Influence of Dietary Inulin on Fecal Microbiota, Cardiometabolic Risk Factors, Eicosanoids, and Oxidative Stress in Rats Fed a High-Fat Diet.

The present study examined the influence of inulin on fecal microbiota, cardiometabolic risk factors, eicosanoids, and oxidative stress in rats on a high-fat (HF) diet. Thirty-six male Wistar-Kyoto rats were divided into three dietary groups: standard diet, HF diet, and HF diet + Inulin diet. After 10 weeks, the HF + Inulin diet promoted high dominance of a few bacterial genera including Blautia and Olsenella in feces while reducing richness, diversity, and rarity compared to the HF diet. These changes in fecal microbiota were accompanied by an increased amount of propionic acid in feces. The HF + Inulin diet decreased cardiometabolic risk factors, decreased the amount of the eicosanoids 11(12)-EET and 15-HETrE in the liver, and decreased oxidative stress in blood compared to the HF diet. In conclusion, increasing consumption of inulin may be a useful nutritional strategy to protect against the onset of obesity and its associated metabolic abnormalities by means of modulation of gut microbiota.

Effects of a Fish Oil Rich in Docosahexaenoic Acid on Cardiometabolic Risk Factors and Oxidative Stress in Healthy Rats.

Omega-3 polyunsaturated fatty acids are associated with a lower risk of cardiometabolic diseases. However, docosahexaenoic acid (DHA) is easily oxidized, leading to cellular damage. The present study examined the effects of an increased concentration of DHA in fish oil (80% of total fatty acids) on cardiometabolic risk factors and oxidative stress compared to coconut oil, soybean oil, and fish oil containing eicosapentaenoic acid (EPA) and DHA in a balanced ratio. Forty healthy male Sprague-Dawley rats were supplemented with corresponding oil for 10 weeks. Supplementation with the fish oil containing 80% DHA decreased plasma fat, plasma total cholesterol and muscle fat compared to the coconut oil and the soybean oil. Increasing concentrations of DHA induced incorporation of DHA and EPA in cell membranes and tissues along with a decrease in ω-6 arachidonic acid. The increase in DHA promoted lipid peroxidation, protein carbonylation and antioxidant response. Taken together, the increased concentration of DHA in fish oil reduced fat accumulation compared to the coconut oil and the soybean oil. This benefit was accompanied by high lipid peroxidation and subsequent protein carbonylation in plasma and in liver. In our healthy framework, the slightly higher carbonylation found after receiving fish oil containing 80% DHA might be a protecting mechanism, which fit with the general improvement of antioxidant defense observed in those rats.

The Effects of the Combination of Buckwheat D-Fagomine and Fish Omega-3 Fatty Acids on Oxidative Stress and Related Risk Factors in Pre-Obese Rats.

The combined supplementation of buckwheat D-fagomine (FG) and fish omega-3 polyunsaturated fatty acids (ω-3 PUFA) attenuates the development of insulin resistance in rats fed a high-fat (HF) diet. This study aimed to examine the effects of combined supplementation with FG and ω-3 PUFA on dyslipidemia, transaminases, interleukin-6, and oxidative stress. Forty-five male Sprague-Dawley rats were fed a standard diet, an HF diet, an HF diet supplemented with FG, an HF diet supplemented with ω-3 PUFA, or an HF diet supplemented with FG and ω-3 PUFA for 21 weeks. Triacylglycerol, cholesterol, aspartate aminotransferase, alanine aminotransferase, and interleukin-6 were measured. The assessment of oxidative stress included plasma antioxidant capacity, antioxidant enzyme activities, glutathione content, lipid peroxidation, and protein carbonylation. The combined supplementation with FG and ω-3 PUFA did not attenuate the slight accumulation of liver cholesterol induced by the HF diet but normalized the plasma alanine aminotransferase activity. Rats fed the HF diet supplemented with the combination showed a lower amount of plasma interleukin-6 than those fed a standard diet. The combination attenuated oxidative damage induced by the HF diet, decreased antioxidant enzyme activities, and enhanced glutathione status. The beneficial effects of the combination of FG and ω-3 PUFA on oxidative stress and related risk factors in pre-obese rats were mainly modulated by ω-3 PUFA.

