Heidi Gruber

Flow cytometric analysis of intracellular IFN-γ, IL-4 and IL-10 in CD3+4+ T-cells from rat spleen

The application of multi-parameter flow cytometry for the assessment of T-cell and cytokine functioning has been used by several groups for studying human and mouse samples, although little has been reported for the rat. Here we report the optimisation of immunofluorescent staining for cell surface and intracellular antigens using three-colour flow cytometric analysis to measure the frequency of rat CD3(+)4(+) T-cells that produce IFN-gamma, IL-4 and IL-10. In vitro stimulation of IFN-gamma production required incubation of splenocytes with PMA and ionomycin in the presence of the protein transport inhibitor brefeldin A for 6 h. Three stimulation protocols for IL-4 and IL-10 production were evaluated. In vitro priming of splenic T-cells with antibodies against CD3 and CD28 and recombinant cytokines (IL-2 and IL-4) for 5 days followed by restimulation with PMA and ionomycin was required to stimulate cells to produce either IL-4 or IL-10. Brefeldin A was found to be a more suitable protein transport inhibitor than monensin. This method will be useful for analysing the nature of individual rat cytokine-producing cells in a variety of experimental model systems.

Increased incorporation of dietary plant sterols and cholesterol correlates with decreased expression of hepatic and intestinal Abcg5 and Abcg8 in diabetic BB rats.

The aim of this study was to determine the impact of dietary plant sterols and stanols on sterol incorporation and sterol-regulatory gene expression in insulin-treated diabetic rats and nondiabetic control rats. Diabetic BioBreeding (BB) and control BB rats were fed a control diet or a diet supplemented with plant sterols or plant stanols (5 g/kg diet) for 4 weeks. Expression of sterol-regulatory genes in the liver and intestine was assessed by real-time quantitative polymerase chain reaction. Diabetic rats demonstrated increased tissue accumulation of cholesterol and plant sterols and stanols compared to control rats. This increase in cholesterol and plant sterols and stanols was associated with a marked decrease in hepatic and intestinal Abcg5 (ATP-binding cassette transporter G5) and Abcg8 (ATP-binding cassette transporter G8) expressions in diabetic rats, as well as decreased mRNA levels of several other genes involved in sterol regulation. Plant sterol or plant stanol supplementation induced the accumulation of plant sterols and stanols in tissues in both rat strains, but induced a greater accumulation of plant sterols and stanols in diabetic rats than in control rats. Surprisingly, only dietary plant sterols decreased cholesterol levels in diabetic rats, whereas dietary plant stanols caused an increase in cholesterol levels in both diabetic and control rats. Therefore, lower expression levels of Abcg5/Abcg8 in diabetic rats may account for the increased accumulation of plant sterols and cholesterol in these rats.

Influence of dietary phytosterols and phytostanols on diastolic blood pressure and the expression of blood pressure regulatory genes in SHRSP and WKY inbred rats.

The aim of the present study was to determine the impact of increased consumption of phytosterols or phytostanols on blood pressure and renal blood pressure regulatory gene expression in stroke-prone spontaneously hypertensive (SHRSP) and normotensive Wistar-Kyoto (WKY) inbred rats. SHRSP and WKY inbred rats (10/group) were fed a control diet or a diet supplemented with phytosterols or phytostanols (2.0 g/kg diet). After 5 weeks, SHRSP rats demonstrated higher systolic and diastolic blood pressures than WKY inbred rats. SHRSP rats that consumed the phytosterol or phytostanol supplemental diets displayed a 2- or 3-fold respective increase in the diastolic blood pressure than those that consumed the control diet. Angiotensinogen (Agt), angiotensin I-converting enzyme 1 (Ace1), nitric oxide synthase (Nos) 1, Nos3, cyclooxygenase 2 (Cox2) and THUMP domain containing 1 were expressed at higher levels in SHRSP compared with WKY inbred rats. Renin and angiotensin II receptor type 1a were expressed at lower levels in SHRSP than WKY inbred rats. Phytostanol supplementation up-regulated the expression of Ace1 and Nos3 in SHRSP rats. Phytosterol supplementation increased the mRNA levels of Nos1 and spondin 1 (Spon1) in SHRSP and WKY inbred rats. Cox2 mRNA levels were elevated in both phytosterol- and phytostanol-supplemented SHRSP and WKY inbred rats. Therefore, the increased blood pressure in SHRSP rats may be partly due to altered renal expression of blood pressure regulatory genes. Specifically, up-regulation of Ace1, Nos1, Nos3, Cox2 and Spon1 were associated with the increased diastolic blood pressure observed in phytosterol- or phytostanol-supplemented SHRSP rats.

