.01) and macroscopic signs triggered by DSS. It can be concluded that Nrf2 activation by higher RA amount contained in En is, at least in part, responsible for the improved protection associated with En intake against DSS-induced colitis.

Short wheat challenge is a reproducible in-vivo assay to detect immune response to gluten.

It has been reported that interferon (IFN)-γ-secreting T cells reactive to gluten can be detected in the peripheral blood of individuals with treated coeliac disease (CD) after a short consumption of wheat-containing food. By contrast, very little is known about the reproducibility of this in-vivo procedure in the same patient cohort which underwent two, or more, gluten consumptions. Fourteen coeliac patients in remission consumed wheat bread for 3 days; 13 underwent a second gluten challenge after a wash-out of 3-10 months on a strict gluten-free diet. Immune reactivity to gluten was analysed in peripheral blood by detecting IFN-γ before and 6 days after commencing a gluten diet. Gliadin-specific IFN-γ-secreting CD4(+) T cells increased significantly on day 6 of the first challenge. These cells resulted as prevalently human leucocyte antigen (HLA)-DQ restricted and with a phenotype of gut homing, as suggested by the expression of ß7-integrin. Similarly, reactiveness to gliadin was observed after the second wheat consumption, although with an individual variability of responses at each challenge. Our findings confirmed that the short wheat challenge is a non-invasive approach to investigate the gluten-related immune response in peripheral blood of subjects intolerant to gluten. Furthermore, we demonstrated that the in-vivo procedure can be reproduced in the same subject cohort after a gluten wash-out of at least 3 months. Our study has important implications for the application of this procedure to clinical practice.

Immunomodulatory effects of Lactobacillus casei administration in a mouse model of gliadin-sensitive enteropathy.

Coeliac disease (CD) is a very common food-sensitive enteropathy, which is triggered by gluten ingestion and is mediated by CD4(+) T cells. In addition, alterations in the intestinal microbiota that is normally involved in the homeostasis of GALT (gut-associated lymphoid tissue) seem to play a role in CD. In accordance with these findings, we previously reported that Lactobacillus casei can induce a strong enhancement of the T cell-mediated response to gliadin without inducing enteropathy. In this study, we analysed the effects of L. casei administration in a mouse model of gliadin-induced villous damage that was recently developed and involves the inhibition of cyclo-oxygenase (COX) activities in gliadin-sensitized HLA-DQ8 transgenic mice. To address the issue, we assessed the weight loss, the intestinal cytokine pattern, the density of CD25(+) cells and morphometry of the gut mucosa. We confirmed that COX inhibition in sensitized mice caused villus blunting, dysregulated expression of tumour necrosis factor (TNF)-α and reduced gliadin-specific IL-2 production. Notably, the administration of probiotic strain induced a complete recovery of villus blunting. This finding was associated with a delay in weight decrease and a recovery of basal TNF-α levels, whereas the numbers of CD25(+) cells and the levels of IL-2 remained unchanged. In conclusion, our data suggest that the administration of L. casei can be effective in rescuing the normal mucosal architecture and GALT homeostasis in a mouse model of gliadin-induced enteropathy.

Effects of gliadin stimulation on bone marrow-derived dendritic cells from HLA-DQ8 transgenic MICE.