Hepatic metabolic adaptation and adipose tissue expansion are altered in mice with steatohepatitis induced by high-fat high sucrose diet.

BACKGROUND: Obesity is a chronic progressive disease with several metabolic alterations. Nonalcoholic fatty liver disease (NAFLD) is an important comorbidity of obesity that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis or hepatocarcinoma. This study aimed at clarifying the molecular mechanisms underlying the metabolic alterations in hepatic and adipose tissue during high-fat high-sucrose diet-induced NAFLD development in mice. METHODS: Twenty-four male mice (C57BL/6J) were randomly allocated into 3 groups (n = 8 mice per group) to receive a chow diet, a high-fat diet (HFD), or a high-fat high-sucrose diet (HF-HSD) for 20 weeks. At sacrifice, liver and adipose tissue were obtained for histopathological, metabolomic, and protein expression analyses. RESULTS: HF-HSD (but not HFD) was associated with NASH and increased oxidative stress. These animals presented an inhibition of hepatic autophagy and alterations in AMP-activated protein kinase/mammalian target of rapamycin activity. We also observed that the ability of metabolic adaptation was adversely affected by the increase of damaged mitochondria. NASH development was associated with changes in adipose tissue dynamics and increased amounts of saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids in visceral adipose tissue. CONCLUSION: HF-HSD led to a metabolic blockage and impaired hepatic mitochondria turnover. In addition, the continuous accumulation of fatty acids produced adipose tissue dysfunction and hepatic fat accumulation that favored the progression to NASH.

Effectiveness of specific stabilization exercise compared with traditional trunk exercise in women with non-specific low back pain: a pilot randomized controlled trial.

BACKGROUND: Non-specific low back pain (LBP) is the leading cause of disability worldwide. The primary physiotherapeutic treatment for LBP is physical exercise, but evidence suggesting a specific exercise as most appropriate for any given case is limited. OBJECTIVE: To determine if specific stabilization exercise (SSE) is more effective than traditional trunk exercise (TTE) in reducing levels of pain, disability and inflammation in women with non-specific low back pain (LBP). DESIGN: A pilot randomized controlled trial was conducted in Rovira i Virgili University, Catalonia. METHODS: Thirty-nine females experiencing non-specific LBP were included in two groups: the TTE program and SSE program, both were conducted by a physiotherapist during twenty sessions. The primary outcome was pain intensity (10-cm Visual Analogue Scale). Secondary outcomes were disability (Roland Morris Disability Questionnaire), and inflammation (IL-6 and TNF-α plasma levels). Measurements were taken at baseline, at half intervention, at post-intervention, and a month later. RESULTS: Mean group differences in change from baseline to post-intervention for TTE were: -4.5 points (CI 3.3 to 5.6) for pain, -5.1 points (CI 3.0 to 7.3) for disability, 0.19 pg/mL (95% CI [-1.6-1.2]) for IL-6 levels, and 46.2 pg/mL (CI 13.0 to 85.3) for TNF-α levels. For SSE, differences were: -4.3 points (CI 3.1 to 5.6) for pain, -6.1 points (CI 3.7 to 8.6) for disability, 1.1 pg/mL (CI 0.0 to 2.1) for IL-6 levels , and 12.8 pg/mL (95% CI [-42.3-16.7]) for TNF-α levels. There were an insignificant effect size and no statistically significant overall mean differences between both groups. CONCLUSION: This study suggests that both interventions (traditional trunk and specific stabilization exercises) are effective in reducing pain and disability in non-specific LBP patients, but the two programs produce different degrees of inflammation change. CLINICAL TRIAL REGISTRATION NUMBER: NCT02103036.

Effects of Fish Oil and Grape Seed Extract Combination on Hepatic Endogenous Antioxidants and Bioactive Lipids in Diet-Induced Early Stages of Insulin Resistance in Rats.

