IL-11 Induces Th17 Cell Responses in Patients with Early Relapsing-Remitting Multiple Sclerosis.

Clinically isolated syndrome (CIS) suggestive of multiple sclerosis (MS) is the earliest clinically evident phase of the disease, which may provide valuable insight into the molecular mechanisms of the initiation of the autoimmune response in MS. Our results introduce IL-11 as a new cytokine that plays a role in the autoimmune response in the early phase of the disease. IL-11 is the highest upregulated cytokine in the sera and cerebrospinal fluid from CIS patients, which is also increased in patients with clinically definitive relapsing-remitting MS in comparison with healthy control subjects. Serum IL-11 levels are significantly increased during clinical exacerbations in comparison with remissions in the same patients. CD4(+) cells represent a predominant cell source of IL-11 in the peripheral circulation, and the percentage of IL-11(+)CD4(+) cells is significantly increased in CIS patients in comparison with healthy control subjects. Furthermore, we have identified IL-11 as a new Th17-promoting cytokine, because it induces a differentiation of naive CD4(+) T cells into Th17 cells, as well as expansion of Th17 memory cells. Because the Th17 cytokines IL-17F, IL-21 and TNF-α, and TGF-ß induce differentiation of naive cells in the IL-11-secreting CD4(+) cells, we propose that cross-talk between IL-11(+)CD4(+) and Th17 cells may play a role in the inflammatory response in relapsing-remitting MS.

The role of endogenous IFN-ß in the regulation of Th17 responses in patients with relapsing-remitting multiple sclerosis.

IFN-ß has been used as a first-line therapy for relapsing-remitting multiple sclerosis (RRMS). Because only a few studies have addressed the role of endogenous IFN-ß in the pathogenesis of the disease, our objective was to characterize its role in the transcriptional regulation of pathogenic Th17 cytokines in patients with RRMS. In vitro studies have demonstrated that IFN-ß inhibits IL-17A, IL-17F, IL-21, IL-22, and IFN-γ secretion in CD4(+) lymphocytes through the induction of suppressor of cytokine secretion 1 and suppressor of cytokine secretion 3. We found that patients with RRMS have increased serum and cerebrospinal fluid Th17 (IL-17A and IL-17F) cytokine levels in comparison with the control subjects, suggesting that deficient endogenous IFN-ß secretion or signaling can contribute to the dysregulation of those pathogenic cytokines in CD4(+) cells. We identified that the endogenous IFN-ß from serum of RRMS patients induced a significantly lower IFN-inducible gene expression in comparison with healthy controls. In addition, in vitro studies have revealed deficient endogenous and exogenous IFN-ß signaling in the CD4(+) cells derived from patients with MS. Interestingly, upon inhibition of the endogenous IFN-ß signaling by silencing IFN regulatory factor (IRF) 7 gene expression, the resting CD4(+) T cells secreted significantly higher level of IL-17A, IL-17F, IL-21, IL-22, and IL-9, suggesting that endogenous IFN-ß suppresses the secretion of these pathogenic cytokines. In vivo recombinant IFN-ß-1a treatment induced IFNAR1 and its downstream signaling molecules' gene expression, suggesting that treatment reconstitutes a deficient endogenous IFN-ß regulation of the CD4(+) T cells' pathogenic cytokine production in patients with MS.

Atf3 negatively regulates Ptgs2/Cox2 expression during acute inflammation.

By generating prostaglandins, cyclooxygenase-2 (Cox-2/Ptgs2) plays a critical role in regulating inflammatory responses. While several inflammatory stimuli have been shown to increase Ptgs2 expression, less is known about how the transcription of this gene is terminated. Here we show that stimulation of macrophages with yeast zymosan, a TLR2/6 and dectin-1 agonist, causes a transient increase in the expression of Ptgs2 accompanied by a simultaneous increase in the expression of the transcriptional repressor, activating transcription factor-3 (Atf3). The expression of Ptgs2 was significantly higher in resident peritoneal macrophages isolated from Atf3(-/-) mice than that from Atf3(+/+) mice and was associated with higher prostaglandin production upon stimulation with zymosan. In activated macrophages, Atf3 accumulated in the nucleus and chromatin-immunoprecipitation analysis showed that Atf3 is recruited to the Ptgs2 promoter region. In acute peritonitis and in cutaneous wounds, there was increased leukocyte accumulation and higher levels of prostaglandins (PGE2/PGD2) in inflammatory exudates of Atf3(-/-) mice compared with WT mice. Collectively, these results demonstrate that during acute inflammation Atf3 negatively regulates Ptgs2 and therefore dysregulation of this axis could potentially contribute to aberrant Ptgs2 expression in chronic inflammatory diseases. Moreover, this axis could be a new therapeutic target for suppressing Ptgs2 expression and the resultant inflammatory responses.

