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Inflammation 2013

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  • 1. InflammaTools: Complete research tools to study Inflammation regulation Raed Samara, Ph.D. R&D Scientist Sample & Assay Technologies
  • 2. Biology-focused solutions for pathway analysis • SABiosciences is now a Company -2- Sample & Assay Technologies
  • 3. Introduction: Inflammation ̣ Definition: a protective tissue response to tissue damage or microbes, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues. Epigenetic Changes Microbes/Infection Tissue Damage Acute Inflammation Infection Clearance Tissue Homeostasis mRNA Changes Cytokines & Chemokines Signaling Pathways Immune system composition Chronic Inflammation Pre-cancer & Cancer Chronic Inflammatory Diseases -3- Sample & Assay Technologies
  • 4. Inflammation experimental design & QIAGEN Gene Expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional Studies Reporter assays siRNA/shRNA Protein Expression Cytokine levels ̣ ̣ ̣ ̣ -4- Sample & Assay Technologies
  • 5. Basic principles of qRT-PCR: Overview Real-Time PCR Amplify and simultaneously quantify target DNA Reverse Transcription Real-Time PCR Amplify and simultaneously quantify mRNA For more information and webinars on real-time PCR, visit: www.sabiosciences.com/seminarlist.php -5- Sample & Assay Technologies
  • 6. Experimental overview: Gene expression analysis Isolate RNA Stimulate Cells RT-PCR Arrays or Assays Isolate DNA Data Analysis -6- Sample & Assay Technologies
  • 7. Gene expression in Inflammation ̣ Definition: a protective tissue response to tissue damage or microbes, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues. Epigenetic Changes Microbes/Infection Tissue Damage Acute Inflammation Infection Clearance Tissue Homeostasis mRNA Changes Cytokines & Chemokines Signaling Pathways Immune system composition Chronic Inflammation Pre-cancer & Cancer Chronic Inflammatory Diseases -7- Sample & Assay Technologies
  • 8. ̣ Inflammation studies: Gene expression The transcription factor cMyb is upregulated in naïve CD4 T cells. -8- Sample & Assay Technologies
  • 9. ̣ Inflammation studies: Gene expression The transcription factor cMyb is upregulated in naïve CD4 T cells. -9- Sample & Assay Technologies
  • 10. ̣ Inflammation studies: Gene expression The transcription factor cMyb is upregulated in naïve CD4 T cells. What is the effect of cMyb on naïve CD4 T cell maturation (to Th1, Th2, or Th3)? - 10 - Sample & Assay Technologies
  • 11. Inflammation studies: Gene expression The transcription factor cMyb is upregulated in naïve CD4 T cells. What is the effect of cMyb on naïve CD4 T cell maturation (to Th1, Th2, or Th3)? ̣ Experiment: Knockdown cMyb in human peripheral CD4+ T cells. Examine gene changes using the Human Th1-Th2-Th3 RT2 Profiler PCR Array (PAHS-034). ̣ - 11 - Sample & Assay Technologies
  • 12. Inflammation studies: Gene expression The transcription factor cMyb is upregulated in naïve CD4 T cells. What is the effect of cMyb on naïve CD4 T cell maturation (to Th1, Th2, or Th3)? ̣ Experiment: Knockdown cMyb in human peripheral CD4+ T cells. Examine gene changes using the Human Th1-Th2-Th3 RT2 Profiler PCR Array (PAHS-034). ̣ Results: ̣ - 12 - Sample & Assay Technologies
  • 13. Inflammation studies: Gene expression The transcription factor cMyb is upregulated in naïve CD4 T cells. What is the effect of cMyb on naïve CD4 T cell maturation (to Th1, Th2, or Th3)? ̣ Experiment: Knockdown cMyb in human peripheral CD4+ T cells. Examine gene changes using the Human Th1-Th2-Th3 RT2 Profiler PCR Array (PAHS-034). ̣ Results: ̣ Conclusions: Knockdown of cMyb in human peripheral CD4+ T cells decreased the expression of Th2 cytokine genes, and negatively affected Th2 cell maturation in primary human peripheral blood T cells. ̣ - 13 - Sample & Assay Technologies
  • 14. Gene expression in Inflammation ̣ Definition: a protective tissue response to tissue damage or microbes, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues. Epigenetic Changes Microbes/Infection Tissue Damage Acute Inflammation Infection Clearance Tissue Homeostasis mRNA Changes Cytokines & Chemokines Signaling Pathways Immune system composition Chronic Inflammation Pre-cancer & Cancer Chronic Inflammatory Diseases - 14 - Sample & Assay Technologies