Dietary phytosterols and phytostanols decrease cholesterol levels but increase blood pressure in WKY inbred rats in the absence of salt-loading

BackgroundThere are safety concerns regarding widespread consumption of phytosterol and phytostanol supplemented food products. The aim of this study was to determine, in the absence of excess dietary salt, the individual effects of excess accumulation of dietary phytosterols and phytostanols on blood pressure in Wistar Kyoto (WKY) inbred rats that have a mutation in the Abcg5 gene and thus over absorb phytosterols and phytostanols.MethodsThirty 35-day old male WKY inbred rats (10/group) were fed a control diet or a diet containing phytosterols or phytostanols (2.0 g/kg diet) for 5 weeks. The sterol composition of the diets, plasma and tissues were analysed by gas chromatography. Blood pressure was measured by the tail cuff method. mRNA levels of several renal blood pressure regulatory genes were measured by real-time quantitative PCR.ResultsCompared to the control diet, the phytosterol diet resulted in 3- to 4-fold increases in the levels of phytosterols in plasma, red blood cells, liver, aorta and kidney of WKY inbred rats (P < 0.05). The phytostanol diet dramatically increased (> 9-fold) the levels of phytostanols in plasma, red blood cells, liver, aorta and kidney of these rats (P < 0.05). The phytosterol diet decreased cholesterol levels by 40%, 31%, and 19% in liver, aorta and kidney, respectively (P < 0.05). The phytostanol diet decreased cholesterol levels by 15%, 16%, 20% and 14% in plasma, liver, aorta and kidney, respectively (P < 0.05). The phytostanol diet also decreased phytosterol levels by 29% to 54% in plasma and tissues (P < 0.05). Both the phytosterol and phytostanol diets produced significant decreases in the ratios of cholesterol to phytosterols and phytostanols in plasma, red blood cells, liver, aorta and kidney. Rats that consumed the phytosterol or phytostanol diets displayed significant increases in systolic and diastolic blood pressure compared to rats that consumed the control diet (P < 0.05). The phytosterol diet increased renal angiotensinogen mRNA levels of these rats.ConclusionThese data suggest that excessive accumulation of dietary phytosterols and phytostanols in plasma and tissues may contribute to the increased blood pressure in WKY inbred rats in the absence of excess dietary salt. Therefore, even though phytosterols and phytostanols lower cholesterol levels, prospective clinical studies testing the net beneficial effects of dietary phytosterols and phytostanols on cardiovascular events for subgroups of individuals that have an increased incorporation of these substances are needed.

Dietary phytosterols and phytostanols alter the expression of sterol-regulatory genes in SHRSP and WKY inbred rats.

Background/Aims: We elucidated the molecular mechanism(s) underlying sterol trafficking by investigating alterations in gene expression in response to increased retention of dietary phytosterols and phytostanols in stroke-prone spontaneously hypertensive (SHRSP) and normotensive Wistar Kyoto (WKY) inbred rats. Methods: SHRSP and WKY inbred rats were fed a control diet or a diet supplemented with phytosterols or phytostanols (2 g/kg diet). Results: Intake of phytosterols and phytostanols increased their incorporation in plasma, red blood cells, liver, aorta and kidney, but decreased cholesterol levels in liver and aorta in both rat strains. Phytosterol intake up-regulated mRNA expression of intestinal Npc1l1 and Abcg8, and hepatic Abcg5, Abca1, Cyp27a1 and Hmgcr. Phytostanol intake up-regulated Npc1l1 and Srebp2, but down-regulated Abcg5 mRNA expression in small intestine. Phytostanols also up-regulated Abca1 expression in SHRSP rats, but down-regulated Abca1 expression in WKY inbred rats. Compared to phytosterols, dietary phytostanols reduced phytosterol levels in plasma, red blood cells, and kidney, as well as altered mRNA levels of hepatic Abca1,Cyp27a1, and Hmgcr and intestinal Abcg5/8, Hmgcr and Srebp2. Conclusions: Altered expression of multiple sterol-regulatory genes may contribute to the incorporation and cholesterol-lowering actions of phytosterols and phytostanols. Phytosterols and phytostanols may act through different mechanism(s) on cholesterol and phytosterol/phytostanol trafficking.