Diacylglycerols (DAG) and ceramides have been suggested as early predictors of insulin resistance. This study was aimed to examine the combined effects of fish oil (FO) and grape seed extract (GSE) on hepatic endogenous antioxidants, DAG and ceramides in diet-induced early stages of insulin resistance. Thirty-five rats were fed one of the following diets: (1) a standard diet (STD group), (2) a high-fat high-sucrose diet (HFHS group), (3) an HFHS diet enriched with FO (FO group), (4) an HFHS diet enriched with GSE (GSE group) or (5) an HFHS diet enriched with FO and GSE (FO + GSE group). In the liver, endogenous antioxidants were measured using spectrophotometric and fluorometric techniques, and non-targeted lipidomics was conducted for the assessment of DAG and ceramides. After 24 weeks, the FO + GSE group showed increased glutathione peroxidase activity, as well as monounsaturated fatty acid and polyunsaturated fatty acid-containing DAG, and long-chain fatty acid-containing ceramides abundances compared to the STD group. The FO and GSE combination induced similar activation of the antioxidant system and bioactive lipid accumulation in the liver than the HFHS diet without supplementation. In addition, the FO and GSE combination increased the abundances of polyunsaturated fatty acid-containing DAG in the liver.

Effects of combined D-fagomine and omega-3 PUFAs on gut microbiota subpopulations and diabetes risk factors in rats fed a high-fat diet.

Food contains bioactive compounds that may prevent changes in gut microbiota associated with Westernized diets. The aim of this study is to explore the possible additive effects of D-fagomine and ω-3 PUFAs (EPA/DHA 1:1) on gut microbiota and related risk factors during early stages in the development of fat-induced pre-diabetes. Male Sprague Dawley (SD) rats were fed a standard diet, or a high-fat (HF) diet supplemented with D-fagomine, EPA/DHA 1:1, a combination of both, or neither, for 24 weeks. The variables measured were fasting glucose and glucose tolerance, plasma insulin, liver inflammation, fecal/cecal gut bacterial subgroups and short-chain fatty acids (SCFAs). The animals supplemented with D-fagomine alone and in combination with ω-3 PUFAs accumulated less fat than those in the non-supplemented HF group and those given only ω-3 PUFAs. The combined supplements attenuated the high-fat-induced incipient insulin resistance (IR), and liver inflammation, while increasing the cecal content, the Bacteroidetes:Firmicutes ratio and the populations of Bifidobacteriales. The functional effects of the combination of D-fagomine and EPA/DHA 1:1 against gut dysbiosis and the very early metabolic alterations induced by a high-fat diet are mainly those of D-fagomine complemented by the anti-inflammatory action of ω-3 PUFAs.

Modulation of the Liver Protein Carbonylome by the Combined Effect of Marine Omega-3 PUFAs and Grape Polyphenols Supplementation in Rats Fed an Obesogenic High Fat and High Sucrose Diet.

Diet-induced obesity has been linked to metabolic disorders such as cardiovascular diseases andtype 2 diabetes. A factor linking diet to metabolic disorders is oxidative stress, which can damagebiomolecules, especially proteins. The present study was designed to investigate the effect of marineomega-3 polyunsaturated fatty acids (PUFAs) (eicosapentaenoic acid (EPA) and docosahexaenoic acid(DHA)) and their combination with grape seed polyphenols (GSE) on carbonyl-modified proteins fromplasma and liver in Wistar Kyoto rats fed an obesogenic diet, namely high-fat and high-sucrose (HFHS)diet. A proteomics approach consisting of fluorescein 5-thiosemicarbazide (FTSC) labelling of proteincarbonyls, visualization of FTSC-labelled protein on 1-DE or 2-DE gels, and protein identification byMS/MS was used for the protein oxidation assessment. Results showed the efficiency of the combinationof both bioactive compounds in decreasing the total protein carbonylation induced by HFHS diet in bothplasma and liver. The analysis of carbonylated protein targets, also referred to as the 'carbonylome',revealed an individual response of liver proteins to supplements and a modulatory effect on specificmetabolic pathways and processes due to, at least in part, the control exerted by the supplements on theliver protein carbonylome. This investigation highlights the additive effect of dietary fish oils and grapeseed polyphenols in modulating in vivo oxidative damage of proteins induced by the consumption ofHFHS diets.

Targeting Hepatic Protein Carbonylation and Oxidative Stress Occurring on Diet-Induced Metabolic Diseases through the Supplementation with Fish Oils.