Increased saturated fatty acids in obesity alter resolution of inflammation in part by stimulating prostaglandin production.

Extensive evidence indicates that nutrient excess associated with obesity and type 2 diabetes activates innate immune responses that lead to chronic, sterile low-grade inflammation, and obese and diabetic humans also have deficits in wound healing and increased susceptibility to infections. Nevertheless, the mechanisms that sustain unresolved inflammation during obesity remain unclear. In this study, we report that saturated free fatty acids that are elevated in obesity alter resolution of acute sterile inflammation by promoting neutrophil survival and decreasing macrophage phagocytosis. Using a targeted mass spectrometry-based lipidomics approach, we found that in db/db mice, PGE2/D2 levels were elevated in inflammatory exudates during the development of acute peritonitis. Moreover, in isolated macrophages, palmitic acid stimulated cyclooxygenase-2 induction and prostanoid production. Defects in macrophage phagocytosis induced by palmitic acid were mimicked by PGE2 and PGD2 and were reversed by cyclooxygenase inhibition or prostanoid receptor antagonism. Macrophages isolated from obese-diabetic mice expressed prostanoid receptors, EP2 and DP1, and contained significantly higher levels of downstream effector, cAMP, compared with wild-type mice. Therapeutic administration of EP2/DP1 dual receptor antagonist, AH6809, decreased neutrophil accumulation in the peritoneum of db/db mice, as well as the accumulation of apoptotic cells in the thymus. Taken together, these studies provide new insights into the mechanisms underlying altered innate immune responses in obesity and suggest that targeting specific prostanoid receptors may represent a novel strategy for resolving inflammation and restoring phagocyte defects in obese and diabetic individuals.

Metabolic remodeling of white adipose tissue in obesity.

Adipose tissue metabolism is a critical regulator of adiposity and whole body energy expenditure; however, metabolic changes that occur in white adipose tissue (WAT) with obesity remain unclear. The purpose of this study was to understand the metabolic and bioenergetic changes occurring in WAT with obesity. Wild-type (C57BL/6J) mice fed a high-fat diet (HFD) showed significant increases in whole body adiposity, had significantly lower V̇(O2), V̇(CO2), and respiratory exchange ratios, and demonstrated worsened glucose and insulin tolerance compared with low-fat-fed mice. Metabolomic analysis of WAT showed marked changes in lipid, amino acid, carbohydrate, nucleotide, and energy metabolism. Tissue levels of succinate and malate were elevated, and metabolites that could enter the Krebs cycle via anaplerosis were mostly diminished in high-fat-fed mice, suggesting altered mitochondrial metabolism. Despite no change in basal oxygen consumption or mitochondrial DNA abundance, citrate synthase activity was decreased by more than 50%, and responses to FCCP were increased in WAT from mice fed a high-fat diet. Moreover, Pgc1a was downregulated and Cox7a1 upregulated after 6 wk of HFD. After 12 wk of high-fat diet, the abundance of several proteins in the mitochondrial respiratory chain or matrix was diminished. These changes were accompanied by increased Parkin and Pink1, decreased p62 and LC3-I, and ultrastructural changes suggestive of autophagy and mitochondrial remodeling. These studies demonstrate coordinated restructuring of metabolism and autophagy that could contribute to the hypertrophy and whitening of adipose tissue in obesity.

Overexpression of endothelial nitric oxide synthase prevents diet-induced obesity and regulates adipocyte phenotype.

