Potential link between the Sphingosine-1-Phosphate (S1P) system and defective alveolar macrophage phagocytic function in Chronic Obstructive Pulmonary Disease (COPD)

We previously reported that alveolar macrophages from patients with chronic obstructive pulmonary disease (COPD) are defective in their ability to phagocytose apoptotic cells, with a similar defect in response to cigarette smoke. The exact mechanisms for this defect are unknown. Sphingolipids including ceramide, sphingosine and sphingosine-1-phosphate (S1P) are involved in diverse cellular processes and we hypothesised that a comprehensive analysis of this system in alveolar macrophages in COPD may help to delineate the reasons for defective phagocytic function.We compared mRNA expression of sphingosine kinases (SPHK1/2), S1P receptors (S1PR1-5) and S1P-degrading enzymes (SGPP1, SGPP2, SGPL1) in bronchoalveolar lavage-derived alveolar macrophages from 10 healthy controls, 7 healthy smokers and 20 COPD patients (10 current- and 10 ex-smokers) using Real-Time PCR. Phagocytosis of apoptotic cells was investigated using flow cytometry. Functional associations were assessed between sphingosine signalling system components and alveolar macrophage phagocytic ability in COPD. To elucidate functional effects of increased S1PR5 on macrophage phagocytic ability, we performed the phagocytosis assay in the presence of varying concentrations of suramin, an antagonist of S1PR3 and S1PR5. The effects of cigarette smoking on the S1P system were investigated using a THP-1 macrophage cell line model.We found significant increases in SPHK1/2 (3.4- and 2.1-fold increases respectively), S1PR2 and 5 (4.3- and 14.6-fold increases respectively), and SGPL1 (4.5-fold increase) in COPD vs. controls. S1PR5 and SGPL1 expression was unaffected by smoking status, suggesting a COPD "disease effect" rather than smoke effect per se. Significant associations were noted between S1PR5 and both lung function and phagocytosis. Cigarette smoke extract significantly increased mRNA expression of SPHK1, SPHK2, S1PR2 and S1PR5 by THP-1 macrophages, confirming the results in patient-derived macrophages. Antagonising SIPR5 significantly improved phagocytosis.Our results suggest a potential link between the S1P signalling system and defective macrophage phagocytic function in COPD and advise therapeutic targets.Jameel Barnawi, Hai Tran, Hubertus Jersmann, Stuart Pitson, Eugene Roscioli, Greg Hodge, Robyn Meech, Rainer Haberberger, Sandra Hodg

Improve Teachers' Ability in Compiling Classroom Action Research Through Workshop Activities

This action research aims to improve the ability of teachers in compiling Classroom Action Research. The subjects of this study were 11 madrasah teachers under the guidance of researchers, namely at MIS Al-Wathoniyah Cantilan. The research was conducted in two cycles where each cycle went through the planning, implementation, observation and reflection stages. Data collection techniques using observation techniques and document study. The collected data were analyzed qualitatively descriptive with the type of percentage. The results showed that teacher activity from 70% in the first cycle increased to 88.89% in the second cycle and the quality of the lesson plans from an average of 77.60 in the first cycle increased to 86.00 in the second cycle. There has been an increase in the quality of PTK by 8.4 points from cycle I to cycle II. Thus it can be concluded that the mentored teachers' abilities in preparing CAR can be improved through workshops. &nbsp

Lung cancer is associated with decreased expression of perforin, granzyme B and interferon (IFN)-gamma by infiltrating lung tissue T cells, natural killer (NK) T-like and NK cells