The present study addressed the ability of long-chain ω-3 polyunsaturated fatty acids (ω-3 PUFA), i.e., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), to ameliorate liver protein damage derived from oxidative stress and induced by consumption of high-caloric diets, typical of Westernized countries. The experimental design included an animal model of Sprague-Dawley rats fed high-fat high-sucrose (HFHS) diet supplemented with ω-3 EPA and DHA for a complete hepatic proteome analysis to map carbonylated proteins involved in specific metabolic pathways. Results showed that the intake of marine ω-3 PUFA through diet significantly decreased liver protein carbonylation caused by long-term HFHS consumption and increased antioxidant system. Fish oil modulated the carbonylation level of more than twenty liver proteins involved in critical metabolic pathways, including lipid metabolism (e.g., albumin), carbohydrate metabolism (e.g., pyruvate carboxylase), detoxification process (e.g., aldehyde dehydrogenase 2), urea cycle (e.g., carbamoyl-phosphate synthase), cytoskeleton dynamics (e.g., actin), or response to oxidative stress (e.g., catalase) among others, which might be under the control of diet marine ω-3 PUFA. In parallel, fish oil significantly changed the liver fatty acid profile given by the HFHS diet, resulting in a more anti-inflammatory phenotype. In conclusion, the present study highlights the significance of marine ω-3 PUFA intake for the health of rats fed a Westernized diet by describing several key metabolic pathways which are protected in liver.

Mechanistically different effects of fat and sugar on insulin resistance, hypertension, and gut microbiota in rats.

Insulin resistance (IR) and impaired glucose tolerance (IGT) are the first manifestations of diet-induced metabolic alterations leading to Type 2 diabetes, while hypertension is the deadliest risk factor of cardiovascular disease. The roles of dietary fat and fructose in the development of IR, IGT, and hypertension are controversial. We tested the long-term effects of an excess of fat or sucrose (fructose/glucose) on healthy male Wistar-Kyoto (WKY) rats. Fat affects IR and IGT earlier than fructose through low-grade systemic inflammation evidenced by liver inflammatory infiltration, increased levels of plasma IL-6, PGE2, and reduced levels of protective short-chain fatty acids without triggering hypertension. Increased populations of gut Enterobacteriales and Escherichia coli may contribute to systemic inflammation through the generation of lipopolysaccharides. Unlike fat, fructose induces increased levels of diacylglycerols (lipid mediators of IR) in the liver, urine F2-isoprostanes (markers of systemic oxidative stress), and uric acid, and triggers hypertension. Elevated populations of Enterobacteriales and E. coli were only detected in rats given an excess of fructose at the end of the study. Dietary fat and fructose trigger IR and IGT in clearly differentiated ways in WKY rats: early low-grade inflammation and late direct lipid toxicity, respectively; gut microbiota plays a role mainly in fat-induced IR, and hypertension is independent of inflammation-mediated IR. The results provide evidence that suggests that the combination of fat and sugar is potentially more harmful than fat or sugar alone when taken in excess.

Effects of the combination of ω-3 PUFAs and proanthocyanidins on the gut microbiota of healthy rats.

ω-3 Polyunsaturated fatty acids (PUFAs) reduce risk factors for cardiovascular diseases (CVD) and other pathologies that involve low-grade inflammation. They have recently been shown to exert complementary functional effects with proanthocyanidins. As the reduction of health-promoting gut bacteria such as lactobacilli and bifidobacteria has been linked to a number of alterations in the host, the aim of this study was to determine whether PUFAs and proanthocyanidins also cooperate in maintaining well-balanced microbiota. To this end, rats were supplemented for 6months with eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) 1:1 (16.6g/kg feed); proanthocyanidin-rich grape seed extract (GSE, 0.8g/kg feed); or both. Plasma adiponectin, cholesterol, and urine nitrites were measured. Gut bacterial subgroups were evaluated in fecal DNA by qRT-PCR. Short-chain fatty acids (SCFAs) were determined in feces by gas chromatography. Body and adipose tissue weights were found to be higher in the animals given ω-3 PUFAs, while their energy intake was lower. Plasma cholesterol was lower in ω-3 PUFA supplemented groups, while adiponectin and urine nitrites were higher. ω-3 PUFAs reduced the population of Lactobacillales and L. acidophilus after 6months of supplementation. GSE significantly reduced L. plantarum and B. longum. The combination of ω-3 PUFAs and GSE maintained the health-promoting bacteria at levels similar to those of the control group. Acetic acid was increased by the ω-3 PUFA individual supplementation, while the combination with GSE kept this value similar to the control value. In conclusion, while individual supplementations with ω-3 PUFAs or GSE modify the populations of Lactobacillus, Bifidobacterium and microbial products (SCFAs), their combination maintains the standard proportions of these bacterial subgroups and their function while also providing the cardiovascular benefits of ω-3 PUFAs.

