ABSTRACT
Recently, we identified in two individuals with intellectual disability (ID) different de novo mutations in DEAF1, which encodes a transcription factor with an important role in embryonic development. To ascertain whether these mutations in DEAF1 are causative for the ID phenotype, we performed targeted resequencing of DEAF1 in an additional cohort of over 2,300 individuals with unexplained ID and identified two additional individuals with de novo mutations in this gene. All four individuals had severe ID with severely affected speech development, and three showed severe behavioral problems. DEAF1 is highly expressed in the CNS, especially during early embryonic development. All four mutations were missense mutations affecting the SAND domain of DEAF1. Altered DEAF1 harboring any of the four amino acid changes showed impaired transcriptional regulation of the DEAF1 promoter. Moreover, behavioral studies in mice with a conditional knockout of Deaf1 in the brain showed memory deficits and increased anxiety-like behavior. Our results demonstrate that mutations in DEAF1 cause ID and behavioral problems, most likely as a result of impaired transcriptional regulation by DEAF1.
Subject(s)
Intellectual Disability/genetics , Mental Disorders/genetics , Nuclear Proteins/genetics , Speech Disorders/genetics , Amino Acid Sequence , Animals , Child , Cohort Studies , DNA Mutational Analysis , DNA-Binding Proteins , Female , Humans , Male , Mice , Mice, Knockout , Molecular Sequence Data , Mutation , Protein Structure, Tertiary/genetics , Transcription FactorsABSTRACT
OBJECTIVE: To determine whether camel articular chondrocytes can be maintained in tissue culture without phenotype loss and whether the response to cytokine stimulation can be modulated. SAMPLE POPULATION: Cartilage from 4 carpal joints of healthy adult dromedary camels (Camelus dromedarius). PROCEDURES: Chondrocytes were evaluated for type II collagen and aggrecan production They were incubated with control media or with 2 test mixtures (alone and then in combination) that have anti-inflammatory activity (avocado-soybean unsaponifiables, glucosamine, and chondroitin sulfate [ie, ASU + GLU + CS] and pentosan polysulfate and N-acetyl glucosamine [ie, PPS + NG]). Cells were then stimulated with interleukin-1ß and tumor necrosis factor-α to determine prostaglandin (PG) E2 production and nuclear factor (NF)-κB activation. RESULTS: Chondrocytes proliferated in media used for propagating equine chondrocytes; they produced type II collagen and aggrecan. Cytokine stimulation induced PGE2 production and translocation of NF-κB. Incubation with each test mixture significantly inhibited PGE2 production. The combination of ASU + GLU + CS and PPS + NG significantly potentiated PGE2 inhibition and disrupted NF-κB translocation, compared with effects for either mixture alone. CONCLUSIONS AND CLINICAL RELEVANCE: Chondrocytes proliferated without loss of the cartilage phenotype. Responses to cytokines were significantly inhibited by the mixtures of ASU + GLU + CS and PPS + NG, which indicated that this response can be modulated. This culture technique can be used to study the functional properties of camel chondrocytes and identify agents that may potentially be used to treat and manage joint inflammation.
Subject(s)
Camelus/physiology , Carpal Joints/cytology , Chondrocytes/drug effects , Chondrocytes/metabolism , Cytokines/pharmacology , Dinoprostone/metabolism , Animals , Cell Proliferation , Cells, Cultured , Chondrocytes/cytology , Gene Expression Regulation , NF-kappa B/genetics , NF-kappa B/metabolism , Protein TransportABSTRACT
We examined whether gastric acidity would affect the activity of myrosinase, co-delivered with glucoraphanin (GR), to convert GR to sulforaphane (SF). A broccoli seed and sprout extract (BSE) rich in GR and active myrosinase was delivered before and after participants began taking the anti-acid omeprazole, a potent proton pump inhibitor. Gastric acidity appears to attenuate GR bioavailability, as evidenced by more SF and its metabolites being excreted after participants started taking omeprazole. Enteric coating enhanced conversion of GR to SF, perhaps by sparing myrosinase from the acidity of the stomach. There were negligible effects of age, sex, ethnicity, BMI, vegetable consumption, and bowel movement frequency and quality. Greater body mass correlated with reduced conversion efficiency. Changes in the expression of 20 genes in peripheral blood mononuclear cells were evaluated as possible pharmacodynamic indicators. When grouped by their primary functions based on a priori knowledge, expression of genes associated with inflammation decreased non-significantly, and those genes associated with cytoprotection, detoxification and antioxidant functions increased significantly with bioavailability. Using principal components analysis, component loadings of the changes in gene expression confirmed these groupings in a sensitivity analysis.