BACKGROUND AND AIM: Gliadin presentation by HLA-DQ2/8 molecules to T cells plays a crucial role in triggering the inflammatory cascade in coeliac disease. We aimed to study the immunological effects of gliadin stimulation on dendritic cells (DCs) from HLA-DQ8 transgenic and BALB/c mice. METHODS: Bone marrow-derived DCs were stimulated with alpha-chymotrypsin-digested gliadin or ovoalbumin (100 microg/ml). Modification of DC maturation, through HLA-DQ8 and MHC class II expression, and activation, by CD80 and CD86, was assessed by flow cytometry. The ability of pulsed and unpulsed DCs to prime T cells was evaluated by mixed leucocyte reaction. The expression of interleukin-4, -10, -12p70 and interferon-alpha, as well as of Toll-like receptor-4, -7, -8, -9 was determined by ELISA and real-time RT-PCR, respectively. RESULTS: Gliadin stimulation induced DC maturation (p<0.001 in BALB/c, p<0.01 in DQ8) but not activation, whereas ovoalbumin upregulated all markers (p<0.01 for maturation and p<0.001 for activation in both DC populations). No increase of T proliferation was elicited by pulsed DCs with respect to unpulsed DCs. Only in DQ8 DCs, gliadin induced Toll-like receptor-4 (p<0.001), -7 (p<0.001), -8 (p<0.005) expression and interferon-alpha (p<0.001) secretion. CONCLUSION: Gliadin resulted unable to activate DC, but stimulated Toll-like receptor expression and interferon-alpha secretion.

Small intestinal enteropathy in non-obese diabetic mice fed a diet containing wheat.

AIMS/HYPOTHESIS: A deranged mucosal immune response and dietary factors may play an important role in the pathogenesis of type 1 diabetes. The aims of our work were to look for the presence of small intestinal enteropathy in non-obese diabetic (NOD) mice in relation to the presence of wheat proteins in the diet, and to assess their role in the risk of developing diabetes. METHODS: Female NOD mice were fed a standard or gluten-free diet or a gluten-free diet with the addition of wheat proteins (MGFD). Small intestine architecture, intraepithelial CD3(+) infiltration, epithelial expression of H2-IA, mRNA for IFN-gamma and IL-4 were assessed. RESULTS: NOD mice fed a standard diet showed reduced villous height, increased intraepithelial infiltration by CD3(+) cells and enhanced expression of H2-IA and IFN-gamma mRNA when compared with mice on the gluten-free diet. The cumulative diabetes incidence at 43 weeks of age was 65% in the latter and 97% in the former (p<0.01). Mice on MGFD also showed increased epithelial infiltration and a higher incidence of diabetes. CONCLUSIONS/INTERPRETATION: Mice fed a wheat-containing diet showed a higher incidence of diabetes, signs of small intestinal enteropathy and higher mucosal levels of proinflammatory cytokines.

Intranasal administration of one alpha gliadin can downregulate the immune response to whole gliadin in mice.

The mucosal lesion present in coeliac disease is an immune-mediated injury triggered by gliadin and restricted by a particular assortment of major histocompatibility complex genes. In view of this, an immunomodulatory approach that induces tolerance to this antigen appears to be a possible alternative to a strict gluten-free diet in treating coeliac disease. We have shown that intranasal administration of multiple doses of whole gliadin is required to specifically inhibit T helper 1-like T-cell reactivity in BALB/c mice immunized parenterally with whole gliadin. However, T-cell activation to multiple antigens, as a consequence of the chemical complexity shown by the antigen gliadin, could hamper efforts to identify single component(s) useful for tolerance induction. In this study, gliadin fractions were purified and administered intranasally to study their ability to induce tolerance to whole gliadin in our animal model. We found that the alpha fraction was particularly effective in downregulating both the in vitro gliadin-specific T-cell proliferation and interferon-gamma production to whole gliadin. In particular, a purified alpha-gliadin was able to suppress the immune response to the entire gliadin mixture. These results demonstrate how an immune response to a complex antigen may be controlled by treatment with a purified component and specifically indicate alpha-gliadin to be a good candidate for further identification of short peptides to be used as tolerogens in this model.

Tissue transglutaminase is the target in both rodent and primate tissues for celiac disease-specific autoantibodies.