RATIONALE: Endothelial dysfunction is a characteristic feature of diabetes and obesity in animal models and humans. Deficits in nitric oxide production by endothelial nitric oxide synthase (eNOS) are associated with insulin resistance, which is exacerbated by high-fat diet. Nevertheless, the metabolic effects of increasing eNOS levels have not been studied. OBJECTIVE: The current study was designed to test whether overexpression of eNOS would prevent diet-induced obesity and insulin resistance. METHODS AND RESULTS: In db/db mice and in high-fat diet-fed wild-type C57BL/6J mice, the abundance of eNOS protein in adipose tissue was decreased without significant changes in eNOS levels in skeletal muscle or aorta. Mice overexpressing eNOS (eNOS transgenic mice) were resistant to diet-induced obesity and hyperinsulinemia, although systemic glucose intolerance remained largely unaffected. In comparison with wild-type mice, high-fat diet-fed eNOS transgenic mice displayed a higher metabolic rate and attenuated hypertrophy of white adipocytes. Overexpression of eNOS did not affect food consumption or diet-induced changes in plasma cholesterol or leptin levels, yet plasma triglycerides and fatty acids were decreased. Metabolomic analysis of adipose tissue indicated that eNOS overexpression primarily affected amino acid and lipid metabolism; subpathway analysis suggested changes in fatty acid oxidation. In agreement with these findings, adipose tissue from eNOS transgenic mice showed higher levels of PPAR-α and PPAR-γ gene expression, elevated abundance of mitochondrial proteins, and a higher rate of oxygen consumption. CONCLUSIONS: These findings demonstrate that increased eNOS activity prevents the obesogenic effects of high-fat diet without affecting systemic insulin resistance, in part, by stimulating metabolic activity in adipose tissue.

Chronic alcohol exposure stimulates adipose tissue lipolysis in mice: role of reverse triglyceride transport in the pathogenesis of alcoholic steatosis.

Alcohol consumption induces liver steatosis; therefore, this study investigated the possible role of adipose tissue dysfunction in the pathogenesis of alcoholic steatosis. Mice were pair-fed an alcohol or control liquid diet for 8 weeks to evaluate the alcohol effects on lipid metabolism at the adipose tissue-liver axis. Chronic alcohol exposure reduced adipose tissue mass and adipocyte size. Fatty acid release from adipose tissue explants was significantly increased in alcohol-fed mice in association with the activation of adipose triglyceride lipase and hormone-sensitive lipase. Alcohol exposure induced insulin intolerance and inactivated adipose protein phosphatase 1 in association with the up-regulation of phosphatase and tensin homolog (PTEN) and suppressor of cytokine signaling 3 (SOCS3). Alcohol exposure up-regulated fatty acid transport proteins and caused lipid accumulation in the liver. To define the mechanistic link between adipose triglyceride loss and hepatic triglyceride gain, mice were first administered heavy water for 5 weeks to label adipose triglycerides with deuterium, and then pair-fed alcohol or control diet for 2 weeks. Deposition of deuterium-labeled adipose triglycerides in the liver was analyzed using Fourier transform ion cyclotron mass spectrometry. Alcohol exposure increased more than a dozen deuterium-labeled triglyceride molecules in the liver by up to 6.3-fold. These data demonstrate for the first time that adipose triglycerides due to alcohol-induced hyperlipolysis are reverse transported and deposited in the liver.

Deficiency of the leukotriene B4 receptor, BLT-1, protects against systemic insulin resistance in diet-induced obesity.

Chronic inflammation is an underlying factor linking obesity with insulin resistance. Diet-induced obesity promotes an increase in circulating levels of inflammatory monocytes and their infiltration into expanding adipose tissue. Nevertheless, the endogenous pathways that trigger and sustain chronic low-grade inflammation in obesity are incompletely understood. In this study, we report that a high-fat diet selectively increases the circulating levels of CD11b(+) monocytes in wild-type mice that express leukotriene B(4) receptor, BLT-1, and that this increase is abolished in BLT-1-null mice. The accumulation of classically activated (M1) adipose tissue macrophages (ATMs) and the expression of proinflammatory cytokines and chemokines (i.e., IL-6 and Ccl2) was largely blunted in adipose tissue of obese BLT-1(-/-) mice, whereas the ratio of alternatively activated (M2) ATMs to M1 ATMs was increased. Obese BLT-1(-/-) mice were protected from systemic glucose and insulin intolerance and this was associated with a decrease in inflammation in adipose tissue and liver and a decrease in hepatic triglyceride accumulation. Deletion of BLT-1 prevented high fat-induced loss of insulin signaling in liver and skeletal muscle. These observations elucidate a novel role of chemoattractant receptor, BLT-1, in promoting monocyte trafficking to adipose tissue and promoting chronic inflammation in obesity and could lead to the identification of new therapeutic targets for treating insulin resistance in obesity.