Excess dietary iodine differentially affects thyroid gene expression in diabetes, thyroiditis-prone versus -resistant BioBreeding (BB) rats

SCOPE To identify genes involved in the susceptibility to iodine-induced autoimmune thyroiditis. METHODS AND RESULTS Diabetes, thyroiditis-prone (BBdp) and -resistant (BBc) rats were fed either a control or a high-iodine diet for 9 wk. Excess iodine intake increased the incidence of insulitis and thyroiditis in BBdp rats. BBdp rats fed the high-iodine diet that did not develop thyroiditis had higher mRNA levels of Fabp4, Cidec, perilipin, Pparγ and Slc36a2 than BBdp rats fed the control diet and BBc rats fed either the control or the high-iodine diet. BBdp rats fed the high-iodine diet that did develop thyroiditis had higher mRNA levels of Cidec, Icam1, Ifitm1, and Slpi than BBdp rats fed the control diet and BBc rats fed either the control or the high-iodine diet. BBdp rats that did develop thyroiditis had lower mRNA levels of Fabp4, perilipin and Slc36a2 but higher mRNA levels of Icam1, Ifitm1 and Slpi than BBdp that did not develop thyroiditis. Excess dietary iodine also increased the protein levels of Fabp4, Cidec and perilipin in BBdp rats. CONCLUSION Differential expression of thyroid genes in BBdp versus BBc rats caused by excess dietary iodine may be implicated in autoimmune thyroiditis and insulitis pathogenesis.

Antioxidant Supplements Improve Profiles of Hepatic Oxysterols and Plasma Lipids in Butter-fed Hamsters.

Hypercholesterolemia diets are associated with oxidative stress that may contribute to hypercholesterolemia by adversely affecting enzymatically-generated oxysterols involved in cholesterol homeostasis. An experiment was conducted to examine whether the cholesterol-lowering effects of the antioxidants selenium and α-tocopherol were related to hepatic oxysterol concentrations. Four groups of male Syrian hamsters (n = 7-8) were fed high cholesterol and saturated fat (0.46% cholesterol, 14.3% fat) hypercholesterolemic semi-purified diets: 1) Control; 2) Control + α-tocopherol (67 IU all-racemic-α-tocopheryl-acetate/kg diet); 3) Control + selenium (3.4 mg selenate/kg diet); and 4) Control + α-tocopherol + selenium. Antioxidant supplementation was associated with lowered plasma cholesterol concentrations, decreased tissue lipid peroxidation and higher hepatic oxysterol concentrations. A second experiment examined the effect of graded selenium doses (0.15, 0.85, 1.7 and 3.4 mg selenate/kg diet) on mRNA expression of the oxysterol-generating enzyme, hepatic 27-hydroxylase (CYP27A1, EC 1.14.13.15), in hamsters (n = 8-9) fed the hypercholesterolemic diets. Supplementation of selenium at 3.4 mg selenate/kg diet was not associated with increased hepatic 27-hydroxylase mRNA. In conclusion, the cholesterol lowering effects of selenium and α-tocopherol were associated with increased hepatic enzymatically generated oxysterol concentrations, which appears to be mediated via improved antioxidant status rather than increased enzymatic production.

High-dose supplemental selenite to male Syrian hamsters fed hypercholesterolaemic diets alters Ldlr , Abcg8 and Npc1l1 mRNA expression and lowers plasma cholesterol concentrations

The aim of the present study was to elucidate possible cholesterol-lowering mechanism(s) of high-dose supplemental Se in the form of selenite, a known hypocholesterolaemic agent. Male Syrian hamsters (four groups, ten per group) were fed semi-purified diets for 4 weeks containing 0.1 % cholesterol and 15 % saturated fat with selenite corresponding to varying levels of Se: (1) Se 0.15 parts per million (ppm), control diet; (2) Se 0.85 ppm; (3) Se 1.7 ppm; (4) Se 3.4 ppm. Lipids were measured in the bile, faeces, liver and plasma. The mRNA expression of several known regulators of cholesterol homeostasis (ATP-binding cassette transporters g5 (Abcg5) and g8 (Abcg8), 7-hydroxylase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, LDL receptor (LdLr) and Nieman-Pick C1-like 1 protein (Npc1l1)) were measured in the liver and/or jejunum. Oxysterols including 24-(S)-hydroxycholesterol, 25-hydroxycholesterol and 27-hydroxycholesterol (27-OHC) were measured in the liver. Significantly lower total plasma cholesterol concentrations were observed in hamsters consuming the low (0.85 ppm) and high (3.4 ppm) Se doses. The two highest doses of Se resulted in decreased plasma LDL-cholesterol concentrations and increased mRNA levels of hepatic Abcg8, Ldlr and jejunal Ldlr. Higher hepatic 27-OHC and TAG concentrations and lower levels of jejunal Npc1l1 mRNA expression were noted in the 1.7 and 3.4 ppm Se-treated hamsters. Overall, Se-induced tissue changes in mRNA expression including increased hepatic Abcg8 and Ldlr, increased jejunal Ldlr and decreased jejunal Npc1l1, provide further elucidation regarding the hypocholesterolaemic mechanisms of action of Se in the form of selenite.