  • 15. ̣ Inflammation studies: Gene expression Limited material Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of inflammation. - 15 - Sample & Assay Technologies
  • 16. ̣ Inflammation studies: Gene expression Limited material Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of inflammation. - 16 - Sample & Assay Technologies
  • 17. ̣ Inflammation studies: Gene expression Limited material Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of inflammation. What roles do TLRs play in MDF? - 17 - Sample & Assay Technologies
  • 18. Inflammation studies: Gene expression Limited material Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of inflammation. What roles do TLRs play in MDF? ̣ Experiment: Treat mice with DMH for 15 weeks to induce MDF. Collect RNA from MDF and normal colon. Convert mRNA to cDNA. Pre-amplify cDNA with the Mouse TLR PreAMP primer mixes (PBM-0018). Analyze differential gene expression with the Mouse TLR RT2 Profiler PCR Arrays (PAMM-018) ̣ - 18 - Sample & Assay Technologies
  • 19. Inflammation studies: Gene expression Limited material Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of inflammation. What roles do TLRs play in MDF? ̣ Experiment: Treat mice with DMH for 15 weeks to induce MDF. Collect RNA from MDF and normal colon. Pre-amplify cDNA with the Mouse TLR PreAMP primer mixes (PBM-0018). Analyze differential gene expression with the Mouse TLR RT2 Profiler PCR Arrays (PAMM018) ̣ Results: ̣ - 19 - Sample & Assay Technologies
  • 20. Inflammation studies: Gene expression Limited material Mucin depleted foci (MDF) are precancerous lesions of the colon that show signs of inflammation. What roles do TLRs play in MDF? ̣ Experiment: Treat mice with DMH for 15 weeks to induce MDF. Collect RNA from MDF and normal colon. Pre-amplify cDNA with the Mouse TLR PreAMP primer mixes (PBM-0018). Analyze differential gene expression with the Mouse TLR RT2 Profiler PCR Arrays (PAMM018) ̣ Results: ̣ Conclusion: MDF induces TLR2 gene expression in MDF compared to normal colon, suggesting a link between TLR2 and MDF. ̣ - 20 - Sample & Assay Technologies
  • 21. Inflammation experimental design & QIAGEN Gene Expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional Studies Reporter assays siRNA/shRNA Protein Expression Cytokine levels ̣ ̣ ̣ ̣ - 21 - Sample & Assay Technologies
  • 22. Epigenetics: Overview Activated Transcription Factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription Initiation Complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA Methylation Histone-DNA Interactions Ac Structural Gene Me Me Me DNA Methylation For more information and webinars on Epigenetics, visit: www.sabiosciences.com/seminarlist.php - 22 - Sample & Assay Technologies
  • 23. miRNA reagents from QIAGEN miRNeasy Isolation Target Identification miRNA Studies Expression miScript miRNA PCR Arrays 3’ UTR Reporters Function miRNA mimics & inhibitors - 23 - Sample & Assay Technologies
  • 24. miRNA in Inflammation ̣ Definition: a protective tissue response to tissue damage or microbes, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues. Microbes/Infection Tissue Damage Epigenetic Changes (miRNA) mRNA Changes Acute Inflammation Infection Clearance Tissue Homeostasis Cytokines & Chemokines Signaling Pathways Immune system composition Chronic Inflammation Pre-cancer & Cancer Chronic Inflammatory Diseases - 24 - Sample & Assay Technologies
  • 25. ̣ Inflammation studies: miRNA function Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1) are a class of players that mediate this resolution. - 25 - Sample & Assay Technologies
  • 26. ̣ Inflammation studies: miRNA function Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1) are a class of players that mediate this resolution. FASEB J. 25, 1–17 (2011) - 26 - Sample & Assay Technologies
