The expression of peroxisome proliferator-activated receptor (ppar1 and ppar2) in naive and memory cd4+ t lymphocytes

Peripheral CD4+ T cells can be divided into two functional groups based on the expression of distinct isoforms of the surface molecule that contains an intracellular twodomain phosphatase portion, known as CD45. Memory T cells express the lowest molecular weight CD45RO isofonn. whereas naive T cells express CD45RA (human) or CD45RB (mouse) isoforms. CD45 is a protein tyrosine phosphatase which plays an important role in TCR-mediated signaling through its activation of Lck by dephosphorylating the regulatory Tyros. Human naive and memory CD4+ T cells differ in the. requirements for activation and magnitude of the cellular responses. The nuclear receptor, peroxisome proliferator-activated receptor 1 {PPARy) has been reported to be involved in regufatinQ the activities of immune cells such as macrophages or monocytes and T lymphocytes. Given their roles in immune regulation, the current study was carried out to determine the expression of PPARy in human naive and memory CD4+ T cells since it is possible that PPARy may be differentially expressed in the different isoforms of CD45. In addition. the differential signaling patterns and cytokine secretion of these subsets of T cells may require engagement with PPARy isoforms ... a possibility that has not been explored thus far. To further dissect the rote of PPARy in the regulation of naive. and memory CD4+ T cell activation, the PP AR.y agonist, ciglitazone, was used to modulate the activation status of naive and memory CD4+ T cells as wetl as the expression of PPARy itself and selected cytokines. Using Real .. Time PCR. unstimulated naive and memory CD4+ T cells were found not to express PPARy1 and PPARy2, whereas stimulated naive and memory CD4+ T cells express high levels of these receptors with PPARy2 expression being higher than PPARy1 in both cell types (p<0.01). In addition, the PPARy1 expression was higher in stimulated memory as compared to stimulated naive C04+ T cells {p<0.05) whereas there was no significant difference between PPARy2 expression in both types of stimulated cells. The addition- of the PPARy agonist, ciglitazone signifiCantly increased the expression of PPARy1 by about 61-fold and 175-fold in stimulated naive and memory CD4+ T cells respectively (p<0.01). In contrast to PPARy1, the addition of ciglitazone significantly decreased the expression of PPARy2 by about 650-fokl and 140-fold in stimulated na"ive and memory CD4+ T cells respectively {p<0.01). In addition, the expression levels of TGF-)3 and· IL-1 J3 gene were higher in unstimulated naive and memory CD4+ T cells but decreased in their stimulated state (p<0.01). lL-8 gene was expressed at low levels in unstimulated but elevated in stimulated naive and memory CD4+ T cells (p<0.01}. However, there were no significant differences in the levels of these cytokines between naive and memory CD4+ T celts between both states. IL-4 lFNy, IL-5, IL-13, TNFa, GM-CSF and IL-6 were only expressed in stimulated naive and memory CD4+ T ceUs but not. in their unstimulated state. The expression levels of IL-2 and IL-13 were significantly higher in stimulated naive as compared to stimulated memory C04+ T cells (p<0 .. 01 ). In contrast, the expression levels of IFNy were significantly higher in stimulated memory as compared to stimulated naive CD4+ T cells (p<0.05). However, there were no significant differences in the expression of IL-5, IL-6, TNFa and GM..CSF between both stimulated cell types. The addition of ciglitazone. decreased the expression levels of TGFp, IL-1p, IL-8, IL-2, IFNy, IL-5, TNFa. and GM-CSF in stimulated memory and na"ive CD4+ T cell& The induction of PPARr1, and suppression of PPARy2 expression in naTve and memory CD4+ T cells in the p~sence of ciglitazone suggest that the PPARy isoforms may have different functions in T cell regulation. The expreqi~n of $elected cytokine genes inactivated naive and memory CD4+ T cells is consistent with previous studies. The exact mechanism of how PPARy inhibit cytokine expression in stimulated naive and memory CD4+ T cells or which PPARy isoforms is responsible for this effect remain uncertain. It is possible that PPARy inhibit the expression of cytokine genes in these stimulated cell subsets. via interacting with NF-KB, AP-1 and STATs. which is important transcription factors for these cytokines as shown by previous studies in other cells

The Modulation of PPARγ1 and PPARγ2 mRNA Expression by Ciglitazone in CD3/CD28-Activated Naïve and Memory CD4+ T Cells