Subject(s)
Brassica , Dietary Supplements , Glucosinolates/administration & dosage , Glycoside Hydrolases/administration & dosage , Imidoesters/administration & dosage , Isothiocyanates/metabolism , Omeprazole/administration & dosage , Plant Extracts/administration & dosage , Proton Pump Inhibitors/administration & dosage , Seedlings , Seeds , Adult , Aged , Biological Availability , Brassica/chemistry , Dietary Supplements/adverse effects , Drug Interactions , Female , Gastric Acid/metabolism , Gene Expression Regulation/drug effects , Glucosinolates/adverse effects , Glucosinolates/isolation & purification , Glucosinolates/metabolism , Glycoside Hydrolases/adverse effects , Glycoside Hydrolases/metabolism , Humans , Hydrogen-Ion Concentration , Imidoesters/adverse effects , Imidoesters/isolation & purification , Imidoesters/metabolism , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Omeprazole/adverse effects , Oximes , Pilot Projects , Plant Extracts/adverse effects , Plant Extracts/isolation & purification , Plant Extracts/metabolism , Proton Pump Inhibitors/adverse effects , Seedlings/chemistry , Seeds/chemistry , Sulfoxides , Young AdultABSTRACT
Objective Pro-inflammatory mediators such as prostaglandin E-2 (PGE2) play major roles in the pathogenesis of osteoarthritis (OA). Although current pharmacologic treatments reduce inflammation, their prolonged use is associated with deleterious side effects prompting the search for safer and effective alternative strategies. The present study evaluated whether chondrocyte production of PGE2 can be suppressed by the combination of avocado/soybean unsaponifiables (ASU) and α-lipoic acid (LA). Design Chondrocytes from articular cartilage of equine joints were incubated for 24 hours with: (1) control media, (2) ASU, (3) LA, or (4) ASU + LA combination. Cells were activated with lipopolysaccharide (LPS), interleukin 1ß (IL-1ß) or hydrogen peroxide (H2O2) for 24 hours and supernatants were immunoassayed for PGE2. Nuclear factor-kappa B (NF-κB) analyses were performed by immunocytochemistry and Western blot following 1 hour of activation with IL-1ß. Results LPS, IL-1ß, or H2O2 significantly increased PGE2 production. ASU or LA alone suppressed PGE2 production in LPS and IL-1ß activated cells. Only LA alone at 2.5 µg/mL was inhibitory in H2O2-activated chondrocytes. ASU + LA inhibited more than either agent alone in all activated cells. ASU + LA also inhibited the IL-1ß induced nuclear translocation of NF-κB. Conclusions The present study provides evidence that chondrocyte PGE2 production can be inhibited by the combination of ASU + LA more effectively than either ASU or LA alone. Inhibition of PGE2 production is associated with the suppression of NF-κB translocation. The potent inhibitory effect of ASU + LA on PGE2 production could offer a potential advantage for a combination anti-inflammatory/antioxidant approach in the management of OA.
Subject(s)
Cells, Cultured/drug effects , Chondrocytes/cytology , Osteoarthritis/metabolism , Persea/adverse effects , Soybean Oil/pharmacology , Thioctic Acid/pharmacology , Animals , Anti-Inflammatory Agents/adverse effects , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Cartilage, Articular/cytology , Cartilage, Articular/drug effects , Cartilage, Articular/metabolism , Chondrocytes/drug effects , Chondrocytes/metabolism , Combined Modality Therapy/methods , Dinoprostone/biosynthesis , Dinoprostone/metabolism , Disease Models, Animal , Horses , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Inflammation/drug therapy , Interleukin-1beta/metabolism , Interleukin-1beta/pharmacology , Lipopolysaccharides/metabolism , Lipopolysaccharides/pharmacology , NF-kappa B/metabolism , NF-kappa B/pharmacology , Osteoarthritis/drug therapy , Osteoarthritis/physiopathology , Persea/metabolism , Plant Extracts/pharmacology , Soybean Oil/adverse effects , Soybean Oil/metabolism , Thioctic Acid/adverse effects , Thioctic Acid/metabolismABSTRACT
BACKGROUND: Osteoarthritis (OA) is characterized by inflammation, joint immobility, and pain. Non-pharmacologic agents modulating pro-inflammatory mediator expression offer considerable promise as safe and effective treatments for OA. We previously determined the anti-inflammatory effect of an avocado/soybean unsaponifiables (ASU) and epigallocatechin gallate (EGCG) combination on prostaglandin E2 (PGE2) production and nuclear factor-kappa B (NF-κB) translocation. The aim of this study was to evaluate the effects of ASU + EGCG on pro-inflammatory gene expression. FINDINGS: Articular chondrocytes from carpal joints of mature horses were pre-incubated for 24 hours with control media alone or ASU (8.3 µg/mL) + EGCG (40 ng/mL), followed by one hour activation with interleukin-1 beta (IL-1ß, 10 ng/mL) and tumor necrosis factor-alpha (TNF-α, 1 ng/mL). Total cellular RNA was isolated and real-time PCR performed to measure IL-1ß, TNF-α, interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and interleukin-8 (IL-8) gene expression. Intracellular localization of NF-κB was analyzed by immunohistochemistry and Western blot. Pre-treatment with ASU + EGCG significantly (P < 0.001) decreased gene expression of IL-1ß, TNF-α, IL-6, COX-2, and IL-8 in cytokine-activated chondrocytes. Western blot and immunostaining confirmed NF-κB translocation inhibition. CONCLUSIONS: We demonstrate that ASU + EGCG inhibits cytokine-induced gene expression of IL-1ß, TNF-α, IL-6, COX-2, and IL-8 through modulation of NF-κB. Our results indicate that the activity of ASU + EGCG affects a wide array of inflammatory molecules in addition to decreasing PGE2 synthesis in activated chondrocytes. The responsiveness of chondrocytes to this combination supports its potential utility for the inhibition of joint inflammation.