  • 27. ̣ Inflammation studies: miRNA function Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1) are a class of players that mediate this resolution. Can RvD1-mediated resolution happen through miRNAs? FASEB J. 25, 1–17 (2011) - 27 - Sample & Assay Technologies
  • 28. Inflammation studies: miRNA function Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1) are a class of players that mediate this resolution. Can RvD1-mediated resolution happen through miRNAs? ̣ ̣ Experiment: Treat mice with zymosan A to induce acute inflammation (murine peritonitis), in the presence or absence of RvD1, for 4, 12, or 24 hours (representing onset, maximum, and resolution phases). Collect RNA from leukocytes. Analyze miRNA expression with the Mouse miRNome miRNA PCR Array (MIMM-216Z) FASEB J. 25, 1–17 (2011) - 28 - Sample & Assay Technologies
  • 29. Inflammation studies: miRNA function Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1) are a class of players that mediate this resolution. Can RvD1-mediated resolution happen through miRNAs? ̣ Experiment: Treat mice with zymosan A to induce acute inflammation (murine peritonitis), in the presence or absence of RvD1, for 4, 12, or 24 hours (representing onset, maximum, and resolution phases). Collect RNA from leukocytes. Analyze miRNA expression with the Mouse miRNome miRNA PCR Array (MIMM-216Z) ̣ ̣ Results: - 29 - Sample & Assay Technologies
  • 30. Inflammation studies: miRNA function Resolution of inflammation is a tightly controlled mechanism. The D-series resolvins (RvD1) are a class of players that mediate this resolution. Can RvD1-mediated resolution happen through miRNAs? ̣ Experiment: Treat mice with zymosan A to induce acute inflammation (murine peritonitis), in the presence or absence of RvD1, for 4, 12, or 24 hours (representing onset, maximum, and resolution phases). Collect RNA from leukocytes. Analyze miRNA expression with the Mouse miRNome miRNA PCR Array (MIMM-216Z) ̣ Results: ̣ ̣ Conclusion: RvD1 regulated resolution by controlling the expression of miR-219 and miR146b. These miRNAs are the first identified miRNAs in resolvin resolution circuits. - 30 - Sample & Assay Technologies
  • 31. Epigenetics: Overview Activated Transcription Factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription Initiation Complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA Methylation Histone-DNA Interactions Ac Structural Gene Me Me Me DNA Methylation - 31 - Sample & Assay Technologies
  • 32. DNA methylation in Inflammation ̣ Definition: a protective tissue response to tissue damage or microbes, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues. Microbes/Infection Tissue Damage Epigenetic Changes (DNA Methylation) mRNA Changes Acute Inflammation Infection Clearance Tissue Homeostasis Cytokines & Chemokines Signaling Pathways Immune system composition Chronic Inflammation Pre-cancer & Cancer Chronic Inflammatory Diseases - 32 - Sample & Assay Technologies
  • 33. Inflammation studies: DNA methylation ANCA Vasculitis is characterized by microvascular inflammation caused by activated neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are silenced in normal individuals. - 33 - Sample & Assay Technologies
  • 34. Inflammation studies: DNA methylation ANCA Vasculitis is characterized by microvascular inflammation caused by activated neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are silenced in normal individuals. - 34 - Sample & Assay Technologies
  • 35. Inflammation studies: DNA methylation ANCA Vasculitis is characterized by microvascular inflammation caused by activated neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are silenced in normal individuals. Does aberrant PR3 and MPO expression result from disrupted epigenetic silencing? - 35 - Sample & Assay Technologies
  • 36. Inflammation studies: DNA methylation ANCA Vasculitis is characterized by microvascular inflammation caused by activated neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are silenced in normal individuals. Does aberrant PR3 and MPO expression result from disrupted epigenetic silencing? Experiment: Isolate gDNA from neutrophils and treat with methylation-sensitive and methylation-dependent restriction enzymes (EpiTect Methyl DNA Restriction Kit 335451). Analyze DNA methylation by real-time PCR using primers specific for CpG islands in PR3 (MePH23428-1A) and MPO (MePH22382-1A). - 36 - Sample & Assay Technologies