Given their roles in immune regulation, the expression of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) 1 and 2 isoforms was investigated in human naïve (CD45RA+) and memory (CD45RO+) CD4+ T cells. Stimulation of both types of cells via the CD3/CD28 pathway resulted in high expression of both PPARγ receptors as measured by real-time PCR. Treatment with the PPARγ agonist, ciglitazone, increased PPARγ1 expression but decreased PPARγ2 expression in stimulated naïve and memory cells. Furthermore, when present, the magnitude of both PPARγ receptors expression was lower in naïve cells, perhaps suggesting a lower regulatory control of these cells. Similar profiles of selected proinflammatory cytokines were expressed by the two cell types following stimulation. The induction of PPARγ1 and suppression of PPARγ2 expressions in naïve and memory CD4+ T cells in the presence of ciglitazone suggest that the PPARγ subtypes may have different roles in the regulation of T-cell function

Usage of traditional medicines among elderly and prednisolone contamination

The elderly consume many medications including traditional medicines. In 1986, it was found that 29% of elderly took traditional medicines although in 1996, the National Health Morbidity survey reported a 2.3% prevalence.However,studies from other countries showed much higher percentages. The Ministry of Health in Malaysia is concerned that some of these preparations maybe contaminated with steroids, antihistamines, hormones and other poisons. The aims of the study were to determine a). the health seeking behaviour of elderly Malays living in rural areas, b). the utilization of both modern and traditional medicines and c).the steroid content of the traditional medicines used. Methodology included interviews using structured questionnaires of elderly Malays living in rural areas of Kelantan, aged above 60 years. Samples of traditional medications ollected were sent to the Pharmacology Department, School of Medical Sciences, Universiti Sains Malaysia, for steroid content analysis using Thin Layer Chromatography. A total of 599 elderly respondents were interviewed comprising 62.4% females and 37.6% males. The 60-69 years cohort group made up 48.7%, followed by 70-79 years at 36.1% and the remainder 15.2% were more than 80 years. There were 82% of elderly taking medicines. The trends of utilization of modern and traditional medicine in the last two weeks among elderly were 59.3% and 40.9% respectively. The utilization of traditional medicine by rural elderly Malays was therefore much higher than that reported in the previous study and nearly similar to that of France and Australian studies. There were 102 samples of traditional medications collected and analysed for steroid content. Results showed that 27.5% were positive for prednisolone, 34.3% positive for unknown steroids (a total of 61.8%) and 38.2% were negative for both steroids. The present study therefore once again confirmed the high usage of traditional medicines where some of which are contaminated with steroids

Preliminary results of electrical characterization of GO towards MCF7 and MCF10a at different concentrations

GO is the 2D carbon sheet with additional functional groups, is more stable in various solvents, easy to be produced and manipulated especially in biological system. At the moment, GO is only utilized as the drug delivery agent during treatment. In this study, the resistivity of GO towards breast cancer cell (MCF7) and normal breast cell (MCF10a) using interdigitated electrodes (IDE) were investigated. The interaction of different concentrations of GO as the sensing material on the tested cells which act as analyte can change electrical response. The tested cell were treated with six different concentrations of GO and was dropped to the IDE with different period of time in order to examine electrical behavior. For MCF10a, at high concentration the resistances of MCF10 remain in the same order of magnitude with increasing time of detection while for MCF7 at high concentration, the resistances were greatly influenced by the time of detection where the value significantly changed after 5 minutes and 10 minutes. The number of viable cell does not give effect to the resistance

Electrical characterization of GO at different pH towards MCF7 and MCF10a: preliminary result

The intracellular pH of cancerous cell is commonly acidic while the intracellular pH of normal cell is neutral. The objective of this study is to study the electrical characterization in terms of resistance between the pH of sensing material with the intracellular pH of the cells. Three different pH of Graphene Oxide (GO) were used as a solvent to analyze their interaction towards breast cancer cells (MCF7) and breast normal cells (MCF10a). GO which produced by Hummer's method was used due to their solubility and biocompatibility characteristics which easily diffuse through the cell. In this experiment, the characteristics of GO were analyzed and confirmed by using Atomic Force Microscopy (AFM) and Fourier Transform Infrared spectroscopy (FTIR). In order to measure the resistance of MCF7 and MCF10a cells after treated with GO for 24 hours, gold electrodes with 10 μ-gaps of interdigitated electrodes (IDEs) were used. The results were obtained for three periods of time which were immediate, 5 minutes and 10 minutes after the treated cells being exposed at room temperature. The results show that the resistance of MCF10a cells increased after treated with higher pH of GO which is pH 7 and the resistances of the MCF7 cells decreased as the pH of GO increased to pH 7. Finally, the viable cells were calculated by using haemocytometer in order to prove that the increased of the resistances were due to the increased number of viable cells

The Mechanism of Th17 Cells Induction and its Role in the Atherosclerotic Plaque Development.