There is a limited understanding how of lung cancer cells evade cytotoxic attack. Previously, we have shown reduced production of the cytotoxic mediator granzyme B by CD8(+) T cells in lung cancer tissue. We hypothesized that lung cancer would be further associated with decreased production of granzyme B, perforin and proinflammatory cytokines by other cytotoxic lymphocytes, natural killer (NK) T-like and NK cells, and that this would result from soluble mediators released by the cancer cells. Lung cancer and non-cancer tissue from five patients was identified by experienced pathologists. Tumour necrosis factor (TNF)-α, interferon (IFN)-γ, granzyme B and perforin were measured in CD4 and CD8(+) T, NK T-like cells and NK cells by flow cytometry. Correlation between cancer stage and granzyme B was analysed retrospectively for 21 patients. The effects of soluble factors released by lung cancer cells on production of cytotoxic mediators and cytokines was assessed, and the role of prostaglandin E2 (PGE)2 /COX investigated using indomethacin inhibition. There were significantly decreased percentages of T, NK T-like and NK cells expressing perforin, TNF-α and IFN-γ in cancer versus non-cancer tissue, and of CD8(+) T cells and CD8(+) NK T-like cells expressing granzyme B (e.g. NK T-like cells: non-cancer 30% ± 7 versus cancer 6% ± 2·5). Cancer cells released soluble factors that inhibited granzyme B, perforin and IFN-γ production that was partially associated with the PGE2 /COX2 pathway. Thus, lung cancer is associated with decreased expression of granzyme B, perforin and IFN-γ by infiltrating T cells, NK T-like and NK cells, possibly as a result of soluble factors produced by the cancer cells including PGE2 . This may be an important immune evasion mechanism.G. Hodge, J. Barnawi, C. Jurisevic, D. Moffat, M. Holmes, P. N. Reynolds, H. Jersmann, and S. Hodg

Cigarette smoke inhibits efferocytosis via deregulation of sphingosine kinase signaling: reversal with exogenous S1P and the S1P analogue FTY720

Alveolar macrophages from chronic obstructive pulmonary disease patients and cigarette smokers are deficient in their ability to phagocytose apoptotic bronchial epithelial cells (efferocytosis). We hypothesized that the defect is mediated via inhibition of sphingosine kinases and/or their subcellular mislocalization in response to cigarette smoke and can be normalized with exogenous sphingosine-1-phosphate or FTY720 (fingolimod), a modulator of sphingosine-1-phosphate signaling, which has been shown to be clinically useful in multiple sclerosis. Measurement of sphingosine kinase 1/2 activities by [(32)P]-labeled sphingosine-1-phosphate revealed a 30% reduction of sphingosine kinase 1 (P < 0.05) and a nonsignificant decrease of sphingosine kinase 2 in THP-1 macrophages after 1 h cigarette smoke extract exposure. By confocal analysis macrophage sphingosine kinase 1 protein was normally localized to the plasma membrane and cytoplasm and sphingosine kinase 2 to the nucleus and cytoplasm but absent at the cell surface. Cigarette smoke extract exposure (24 h) led to a retraction of sphingosine kinase 1 from the plasma membrane and sphingosine kinase 1/2 clumping in the Golgi domain. Selective inhibition of sphingosine kinase 2 with 25 µM ABC294640 led to 36% inhibition of efferocytosis (P < 0.05); 10 µM sphingosine kinase inhibitor/5C (sphingosine kinase 1-selective inhibitor) induced a nonsignificant inhibition of efferocytosis, but its combination with ABC294640 led to 56% inhibition (P < 0.01 vs. control and < 0.05 vs. single inhibitors). Cigarette smoke-inhibited efferocytosis was significantly (P < 0.05) reversed to near-control levels in the presence of 10-100 nM exogenous sphingosine-1-phosphate or FTY720, and FTY720 reduced cigarette smoke-induced clumping of sphingosine kinase 1/2 in the Golgi domain. These data strongly support a role of sphingosine kinase 1/2 in efferocytosis and as novel therapeutic targets in chronic obstructive pulmonary disease.Hai B. Tran, Jameel Barnawi, Miranda Ween, Rhys Hamon, Eugene Roscioli, Greg Hodge, Paul N. Reynolds, Stuart M. Pitson, Lorena T. Davies, Rainer Haberberger and Sandra Hodg