  • 37. Inflammation studies: DNA methylation ANCA Vasculitis is characterized by microvascular inflammation caused by activated neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are silenced in normal individuals. Does aberrant PR3 and MPO expression result from disrupted epigenetic silencing? Experiment: Isolate gDNA from neutrophils and treat with methylation-sensitive and methylation-dependent restriction enzymes (EpiTect Methyl DNA Restriction Kit 335451). Analyze DNA methylation by real-time PCR using primers specific for CpG islands in PR3 (MePH23428-1A) and MPO (MePH22382-1A). Results: - 37 - Sample & Assay Technologies
  • 38. Inflammation studies: DNA methylation ANCA Vasculitis is characterized by microvascular inflammation caused by activated neutrophils. These activated neutrophils aberrantly express PR3 and MPO, which are silenced in normal individuals. Does aberrant PR3 and MPO expression result from disrupted epigenetic silencing? Experiment: Isolate gDNA from neutrophils and treat with methylation-sensitive and methylation-dependent restriction enzymes (EpiTect Methyl DNA Restriction Kit 335451). Analyze DNA methylation by real-time PCR using primers specific for CpG islands in PR3 (MePH23428-1A) and MPO (MePH22382-1A). Results: Conclusion: Methylation of CpG islands is a mechanism that controls the expression of MPO, but not the expression of PR3. The EpiTect Methyl System uses the MethylScreen™ Technology provided under license from Orion Genomics, LLC. - 38 - Sample & Assay Technologies
  • 39. Epigenetics: Overview Activated Transcription Factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription Initiation Complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA Methylation Histone-DNA Interactions Ac Structural Gene Me Me Me DNA Methylation - 39 - Sample & Assay Technologies
  • 40. Histone modification in Inflammation ̣ Definition: a protective tissue response to tissue damage or microbes, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues. Microbes/Infection Tissue Damage Epigenetic Changes (Histone Modifications) mRNA Changes Acute Inflammation Infection Clearance Tissue Homeostasis Cytokines & Chemokines Signaling Pathways Immune system composition Chronic Inflammation Pre-cancer & Cancer Chronic Inflammatory Diseases - 40 - Sample & Assay Technologies
  • 41. Inflammation studies: Histone modification Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays an important role in T cell differentiation in the periphery. - 41 - Sample & Assay Technologies
  • 42. Inflammation studies: Histone modification Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays an important role in T cell differentiation in the periphery. - 42 - Sample & Assay Technologies
  • 43. Inflammation studies: Histone modification Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays an important role in T cell differentiation in the periphery. Does TCF-1 control the differentiation of Th17 cells, and how? - 43 - Sample & Assay Technologies
  • 44. Inflammation studies: Histone modification Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays an important role in T cell differentiation in the periphery. Does TCF-1 control the differentiation of Th17 cells, and how? Experiment: Isolate naïve T cells from spleens of mice. Induce Th17 cell differentiation. Collect chromatin and process with the EpiTect ChIP one-day kit (334471) for chromatin immunoprecipitation with anti-histone antibodies. Detect precipitated DNA with qRT-PCR. - 44 - Sample & Assay Technologies
  • 45. Inflammation studies: Histone modification Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays an important role in T cell differentiation in the periphery. Does TCF-1 control the differentiation of Th17 cells, and how? Experiment: Isolate naïve T cells from spleens of mice. Induce aTh17 cell differentiation. Collect chromatin and process with the EpiTect ChIP one-day kit for chromatin immunoprecipitation with anti-histone antibodies. Detect precipitated DNA with qRT-PCR. Results: Open chromatin = increased expression - 45 - Sample & Assay Technologies