The role of glycogen synthase kinase 3 in regulating IFN-ß-mediated IL-10 production.

The ability of IFN-ß to induce IL-10 production from innate immune cells is important for its anti-inflammatory properties and is believed to contribute to its therapeutic value in treating multiple sclerosis patients. In this study, we identified that IFN-ß stimulates IL-10 production by activating the JAK1- and PI3K-signaling pathways. JAK1 activity was required for IFN-ß to activate PI3K and Akt1 that resulted in repression of glycogen synthase kinase 3 (GSK3)-ß activity. IFN-ß-mediated suppression of GSK3-ß promoted IL-10, because IL-10 production by IFN-ß-stimulated dendritic cells (DC) expressing an active GSK3-ß knockin was severely reduced, whereas pharmacological or genetic inhibition of GSK3-ß augmented IL-10 production. IFN-ß increased the phosphorylated levels of CREB and STAT3 but only CREB levels were affected by PI3K. Also, a knockdown in CREB, but not STAT3, affected the capacity of IFN-ß to induce IL-10 from DC. IL-10 production by IFN-ß-stimulated DC was shown to suppress IFN-γ and IL-17 production by myelin oligodendrocyte glycoprotein-specific CD4(+) T cells, and this IL-10-dependent anti-inflammatory effect was enhanced by directly targeting GSK3 in DC. These findings highlight how IFN-ß induces IL-10 production and the importance that IL-10 plays in its anti-inflammatory properties, as well as identify a therapeutic target that could be used to increase the IL-10-dependent anti-inflammatory properties of IFN-ß.

Activation of peroxisome proliferator-activated receptor-γ by rosiglitazone improves lipid homeostasis at the adipose tissue-liver axis in ethanol-fed mice.

The development of alcohol-induced fatty liver is associated with a reduction of white adipose tissue (WAT). Peroxisome proliferator-activated receptor (PPAR)-γ prominently distributes in the WAT and plays a crucial role in maintaining adiposity. The present study investigated the effects of PPAR-γ activation by rosiglitazone on lipid homeostasis at the adipose tissue-liver axis. Adult C57BL/6 male mice were pair fed liquid diet containing ethanol or isocaloric maltose dextrin for 8 wk with or without rosiglitazone supplementation to ethanol-fed mice for the last 3 wk. Ethanol exposure downregulated adipose PPAR-γ gene and reduced the WAT mass in association with induction of inflammation, which was attenuated by rosiglitazone. Ethanol exposure stimulated lipolysis but reduced fatty acid uptake capacity in association with dysregulation of lipid metabolism genes. Rosiglitazone normalized adipose gene expression and corrected ethanol-induced lipid dyshomeostasis. Ethanol exposure induced steatosis and upregulated inflammatory genes in the liver, which were attenuated by rosiglitazone. Hepatic peroxisomal fatty acid ß-oxidation was suppressed by ethanol in associated with inhibition of acyl-coenzyme A oxidase 1. Rosiglitazone elevated plasma adiponectin level and normalized peroxisomal fatty acid ß-oxidation rate. However, rosiglitazone did not affect ethanol-reduced very low-density lipoprotein secretion from the liver. These results demonstrated that activation of PPAR-γ by rosiglitazone reverses ethanol-induced adipose dysfunction and lipid dyshomeostasis at the WAT-liver axis, thereby abrogating alcoholic fatty liver.

Resolvin D1 decreases adipose tissue macrophage accumulation and improves insulin sensitivity in obese-diabetic mice.