Acknowledgement to the 2009 Reviewers

Helmut Heseker, Paderborn, Germany Manfred Hüttinger, Vienna, Austria Mohsen Janghorbani, Isfahan, Iran Miroslaw Jarosz, Warsaw, Poland Majken K. Jensen, Boston, Mass., USA Alex Johnstone, Aberdeen, UK Anthony Kafatos, Heraklion, Greece Andre Pascal Kengne, Camperdown, N.S.W., Australia Arthur Klatsky, Oakland, Calif., USA Nanne Kleefstra, Zwolle, The Netherlands Marlena Kruger, Palmerston North, New Zealand Anura Kurpad, Bangalore, India Denis Lairon, Marseille, France Wolfgang Langhans, Zürich, Switzerland Mark Lawrence, Melbourne, Vic., Australia Donald K. Layman, Urbana, Ill., USA James V. Leonard, London, UK Lars Libuda, Dortmund, Germany Xu Lin, Shanghai, China Jakob Linseisen, Munich, Germany Dieter Luetjohann, Bonn, Germany Silvia Maggini, Basel, Switzerland Philippe Marmillot, Washington, D.C., USA Franscesco Marotta, Milano, Italy Eva Martos, Budapest, Hungary Velimir Matkovic, Columbus, Ohio, USA Regis Moreau, Corvallis, Oreg., USA Luis A. Moreno, Zaragoza, Spain Yuji Naito, Kyoto, Japan Serge Nef, Geneva, Switzerland Hee Young Paik, Seoul, Korea Mihalis I. Panagiotidis, Reno, Nev., USA Sun Ming Park, Asan-si, Korea Kristina Pentieva, Ulster, UK Carmen Perez-Rodrigo, Bilbao, Spain Nancy M. Petry, Farmington, Conn., USA John M. Pettifor, Johannesburg, South Africa Spencer Proctor, Edmonton, Alta., Canada Ana Maria Proenza Arenas, Palma de Mallorca, Spain Giuliano Ramadori, Göttingen, Germany Aune Rehema, Tartu, Estonia Lajos Rethy, Budapest, Hungary Gerald Rimbach, Kiel, Germany Glorimar Rosa, Rio de Janeiro, Brazil Catherine A. Ross, University Park, Pa., USA Elaine Rush, Auckland, NZ Lydia Afman, Wageningen, The Netherlands Tasnime Akbaraly, London, UK Anthony Alberg, Baltimore, Md., USA Hooman Allayee, Los Angeles, Calif., USA James W. Anderson, Lexington, Ky., USA Bahram Arjmandi, Tallahassee, Fla., USA Georgianne L. Arnold, Rochester, N.Y., USA Benoit Arsenault, Québec, Qué., Canada Fereidoun Azizi, Tehran, Iran Matthias Baum, Kaiserslautern, Germany Giorgio Bedogni, Milano, Italy Joe Begley, Poole, UK Aloys Berg, Freiburg, Germany Renate Bergmann, Berlin, Germany Odilia I. Bermudez, Boston, Mass., USA Francisco Blanco-Vaca, Barcelona, Spain William S. Blaner, New York, N.Y., USA Pontus Boström, Göteborg, Sweden Regina Brigelius-Flohé, Nuthetal, Germany Veronika Butterweck, Gainesville, Fla., USA Anette Buyken, Dortmund, Germany Philip C. Calder, Southampton, UK Dexter Canoy, Manchester, UK Namsoo Chang, Seoul, Korea Supranee Changbumrung, Bangkok, Thailand Marie-Aline Charles, Villejuif, France Armand B. Christophe, Ghent, Belgium Nain-Feng Chu, Taipei, Taiwan Dolores Corella, Valencia, Spain Lindsey Darrow, Atlanta, Ga., USA Tamás Decsi, Pécs, Hungary Antonio Di Stefano, Chieti, Italy Marie-Claude Dop, Rome, Italy Klaus Eder, Freising, Germany Mathias Fasshauer, Leipzig, Germany Annie Ferland, Québec, Qué., Canada Jose Fernandez, Birmingham, Ala., USA Nicola Fuiano, Foggia, Italy Claudio Galli, Milano, Italy Dieter Genser, Vienna, Austria Frank Greer, Madison, Wisc., USA M.H. Hamdaoui, Tunis, Tunisia Igor Harsch, Erlangen, Germany Hans Hauner, Munich, Germany Alan S. Hazell, Montréal, Qué., Canada Erik Hemmingsson, Stockholm, Sweden