  • 46. Inflammation studies: Histone modification Th17 cells are a subset of CD4 T cells that differentiate from naïve T cells, secrete IL-17, and induce massive tissue inflammation. T cell factor 1 (TCF-1) is a transcription factor that plays an important role in T cell differentiation in the periphery. Does TCF-1 control the differentiation of Th17 cells, and how? Experiment: Isolate naïve T cells from spleens of mice. Induce Th17 cell differentiation. Collect chromatin and process with the EpiTect ChIP one-day kit (334471) for chromatin immunoprecipitation with anti-histone antibodies. Detect precipitated DNA with qRT-PCR. Results: Conclusion: TCF-1 mediates the repression of IL-17 locus during T cell development by chromatin modifications. - 46 - Sample & Assay Technologies
  • 47. Inflammation experimental design & QIAGEN Gene Expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional Studies Reporter assays siRNA/shRNA Protein Expression Cytokine levels ̣ ̣ ̣ ̣ - 47 - Sample & Assay Technologies
  • 48. Cignal Reporter Assays: Complete Solution Reporter assays: Overview Transcriptional Regulatory Elements (TRE), which establish the specificity of each reporter TATA box Reporter Construct GFP/firefly luciferase Tandem repeats of TRE EGFP TF FL Upstream Signaling Events - 48 - Sample & Assay Technologies
  • 49. Reporter assays for Inflammation ̣ Definition: a protective tissue response to tissue damage or microbes, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues. Epigenetic Changes Microbes/Infection Tissue Damage Acute Inflammation Infection Clearance Tissue Homeostasis mRNA Changes Cytokines & Chemokines Signaling Pathways Immune system composition Chronic Inflammation Pre-cancer & Cancer Chronic Inflammatory Diseases - 49 - Sample & Assay Technologies
  • 50. ̣ Inflammation studies: Reporter assay Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory activity of CPS is largely unknown. - 50 - Sample & Assay Technologies
  • 51. ̣ Inflammation studies: Reporter assay Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory activity of CPS is largely unknown. - 51 - Sample & Assay Technologies
  • 52. ̣ Inflammation studies: Reporter assay Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory activity of CPS is largely unknown. What signaling pathways are induced upon CPS recognition? - 52 - Sample & Assay Technologies
  • 53. Inflammation studies: Reporter assay Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory activity of CPS is largely unknown. What signaling pathways are induced upon CPS recognition? ̣ Experiment: Reverse-transfect cells with 10 dual-luciferase reporter assays, individually, on a reporter array (Cignal Finder 10-pathway Reporter Array; CCA-108L). Induce transfected cells with CPS, and measure luciferase levels to determine which pathway(s) is(are) activated by CPS ̣ - 53 - Sample & Assay Technologies
  • 54. Inflammation studies: Reporter assay Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory activity of CPS is largely unknown. What signaling pathways are induced upon CPS recognition? ̣ Experiment: Reverse-transfect cells with 10 dual-luciferase reporter assays, individually, on a reporter array (Cignal Finder 10-pathway Reporter Array; CCA-108L). Induce transfected cells with CPS, and measure luciferase levels to determine which pathway(s) is(are) activated by CPS ̣ Results: ̣ - 54 - Sample & Assay Technologies
  • 55. Inflammation studies: Reporter assay Neisseria Meningitidis infections can be rapidly fatal due to acute inflammatory responses that are mediated by capsular polysaccharides (CPS), but the innate immunostimulatory activity of CPS is largely unknown. What signaling pathways are induced upon CPS recognition? ̣ Experiment: Reverse-transfect cells with 10 dual-luciferase reporter assays, individually, on a reporter array (Cignal Finder 10-pathway Reporter Array; CCA-108L). Induce transfected cells with CPS, and measure luciferase levels to determine which pathway(s) is(are) activated by CPS ̣ Results: ̣ Conclusions: NFkB is the major signaling pathway activated in response to CPS. ̣ - 55 - Sample & Assay Technologies