A lipidomic study on the regulation of inflammation and oxidative stress targeted by marine ω-3 PUFA and polyphenols in high-fat high-sucrose diets.

The ability of polyphenols to ameliorate potential oxidative damage of ω-3 PUFAs when they are consumed together and then, to enhance their potentially individual effects on metabolic health is discussed through the modulation of fatty acids profiling and the production of lipid mediators. For that, the effects of the combined consumption of fish oils and grape seed procyanidins on the inflammatory response and redox unbalance triggered by high-fat high-sucrose (HFHS) diets were studied in an animal model of Wistar rats. A standard diet was used as control. Results suggested that fish oils produced a replacement of ω-6 by ω-3 PUFAs in membranes and tissues, and consequently they improved inflammatory and oxidative stress parameters: favored the activity of 12/15-lipoxygenases on ω-3 PUFAs, enhanced glutathione peroxidases activity, modulated proinflammatory lipid mediators synthesis through the cyclooxygenase (COX) pathways and down-regulated the synthesis de novo of ARA leaded by Δ5 desaturase. Although polyphenols exerted an antioxidative and antiinflammatory effect in the standard diet, they were less effective to reduce inflammation in the HFHS dietary model. Contrary to the effect observed in the standard diet, polyphenols up-regulated COX pathways toward ω-6 proinflammatory eicosanoids as PGE2 and 11-HETE and decreased the detoxification of ω-3 hydroperoxides in the HFHS diet. As a result, additive effects between fish oils and polyphenols were found in the standard diet in terms of reducing inflammation and oxidative stress. However, in the HFHS diets, fish oils seem to be the one responsible for the positive effects found in the combined group.

Lipidomics to analyze the influence of diets with different EPA:DHA ratios in the progression of Metabolic Syndrome using SHROB rats as a model.

The role of specific proportions of ω-3 EPA and DHA, in the modulation of inflammation and oxidative stress markers associated to the progression of Metabolic Syndrome was investigated. Potential inflammatory eicosanoids and docosanoids were discussed together to biomarkers of CVD, obesity, inflammation and oxidative stress in an animal model of metabolic disorders. Results evidenced a noteworthy health effect of 1:1 and 2:1 EPA:DHA proportions over 1:2 EPA:DHA based diets through a down-regulation in the production of strong pro-inflammatory ω-6 eicosanoids, a decrement of biomarkers of oxidative stress, and a modulation of fatty acid desaturase activities and plasma and membrane PUFAs towards greater anti-inflammatory profiles. Outcomes contribute to the general knowledge on the health benefits of marine lipids and their role on the progress of MetS, inflammation and oxidative stress. Results shed light on controversial protective mechanisms of EPA and DHA to better design dietary interventions aimed at reducing MetS.

D-Fagomine attenuates metabolic alterations induced by a high-energy-dense diet in rats.

d-Fagomine is a natural iminosugar that counteracts the short-term effects of a high-energy-dense diet on body weight, fasting blood glucose levels and the proportion of gut Enterobacteriales. This suggests that supplementation with d-fagomine for longer periods may delay the onset of other factors related to metabolic syndrome. Here we evaluate the effects of d-fagomine dietary supplementation on relevant metabolic hormones and lipid peroxidation. Adult Sprague-Dawley rats were fed a high-fat high-sucrose diet supplemented or not with d-fagomine (0.065% w/w) for 9 weeks. Weight gain, plasma triglycerides, glucose, insulin, glucagon, ghrelin, leptin, and urine F2-isoprostanes were evaluated. d-Fagomine attenuated the changes induced by the high-energy-dense diet in triglycerides and all the hormones tested. These results suggest that d-fagomine may help to avert the complications associated with unhealthy eating by counteracting the effects of high-energy-dense diets during the early stages of the development of metabolic disorders.

Diet, iron biomarkers and oxidative stress in a representative sample of Mediterranean population.