Atherosclerosis is a chronic inflammatory disease regulated by T-cell subsets. Recently, Th17 cells have been shown to be critical in exacerbating atherosclerosis in humans and mice. Possible mechanisms of Th17 cell differentiation from mouse CD4+ T-cells via oxLDL-treated dendritic cells (DCs) and smooth muscle cells (SMCs) were investigated. The possibility of direct interaction of oxLDL with Th17 cells followed by the effect of Th17 cells induction in vivo on the atherosclerotic plaque development in apoE-/- were examined. It was demonstrated that an atherogenic stimuli, oxLDL induced maturation of DCs by up-regulating the expression of maturation markers CD40, CD86 and MHC class II which facilitated the differentiation of CD4+ T-cells into Th17 cells. The addition of IL-6 and TGF-&beta; during CD4+ T-cells co-cultured with oxLDL-treated DCs increased the expression of IL-17A on CD4+RORyt+ expressing cells. On the other hand, Mitomycin C (MMC) treatment on oxLDL-treated DCs and co-cultured with CD4+ T-cells reduced the expression of IL-17A on CD4+RORyt+ expressing cells. The current study also showed that oxLDL induced the secretion of IL-6, TGF-&beta; and IL-23 by SMCs after 24 hours of incubation. Exogenous IL-6 and TGF-&beta; increased IL-17A expression on CD4+CD25- expressing cells and also IL-17F expression on both CD4+CD25-expressing cells and CD4+RORyt+ expressing cells when CD4+ T-cells co-cultured with oxLDL-treated SMCs. We then investigated the possible direct interaction between oxLDL with Th17 cells. The results showed that treated Th17 cells induced expression of CD36. The current study also demonstrated that treated Th17 cell induced culture also increased the expression of p-STAT-3 and secretion of Th17 cell related cytokines, IL-17A and IL-17F. Finally, the current study demonstrated that pertussis toxin (PTx), anti-CD25 (PC61) and a combination of both treatments increased Th17 cells in the splenocytes of female apoE-/- mice fed with high fat diet (HFD). The serum cholesterol also was significantly higher in the treated mice. Furthermore, the plaque size significantly increased in mice that were treated with PC61 and combinations of PTx and PC61, but no changes were observed in the collagen content. Taken together, the findings suggest that the level of IL-6 and TGF-&beta; secreted by oxLDL-treated DCs and SMCs may influence Th17 cell differentiation. The possible engagement of oxLDL by Th17 cells via CD36 may activate STAT-3, which in turn may mediate the release of IL-17A and IL-17F. The induction of Th17 cells following respective treatments in the apoE-/- mice fed with HFD displayed close correlation between high cholesterol levels and thus increase atherosclerotic plaque development. Therefore, Th17 cells may be a potential target for modulation of atherogenesis

The Mechanism of Th17 Cells Induction and its Role in the Atherosclerotic Plaque Development.

Atherosclerosis is a chronic inflammatory disease regulated by T-cell subsets. Recently, Th17 cells have been shown to be critical in exacerbating atherosclerosis in humans and mice. Possible mechanisms of Th17 cell differentiation from mouse CD4+ T-cells via oxLDL-treated dendritic cells (DCs) and smooth muscle cells (SMCs) were investigated. The possibility of direct interaction of oxLDL with Th17 cells followed by the effect of Th17 cells induction in vivo on the atherosclerotic plaque development in apoE-/- were examined. It was demonstrated that an atherogenic stimuli, oxLDL induced maturation of DCs by up-regulating the expression of maturation markers CD40, CD86 and MHC class II which facilitated the differentiation of CD4+ T-cells into Th17 cells. The addition of IL-6 and TGF-&beta; during CD4+ T-cells co-cultured with oxLDL-treated DCs increased the expression of IL-17A on CD4+RORyt+ expressing cells. On the other hand, Mitomycin C (MMC) treatment on oxLDL-treated DCs and co-cultured with CD4+ T-cells reduced the expression of IL-17A on CD4+RORyt+ expressing cells. The current study also showed that oxLDL induced the secretion of IL-6, TGF-&beta; and IL-23 by SMCs after 24 hours of incubation. Exogenous IL-6 and TGF-&beta; increased IL-17A expression on CD4+CD25- expressing cells and also IL-17F expression on both CD4+CD25-expressing cells and CD4+RORyt+ expressing cells when CD4+ T-cells co-cultured with oxLDL-treated SMCs. We then investigated the possible direct interaction between oxLDL with Th17 cells. The results showed that treated Th17 cells induced expression of CD36. The current study also demonstrated that treated Th17 cell induced culture also increased the expression of p-STAT-3 and secretion of Th17 cell related cytokines, IL-17A and IL-17F. Finally, the current study demonstrated that pertussis toxin (PTx), anti-CD25 (PC61) and a combination of both treatments increased Th17 cells in the splenocytes of female apoE-/- mice fed with high fat diet (HFD). The serum cholesterol also was significantly higher in the treated mice. Furthermore, the plaque size significantly increased in mice that were treated with PC61 and combinations of PTx and PC61, but no changes were observed in the collagen content. Taken together, the findings suggest that the level of IL-6 and TGF-&beta; secreted by oxLDL-treated DCs and SMCs may influence Th17 cell differentiation. The possible engagement of oxLDL by Th17 cells via CD36 may activate STAT-3, which in turn may mediate the release of IL-17A and IL-17F. The induction of Th17 cells following respective treatments in the apoE-/- mice fed with HFD displayed close correlation between high cholesterol levels and thus increase atherosclerotic plaque development. Therefore, Th17 cells may be a potential target for modulation of atherogenesis