  • 56. shRNA/siRNA function Activated Transcription Factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription Initiation Complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA Methylation Histone-DNA Interactions Ac Structural Gene Me Me Me DNA Methylation - 56 - Sample & Assay Technologies
  • 57. Gene knockdown for Inflammation ̣ Definition: a protective tissue response to tissue damage or microbes, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues. Epigenetic Changes Microbes/Infection Tissue Damage Acute Inflammation Infection Clearance Tissue Homeostasis mRNA Changes Cytokines & Chemokines Signaling Pathways Immune system composition Chronic Inflammation Pre-cancer & Cancer Chronic Inflammatory Diseases - 57 - Sample & Assay Technologies
  • 58. ̣ Inflammation studies: siRNA knockdown Resident alveolar macrophages (AM) constitute the first line of defense against invading lung Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience increased mortality and bacterial outgrowth and dissemination. - 58 - Sample & Assay Technologies
  • 59. ̣ Inflammation studies: siRNA knockdown Resident alveolar macrophages (AM) constitute the first line of defense against invading lung Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience increased mortality and bacterial outgrowth and dissemination. The Journal of Immunology, 2008, 180: 3594–3600. - 59 - Sample & Assay Technologies
  • 60. ̣ Inflammation studies: siRNA knockdown Resident alveolar macrophages (AM) constitute the first line of defense against invading lung Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience increased mortality and bacterial outgrowth and dissemination. What is the mechanism by which morphine does this? The Journal of Immunology, 2008, 180: 3594–3600. - 60 - Sample & Assay Technologies
  • 61. Inflammation studies: siRNA knockdown Resident alveolar macrophages (AM) constitute the first line of defense against invading lung Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience increased mortality and bacterial outgrowth and dissemination. What is the mechanism by which morphine does this? ̣ Experiment: MH-S cells (murine AM cells) were transfected with either a negative control SureSilencing shRNA or a SureSilencing shRNA plasmid for mouse TLR9. Levels of MIP-2 were measured after treating transfected cells with morphine and Streptococcus pneumoniae ̣ The Journal of Immunology, 2008, 180: 3594–3600. - 61 - Sample & Assay Technologies
  • 62. Inflammation studies: siRNA knockdown Resident alveolar macrophages (AM) constitute the first line of defense against invading lung Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience increased mortality and bacterial outgrowth and dissemination. What is the mechanism by which morphine does this? ̣ Experiment: MH-S cells (murine AM cells) were transfected with either a negative control SureSilencing shRNA or a SureSilencing shRNA plasmid for mouse TLR9. Levels of MIP-2 were measured after treating transfected cells with morphine and Streptococcus pneumoniae. ̣ Results: ̣ - 62 - Sample & Assay Technologies
  • 63. Inflammation studies: siRNA knockdown Resident alveolar macrophages (AM) constitute the first line of defense against invading lung Streptococcus pneumoniae. Studies have shown that morphine-treated mice experience increased mortality and bacterial outgrowth and dissemination. What is the mechanism by which morphine does this? ̣ Experiment: MH-S cells (murine AM cells) were transfected with either a negative control SureSilencing shRNA or a SureSilencing shRNA plasmid for mouse TLR9. Levels of MIP-2 were measured after treating transfected cells with morphine and Streptococcus pneumoniae. ̣ Results: ̣ Conclusion: Morphine reduces levels of Streptococcus pneumoniae -induced MIP-2 from AM cells in a TLR9-dependent manner. ̣ - 63 - Sample & Assay Technologies