BACKGROUND: Endomysial antibodies have recently been shown to react with tissue transglutaminase. This study was undertaken to investigate whether the tissue distribution of transglutaminase is also compatible with reticulin, jejunal, and fibroblast autoantibody binding patterns. METHODS: Sera from patients with and without celiac disease, monoclonal tissue transglutaminase antibodies, and sera from mice parenterally immunized against commercially available tissue transglutaminase, transglutaminase complexed with gliadin, or gliadin were used in indirect immunofluorescence and double-staining studies using both rodent and primate tissues as substrates. Also, antibody competition, affinity chromatography, and potassium thiocyanate extraction studies were undertaken. RESULTS: Tissue transglutaminase antibody binding patterns were identical with the extracellular binding patterns seen with celiac patient sera. Human umbilical cord-derived fibroblasts exhibited both cytoplasmic and extracellular matrix staining. Double staining with patients' sera and tissue transglutaminase antibodies showed complete overlapping. Tissue transglutaminase effectively absorbed reticulin-endomysial antibodies from celiac sera, and patients' sera blocked the staining of the monoclonal tissue transglutaminase antibodies. Potassium thiocyanate extraction abolished the staining patterns, but they were elicited again after readdition of tissue transglutaminase. CONCLUSIONS: Reticulin, endomysial, and jejunal antibodies detect transglutaminase in both rodent and primate tissues, indicating that these tissue autoantibodies are identical.

Intravenous or intranasal administration of gliadin is able to down-regulate the specific immune response in mice.

The mucosal lesion in coeliac disease (CD) represents an immunologically mediated injury triggered by gliadin and is restricted by a particular assortment of major histocompatibility complex (MHC) class II genes. Therefore, immunomodulatory strategies to tolerize gliadin-specific, class II-restricted T-cell responses could represent an alternative to current treatments of CD, which are based on a gluten-free diet. In this study, BALB/c mice derived from a gluten-free diet colony were tolerized by either intranasal (i.n.) or intravenous (i.v.) administration of single or multiple doses of gliadin. While a single dose failed to induce tolerance, a significant decrease in gliadin-specific T-cell proliferation was detected (P < 0.001) after multiple i.n. or i.v. administrations. No significant difference in antibody titre was detected for antigen-specific immunoglobulin G (IgG) or the IgG1 subclass, but a lower IgG2a-specific titre was observed. Both interferon-gamma (IFN-gamma) and interleukin (IL)-2 expression, measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR), were reduced on antigen administration, both i.v. and i.n. Neither regimen showed a regulatory effect on IL-4 production. As T helper 1 (Th1) cytokines seem to be important in the pathogenesis of CD, our data therefore highlight the potential of i.n. and i.v. routes for the design of useful immunomodulatory strategies for CD.

IgA antibodies to tissue transglutaminase: An effective diagnostic test for celiac disease.

OBJECTIVE: Tissue transglutaminase (tTG) is the main autoantigen recognized by endomysial antibodies. The aim of this study was to assess sensitivity, specificity, and predictive value of IgA and IgG antibodies to tTG in the diagnosis of celiac disease compared with endomysial antibodies. STUDY DESIGN: We established enzyme-linked immunosorbent assay procedures to measure IgA and IgG antibodies to tTG in sera from 48 untreated and 33 treated patients with celiac disease and from 63 patients with gastrointestinal disease who were in a control group. Sera from 10 patients with celiac disease were examined at various times after gluten was reintroduced into the patients' diet. RESULTS: Both IgA and IgG to tTG were significantly (P <.001) higher in serum of untreated patients with celiac disease versus those in the control group; IgA but not IgG was significantly (P <.001) higher in untreated versus treated patients with celiac disease. IgA and IgG antitissue tTG had a diagnostic sensitivity, specificity, and positive predictive value of 92% and 21%, 98% and 97%, and 98% and 83%, respectively. The concordance rate of IgA anti-tTG with IgA antiendomysial antibodies was 95%. In 5 of the 10 patients undergoing gluten challenge, IgA antiendomysium antibodies were detected earlier than IgA anti-tTG antibodies. CONCLUSIONS: tTG-based enzyme-linked immunosorbent assay is an effective diagnostic test, although immunofluorescent-based assays are more sensitive, particularly during gluten challenge.

Coeliac disease in the year 2000.