Type 2 diabetes and obesity have emerged as global public health crises. Adipose tissue expansion in obesity promotes accumulation of classically activated macrophages that perpetuate chronic inflammation and sustain insulin resistance. Acute inflammation normally resolves in an actively orchestrated series of molecular and cellular events that ensures return to homeostasis after an inflammatory insult, a process regulated in part by endogenous lipid mediators such as the resolvins. In this study, we sought to determine whether stimulating resolution with resolvin D1 (RvD1) improves insulin sensitivity by resolving chronic inflammation associated with obesity. In male leptin receptor-deficient (db/db) mice, treatment with RvD1 (2 µg/kg) improved glucose tolerance, decreased fasting blood glucose, and increased insulin-stimulated Akt phosphorylation in adipose tissue relative to vehicle-treated mice. Treatment with RvD1 increased adiponectin production, while expression of IL-6 in adipose tissue was decreased. The formation of crown-like structures rich in inflammatory F4/80(+)CD11c(+) macrophages was reduced by >50% in adipose tissue by RvD1 and was associated with an increased percentage of F4/80(+) cells expressing macrophage galactose-type C-type lectin 1 (MGL-1), a marker of alternatively activated macrophages. These results suggest that stimulating resolution with the endogenous proresolving mediator RvD1 could provide a novel therapeutic strategy for treating obesity-induced diabetes.

90-day nose-only inhalation toxicity study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Sprague-Dawley rats.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. Repeated dose inhalation toxicity data on NNK, particularly relevant to cigarette smoking, however, is surprisingly limited. Hence, there is a lack of direct information available on the carcinogenic and potential non-carcinogenic effects of NNK via inhalational route exposure. In the present study, the subchronic inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 23 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.2, 0.8, 3.2, or 7.8 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.0066, 0.026, 0.11, or 0.26 mg/L air) for 1 h/day for 90 consecutive days. Toxicity was evaluated by assessing body weights; food consumption; clinical pathology; histopathology; organ weights; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); tissue levels of the DNA adduct O6-methylguanine; blood and bone marrow micronucleus (MN) frequency; and bone marrow DNA strand breaks (comet assay). The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic lesions in the nose. Although the genotoxic biomarker O6-methylguanine was detected, genotoxicity from NNK exposure was negative in the MN and comet assays. The Lowest-Observed-Adverse-Effect-Level (LOAEL) was 0.8 mg/kg BW/day or 0.026 mg/L air of NNK for 1 h/day for both sexes. The No-Observed-Adverse-Effect-Level (NOAEL) was 0.2 mg/kg BW/day or 0.0066 mg/L air of NNK for 1 h/day for both sexes. The results of this study provide new information relevant to assessing the human exposure hazard of NNK.

MetSign: a computational platform for high-resolution mass spectrometry-based metabolomics.

Data analysis in metabolomics is currently a major challenge, particularly when large sample sets are analyzed. Herein, we present a novel computational platform entitled MetSign for high-resolution mass spectrometry-based metabolomics. By converting the instrument raw data into mzXML format as its input data, MetSign provides a suite of bioinformatics tools to perform raw data deconvolution, metabolite putative assignment, peak list alignment, normalization, statistical significance tests, unsupervised pattern recognition, and time course analysis. MetSign uses a modular design and an interactive visual data mining approach to enable efficient extraction of useful patterns from data sets. Analysis steps, designed as containers, are presented with a wizard for the user to follow analyses. Each analysis step might contain multiple analysis procedures and/or methods and serves as a pausing point where users can interact with the system to review the results, to shape the next steps, and to return to previous steps to repeat them with different methods or parameter settings. Analysis of metabolite extract of mouse liver with spiked-in acid standards shows that MetSign outperforms the existing publically available software packages. MetSign has also been successfully applied to investigate the regulation and time course trajectory of metabolites in hepatic liver.

IFN-beta1a inhibits the secretion of Th17-polarizing cytokines in human dendritic cells via TLR7 up-regulation.

IFN-beta, an effective therapy against relapsing-remitting multiple sclerosis, is naturally secreted during the innate immune response against viral pathogens. The objective of this study was to characterize the immunomodulatory mechanisms of IFN-beta targeting innate immune response and their effects on dendritic cell (DC)-mediated regulation of T cell differentiation. We found that IFN-beta1a in vitro treatment of human monocyte-derived DCs induced the expression of TLR7 and the members of its downstream signaling pathway, including MyD88, IL-1R-associated kinase 4, and TNF receptor-associated factor 6, while it inhibited the expression of IL-1R. Using small interfering RNA TLR7 gene silencing, we confirmed that IFN-beta1a-induced changes in MyD88, IL-1R-associated kinase 4, and IL-1R expression were dependent on TLR7. TLR7 expression was also necessary for the IFN-beta1a-induced inhibition of IL-1beta and IL-23 and the induction of IL-27 secretion by DCs. Supernatant transfer experiments confirmed that IFN-beta1a-induced changes in DC cytokine secretion inhibit Th17 cell differentiation as evidenced by the inhibition of retinoic acid-related orphan nuclear hormone receptor C and IL-17A gene expression and IL-17A secretion. Our study has identified a novel therapeutic mechanism of IFN-beta1a that selectively targets the autoimmune response in multiple sclerosis.