USAGE OF TRADITIONAL MEDICINES AMONG ELDERLY AND THE PREVALENCE OF PREDNISOLONE CONTAMINATION

The elderly consume many medications including traditional medicines. In 1986, it was found that 29% of elderly took traditional medicines although in 1996, the National Health Morbidity survey reported a 2.3% prevalence. However, studies from other countries showed much higher percentages. The Ministry of Health in Malaysia is concerned that some of these preparations maybe contaminated with steroids, antihistamines, hormones and other poisons. The aims of the study were to determine a). the health seeking behaviour of elderly Malays living in rural areas, b). the utilization of both modern and traditional medicines and c). the steroid content of the traditional medicines used. Methodology included interviews using structured questionnaires of elderly Malays living in rural areas of Kelantan, aged above 60 years. Samples of traditional medications collected were sent to the Pharmacology Department, School of Medical Sciences, Universiti Sains Malaysia, for steroid content analysis using Thin Layer Chromatography. A total of 599 elderly respondents were interviewed comprising 62.4% females and 37.6% males. The 60-69 years cohort group made up 48.7%, followed by 70-79 years at 36.1% and the remainder 15.2% were more than 80 years. There were 82% of elderly taking medicines. The trends of utilization of modern and traditional medicine in the last two weeks among elderly were 59.3% and 40.9% respectively. The utilization of traditional medicine by rural elderly Malays was therefore much higher than that reported in the previous study and nearly similar to that of France and Australian studies. There were 102 samples of traditional medications collected and analysed for steroid content. Results showed that 27.5% were positive for prednisolone, 34.3% positive for unknown steroids (a total of 61.8%) and 38.2% were negative for both steroids. The present study therefore once again confirmed the high usage of traditional medicines where some of which are contaminated with steroids

Phytoconstituents of the Gynura procumbens ethanol leaf extract and its fractions and their effects on viability of macrophages

Introduction: Gynura procumbens (GP) is a medicinal plant with numerous beneficial pharmacological activities. The aim of this study was to identify the bioactive phytoconstituents in GP ethanol extract and hexane, chloroform, ethyl acetate, and aqueous fractions of GP, and also to evaluate the cell viability of GP ethanol extract and its fractions-treated RAW264.7 cells.Methods: The ethanol GP leaf extract was prepared and further subjected to fractionation. The cell viability of GP ethanol extract and its fractions-treated RAW264.7 cells were measured by PrestoBlue. The phytoconstituents of GP ethanol extract and its fraction were determined by using liquid chromatography-mass spectrometry (LC-MS).Results: RAW264.7 cells exposed to the GP ethanol extract and its fractions showed significantly high proliferation and weak cytotoxic effect on the macrophages, with an average inhibitory concentration of 90% at 24, 48, and 72 hours of incubation. However, at a concentration of 10 μg/mL, the aqueous GP fraction clearly displayed anti-proliferative properties because the cell viability of aqueous GP fraction-treated RAW264.7 cells reduced to 64%, 29% and 4% after 24, 48 and 72 hours of incubation, respectively. The GP extracts and its fractions contained mainly fatty acids, flavonoids, sesquiterpenoids, and products of chlorophyll breakdown.Conclusion: GP ethanol extract and its fractions at certain concentrations may act as immunomodulators, as they induced promising proliferation activity of macrophages. Further studies are needed to determine either the identified chemical compounds influenced on the proliferation of macrophages solely or cooperatively