Coeliac disease is a permanent intolerance to wheat gliadins and related prolamines. Patients who have an obvious malabsorption syndrome form only a small minority of the total number of people with coeliac disease. There are, in fact, no pathognomonic clinical features, and the condition is defined and diagnosed by the presence of pathological changes in the small bowel mucosa related to the presence of toxic prolamines. Susceptibility to coeliac disease is determined to a significant extent by genetic factors. A large part of the genetic susceptibility maps to the HLA region on chromosome 6, as approximately 95% of coeliac disease patients carry an almost identical HLA DQ2/heterodimer; a role of non-HLA genes has also been postulated. From a pathogenetic point of view, most evidence supports the notion of a DQ-restricted gluten-specific Th1 response in the lamina propria; nonetheless, it is possible that, in coeliac subjects, gluten, prior to T cell activation, could exert a direct toxic effect leading to the production of proinflammatory signals.

Membrane events and ionic processes involved in dopamine release from tuberoinfundibular neurons. I. Effect of the inhibition of the Na+,K+-adenosine triphosphatase pump by ouabain.

In the present study we investigated the membrane events and the ionic processes which mediate the stimulatory effect of ouabain on the release of endogenous dopamine (DA) and "previously taken-up" [3H]DA release from rat hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons. Ouabain (0.1-1 mM) dose-dependently stimulated endogenous DA and "newly taken-up" [3H]DA release. This effect was counteracted partially by nomifensine (10 microM). Removal of Ca++ ions from the extracellular space in the presence of the Ca++-chelator ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid prevented completely ouabain-elicited [3H]DA release. Lanthanum (1 mM) and cobalt (2 mM), two inorganic Ca++-entry blockers, were able to inhibit this stimulatory effect, whereas verapamil (10 microM) and nitrendipine (50 microM), two organic antagonists of the voltage-operated channel for Ca++ ions, failed to affect ouabain-induced [3H]DA release. By contrast, adriamycin (100-300 microM), a putative inhibitor of cardiac Na+-Ca++ antiporter, dose-dependently prevented ouabain-induced [3H]DA release from TIDA neurons. Finally, tetrodotoxin reduced digitalis-stimulated [3H]DA release. In conclusion, these results seem to be compatible with the idea that the inhibition of Na+,K+-adenosine triphosphatase by ouabain stimulates the release of [3H]DA from a central neuronal system like the TIDA tract and that this effect is critically dependent on the entrance of Ca++ ions into the nerve terminals of these neurons. In addition the Na+-Ca++ exchange antiporter appears to be the membrane system which transports Ca++ ions into the neuronal cytoplasm during Na+,K+-adenosine triphosphatase inhibition. The enhanced intracellular Ca++ availability triggers DA release which could occur partially through a carrier-dependent process.

Lack of evidence for an impairment of tuberoinfundibolar dopaminergic neurons in aged male rats of the Sprague-Dawley strain.

Circulating prolactin (PRL) levels, dopamine (DA) content, in vitro basal and stimulus-evoked endogenous DA release from arcuate-periventricular nuclei median-eminence fragments were studied in young (4 months) and old (24-25 months) male rats of Sprague-Dawley strain. Serum PRL levels did not differ in young and aged animals. In addition DA tissue content, basal and K+- or d-amphetamine evoked endogenous DA release did not show age-related differences. These results suggest that in male rats of the Sprague-Dawley strain the activity of tuberoinfundibular dopaminergic (TIDA) neurons does not change during senescence, unlike what happens in other strains of rats.

Possible inhibitory role of histamine H2 receptors in the control of basal TSH secretion in male rats: studies with dimaprit, a selective H2 receptor agonist.

Dimaprit,a selective H2 receptor agonist, induced a dose-related decrease of serum TSH basal levels in male rats. This effect was blocked in animals pretreated with cimetidine, a blocker of H2 receptors. Furthermore, dimaprit, added to the anterior pituitary in vitro, failed to induce any change of TSH release. These results are consistent with the hypothesis of an inhibitory role played by H2 receptors located on the median eminence in the control of TSH secretion in male rats.