14-Day Nose-Only Inhalation Toxicity and Haber's Rule Study of NNK in Sprague-Dawley Rats.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. However, repeated inhalation toxicity data on NNK, which is more directly relevant to cigarette smoking, are currently limited. In the present study, the subacute inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 16 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.8, 3.2, 12.5, or 50 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.03, 0.11, 0.41, or 1.65 mg/L air) for 1 h/day for 14 consecutive days. Toxicity was evaluated by assessing body and organ weights; food consumption; clinical pathology; histopathology observations; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); O6-methylguanine DNA adduct formation; and blood and bone marrow micronucleus frequency. Whether the subacute inhalation toxicity of NNK followed Haber's Rule was also determined using additional animals exposed 4 h/day. The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic histopathological lesions in the nose. The lowest-observed-adverse-effect level (LOAEL) was 0.8 mg/kg BW/day or 0.03 mg/L air for 1 h/day for both sexes. An assessment of Haber's Rule indicated that 14-day inhalation exposure to the same dose at a lower concentration of NNK aerosol for a longer time (4 h daily) resulted in greater adverse effects than exposure to a higher concentration of NNK aerosol for a shorter time (1 h daily).

Toxicokinetic and Genotoxicity Study of NNK in Male Sprague Dawley Rats Following Nose-Only Inhalation Exposure, Intraperitoneal Injection, and Oral Gavage.

The tobacco-specific nitrosamine NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] is found in tobacco products and tobacco smoke. NNK is a potent genotoxin and human lung carcinogen; however, there are limited inhalation data for the toxicokinetics (TK) and genotoxicity of NNK in vivo. In the present study, a single dose of 5 × 10-5, 5 × 10-3, 0.1, or 50 mg/kg body weight (BW) of NNK, 75% propylene glycol (vehicle control), or air (sham control) was administered to male Sprague-Dawley (SD) rats (9-10 weeks age) via nose-only inhalation (INH) exposure for 1 h. For comparison, the same doses of NNK were administered to male SD rats via intraperitoneal injection (IP) and oral gavage (PO). Plasma, urine, and tissue specimens were collected at designated time points and analyzed for levels of NNK and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and tissue levels of DNA adduct O6-methylguanine by LC/MS/MS. TK data analysis was performed using a non-linear regression program. For the genotoxicity subgroup, tissues were collected at 3 h post-dosing for comet assay analysis. Overall, the TK data indicated that NNK was rapidly absorbed and metabolized extensively to NNAL after NNK administration via the three routes. The IP route had the greatest systemic exposure to NNK. NNK metabolism to NNAL appeared to be more efficient via INH than IP or PO. NNK induced significant increases in DNA damage in multiple tissues via the three routes. The results of this study provide new information and understanding of the TK and genotoxicity of NNK.

Negative functional interaction of retinoic acid and TGF-beta signaling mediated by TG-interacting factor during chondrogenesis.