RNA-based thermoregulation of a Campylobacter jejuni zinc resistance determinant

RNA thermometers (RNATs) trigger bacterial virulence factor expression in response to the temperature shift on entering a warm-blooded host. At lower temperatures these secondary structures sequester ribosome-binding sites (RBSs) to prevent translation initiation, whereas at elevated temperatures they "melt" allowing translation. Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide yet little is known about how it interacts with the host including host induced gene regulation. Here we demonstrate that an RNAT regulates a C. jejuni gene, Cj1163c or czcD, encoding a member of the Cation Diffusion Facilitator family. The czcD upstream untranslated region contains a predicted stem loop within the mRNA that sequesters the RBS to inhibit translation at temperatures below 37°C. Mutations that disrupt or enhance predicted secondary structure have significant and predictable effects on temperature regulation. We also show that in an RNAT independent manner, CzcD expression is induced by Zn(II). Mutants lacking czcD are hypersensitive to Zn(II) and also over-accumulate Zn(II) relative to wild-type, all consistent with CzcD functioning as a Zn(II) exporter. Importantly, we demonstrate that C. jejuni Zn(II)-tolerance at 32°C, a temperature at which the RNAT limits CzcD production, is increased by RNAT disruption. Finally we show that czcD inactivation attenuates larval killing in a Galleria infection model and that at 32°C disrupting RNAT secondary structure to allow CzcD production can enhance killing. We hypothesise that CzcD regulation by metals and temperature provides a mechanism for C. jejuni to overcome innate immune system-mediated Zn(II) toxicity in warm-blooded animal hosts

Genetic Determinants of Cardiovascular Disease: The Endothelial Nitric Oxide Synthase 3 (eNOS3), Krüppel-Like Factor-14 (KLF-14), Methylenetetrahydrofolate Reductase (MTHFR), MiRNAs27a and Their Association with the Predisposition and Susceptibility to Coronary Artery Disease

Coronary artery disease (CAD) is an important cause of death worldwide. CAD is caused by genetic and other factors including hypertension, hyperlipidemia, obesity, stress, unhealthy diet, physical inactively, smoking and Type 2 diabetes (T2D). The genome wide association studies (GWASs) have revealed the association of many loci with risk to diseases such as cancers, T2D and CAD. Nitric oxide (NO) is a potent vasodilator and is required for normal vascular health. It is produced in the endothelial cells in a reaction catalyzed by the endothelial NO synthase (eNOS). Methylenetetrahydrofolate reductase (MTHFR) is a very important enzyme involved in metabolism of folate and homocysteine, and its reduced function leads to cardiovascular disease. The Krüppel-like factor-14 (KLF-14) is an important transcriptional regulator that has been implicated in metabolic syndrome. MicroRNA (MiRNAs) are short non-coding RNAs that regulate the gene expression of proteins involved in important physiological processes including cell cycle and metabolism. In the present study, we have investigated the potential impact of germline pathogenic variants of endothelial eNOS, KLF-14, MTHFR, MiRNA-27a and their association with risk to CAD in the Saudi population. Methods: Amplification Refractory Mutation System (ARMS) PCR was used to detect MTHFR, KLF-14, miRNA-27a and eNOS3 genotyping in CAD patients and healthy controls. About 125 CAD cases and 125 controls were enrolled in this study and statistical associations were calculated including p-value, risk ratio (RR), and odds ratio (OD). Results: There were statistically significant differences (p T, KLF-14 rs972283 G>A, miRNAs27a rs895819 A>G and eNOS3 rs1799983 G>T between CAD patients and controls. In addition, our results indicated that the MTHFR-TT genotype was associated with increased CAD susceptibility with an OR 2.75 (95%) and p p p = 0.016. Our results also indicated that eNOS3 -GT genotype is associated with CAD susceptibility with an OR = 2.65, and p T, KLF14 rs972283 G>A, miRNAs27a A>G, and eNOS3 rs1799983 G>T genotypes were associated with CAD susceptibility (p < 0.05). These findings require verification in future large-scale population based studies before these loci are used for the prediction and identification of individuals at risk to CAD. Weight control, physical activity, and smoking cessation are very influential recommendations given by clinicians to the at risk individuals to reduce or delay the development of CAD