BACKGROUND: The consumption pattern characterized by high consumption of vegetables, fruit, fish, olive oil and red wine has been associated with improvements in the total antioxidant capacity of individuals and reduced incidence of diseases related to oxidation. Also, high body iron levels may contribute to increase the oxidative stress by the generation of reactive oxygen species. The objective of this study is to analyze the relationship between antioxidant and pro-oxidant factors obtained from the diet and iron biomarkers on lipoprotein oxidation and total antioxidant capacity in a representative sample of the Mediterranean population. METHODS: Cross-sectional prospective study, carried out with 815 randomly selected subjects (425 women and 390 men). Dietary assessment (3-day food records), iron biomarkers (serum ferritin, serum iron and transferrin saturation), biochemical markers of lipoperoxidation (TBARS), antioxidant capacity (ORAC) and CRP (C-Reactive Protein) were determined. Multiple Linear Regression (MLR) models were applied to analyze the association between diet factors and iron biomarkers on TBARS and ORAC levels. RESULTS: We observed that lipoperoxidation measured by TBARS increased by age but no differences were observed by sex. Antioxidant capacity measured by ORAC is independent of age and sex. In general, increasing age, tobacco, heme iron intake from meat and fish and transferrin saturation were independently and positively associated with TBARS, while non-heme iron was negatively associated. Vegetables, vitamin C intake and serum ferritin were positively associated with ORAC, whereas saturated fatty acids and meat intake were negatively associated. CONCLUSIONS: In our general population, we observed that oxidative stress is related to aging, but antioxidant capacity is not. The highest intake of dietary non-heme iron, vegetables and vitamin C intake exerts a protective effect against oxidation while the highest intake of dietary heme iron from meat and fish and saturated fatty acids are associated with increased oxidative stress. High levels of circulating iron measured by transferrin saturation are associated with increased oxidative stress in women however its association with the higher levels of serum ferritin is controversial.

Polypodium leucotomos extract inhibits glutathione oxidation and prevents Langerhans cell depletion induced by UVB/UVA radiation in a hairless rat model.

In this report, we have addressed the effect of oral administration of a hydrophilic extract of the fern Polypodium leucotomos (PL) on the deleterious effects of ultraviolet radiation (UVR) on the levels of epidermal and plasmatic antioxidants in hairless rats. We have found that pretreatment with PL effectively reduced glutathione oxidation in both blood and epidermis, suggesting a potent systemic antioxidant effect. In addition, PL inhibited UVR-mediated Langerhans cell (LC) depletion. Our results demonstrate the efficacy of PL as an oral antioxidant and photoimmunoprotective agent and support its employment as a complement to topical sunscreens.

Oxidative stress-related markers and langerhans cells in a hairless rat model exposed to UV radiation.

Biomarkers related to the oxidative stress in blood and epidermis and the number of Langerhans cells were determined in hairless rats after acute irradiation with 1.54, 1.93, or 2.41 J/cm2 of ultraviolet (UV) light and chronic exposure to 13 suberythemal UV doses of 1.1 J/cm2 for 2 mo. After acute UV irradiation, in epidermis, the thiobarbituric acid-reactive substances (TBARS) content increased at the highest UV dose, whereas the activities of glutathione S-transferase and catalase rose and the oxidized glutathione (GSSG) content diminished at all UV doses. In erythrocytes, glutathione S-transferase activity increased at the two lowest UV doses, glutathione peroxidase activity rose at all UV doses, and catalase activity increased after the highest UV dose. In plasma, the TBARS content and the reduced glutathione (GSH)/GSSG ratio increased at the highest UV dose; the number of Langerhans cells decreased at all UV doses. Linear Pearson correlation analysis revealed many relationships between different biomarkers, and multiple linear regression analysis indicated that the number of Langerhans cells was predicted by epidermal GSSG and catalase (R2 = .64) and by erythrocytic glutathione peroxidase and GSSG (R2 = .72). After suberythemal UV radiation, in epidermis, the GST activity and the content of GSH and GSSG increased; in erythrocytes, the GST activity decreased and the GSH/GSSG ratio increased. Thus, the hairless rat appears to be a useful model for studying the oxidative stress-related mechanisms after UV radiation, which are involved in the loss of the immune capacity mediated by Langerhans cells, even at suberythemal doses.