We previously showed that atRA (all-trans Retinoic Acid, atRA) inhibites chondrogenesis by downregulation of TGF-beta/Smad signaling. However, the molecular link between RA and TGF-beta signaling is unknown. Using a mouse embryo palate mesenchyme micomass cultures (MMCs) system, we explored interactions of RA and TGF-beta signaling during chondrogenesis. We found that atRA suppressed chondrogenesis and Smad2/3 phosphorylation regardless of the presence of TGF-beta3. Functional assays indicated that TGF-beta3 treatment or co-transfection of expressing Smad2/3 vectors suppressed atRA-induced RARE-tk-Luc activity. Conversely, atRA or RAR-overexpression repressed TGF-beta3-induced transactivation of the TGF-beta-responsive reporter, p3TP-Lux. ChIP assay revealed the binding of the Smad transcriptional co-repressor TGIF (TG-interacting factor, TGIF) to RARbeta promoter in control MMCs, but this association was decreased by the addition of RA and increased by TGF-beta3, respectively. Further examinations revealed that TGIF exerted a pivotal role in regulating crosstalk of RA and TGF-beta signaling, since siRNA knockdown of TGIF partially abolished the ability of atRA to suppress TGF-beta3-induced chondrogenesis, whereas forced expression of TGIF blocked the ability of TGF-beta3 to relieve atRA-mediated the suppression of chondrogenesis. Furthermore, we demonstrated that the effects of atRA on TGF-beta-dependent gene activation and of TGF-beta on RA-dependent gene activation are mediated by TGIF with siRNA to downregulate TGIF. Collectively, these findings indicated a negative functional interplay of RA and TGF-beta signaling mediated by TGIF to modulate chondrogenesis in MMCs.

Evaluation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) mutagenicity using in vitro and in vivo Pig-a assays.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a genotoxic carcinogen found in tobacco and tobacco smoke. Several in vitro and in vivo assays have been used for evaluating the genotoxicity of tobacco smoke and tobacco smoke constituents like NNK, yet it is not clear which in vitro assays are most appropriate for extrapolating the in vitro responses of these test agents to animal models and humans. The Pig-a gene mutation assay can be performed in vitro, in laboratory animals, and in humans, a potential benefit in estimating in vivo responses from in vitro data. In the current study we used Pig-a as a reporter of gene mutation both in vitro, in L5178Y/Tk+/- cells, and in vivo, in Sprague-Dawley rats. NNK significantly increased Pig-a mutant frequency in L5178Y/Tk+/- cells, but only at concentrations of 100 µg/ml and greater, and only in the presence of S9 activation. Pig-a mutations in L5178Y/Tk+/- cells were detected in 80% of the NNK-induced mutants, with the predominate mutation being G→A transition; vehicle control mutants contained deletions. In the in vivo study, rats were exposed to NNK daily for 90 days by inhalation, a common route of exposure to NNK for humans. Although elevated mutant frequencies were detected, these responses were not clearly associated with NNK exposure, so that overall, the in vivo Pig-a assays were negative. Thus, while NNK induces mutations in the in vitro Pig-a assay, the in vivo Pig-a assay has limited ability to detect NNK mutagenicity under conditions relevant to NNK exposure in smokers.

Immunomodulatory effects of 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitors, potential therapy for relapsing remitting multiple sclerosis.

This study characterized immunomodulatory targets of statins in humans and their potential for treatment of relapsing remitting multiple sclerosis (RR MS). Statins inhibited the proliferative response of mononuclear cells. Simvastatin, the statin with the strongest antiproliferative effect, inhibited IFN-gamma-induced expression of MHC class II DR on monocytes and decreased their antigen presenting capacity. As for T lymphocytes, it inhibited their activation and expression of the Th1 lineage differentiation markers. Simvastatin inhibited IFN-gamma, TNF-alpha, and IL-2 secretion, as well as the expression of T-bet, a transcription factor that regulates Th1 cell differentiation.

The maternal combined supplementation of folic acid and Vitamin B(12) suppresses ethanol-induced developmental toxicity in mouse fetuses.

Maternal ethanol consumption during pregnancy can induce developmental defects in the fetus. The objective of this study was to assess whether combined supplementation of folic acid (FA) and Vitamin B(12) (VB(12)) in dams would suppress ethanol-induced developmental toxicity in CD-1 mice. Ethanol (5.0 g/kg) was given intragastrically from gestational day (GD) 6 to GD15. Vitamin supplementation groups were additionally given 60.0 mg/kg FA, 1.0 mg/kg VB(12), or 60.0 mg/kg FA+1.0 mg/kg VB(12) during GD1-16. The control group received distilled water only. Results of litter evaluation on GD18 showed that combined supplementation of FA and VB(12) ameliorated many of the adverse effects of ethanol. In contrast, the single vitamin supplementation groups showed little or no amelioration. These results suggest that combined supplementation of FA and VB(12) was more effective than each vitamin toward suppressing ethanol-induced developmental toxicity in CD-1 mice.