  • 64. Inflammation experimental design & QIAGEN Gene Expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional Studies Reporter assays siRNA/shRNA Protein Expression Cytokine levels ̣ ̣ ̣ ̣ - 64 - Sample & Assay Technologies
  • 65. Chemokine expression in Inflammation ̣ Definition: a protective tissue response to tissue damage or microbes, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues. Epigenetic Changes Microbes/Infection Tissue Damage Acute Inflammation Infection Clearance Tissue Homeostasis mRNA Changes Cytokines & Chemokines Signaling Pathways Immune system composition Chronic Inflammation Pre-cancer & Cancer Chronic Inflammatory Diseases - 65 - Sample & Assay Technologies
  • 66. ̣ Inflammation studies: Chemokine levels Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective immunity responsible for this has not been defined. - 66 - Sample & Assay Technologies
  • 67. ̣ Inflammation studies: Chemokine levels Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective immunity responsible for this has not been defined. The Journal of Immunology, 2010, 184: 6275–6282 - 67 - Sample & Assay Technologies
  • 68. ̣ Inflammation studies: Chemokine levels Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective immunity responsible for this has not been defined. Could this be due to defective chemokine secretion leading to delayed priming of adaptive immunity? The Journal of Immunology, 2010, 184: 6275–6282 - 68 - Sample & Assay Technologies
  • 69. Inflammation studies: Chemokine levels Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective immunity responsible for this has not been defined. Could this be due to defective chemokine secretion leading to delayed priming of adaptive immunity? ̣ Experiment: Lung homogenates from TB-infected Diabetic mice were tested for chemokine levels using the Multi-Analyte ELISArray Kit. ̣ The Journal of Immunology, 2010, 184: 6275–6282 - 69 - Sample & Assay Technologies
  • 70. Inflammation studies: Chemokine levels Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective immunity responsible for this has not been defined. Could this be due to defective chemokine secretion leading to delayed priming of adaptive immunity? ̣ Experiment: Lung homogenates from TB-infected Diabetic mice were tested for chemokine levels using the ELISArray. ̣ Results: ̣ - 70 - Sample & Assay Technologies
  • 71. Inflammation studies: Chemokine levels Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective immunity responsible for this has not been defined. Could this be due to defective chemokine secretion leading to delayed priming of adaptive immunity? ̣ Experiment: Lung homogenates from TB-infected Diabetic mice were tested for chemokine levels using the Multi-Analyte ELISArray Kit. ̣ Results: ̣ Conclusion: Reduced levels of leukocyte chemoattractant factors including CCL2 and CCL5 at early timepoints post-infection could explain why diabetic mice are more prone to TB. ̣ - 71 - Sample & Assay Technologies
  • 72. Conclusions QIAGEN offers many methods to study molecular and cellular mechanisms involved in Inflammation: Gene Expression RT2 Profiler PCR Arrays & Assays RT2 PreAMP Primer Mixes Epigenetics miScript miRNA PCR System EpiTect Methyl qPCR Arrays EpiTect ChIP qPCR Arrays Functional Studies Cignal Reporter Assays SureSilencing shRNA Plasmid Protein expression ELISArray ̣ www.sabiosciences.com ̣ - 72 - Sample & Assay Technologies
  • 73. Keep up to date: Follow Pathway focused biology on Facebook Latest information on pathway and disease research, resources and demos. - 73 - Sample & Assay Technologies
  • 74. Thank you for attending! Would you like to try an Inflammation-related PCR Array? RT2 Profiler PCR Array Starter Pack miScript PCR Array Starter Pack PCR Arrays of any Pathway (FREE) • 2 96-well/100-well (2 samples) OR • 1 384-well (4 samples) • Required Reagents (w/ Purchase) • RT2 First-Strand cDNA Synthesis Kit, OR • RT2 SYBR Green Mastermix (2-Pack) Call 1-888-503-3187 for more information Email: support@SABiosciences.com (2012 US and Canada only) Webinar-related question? QIAWEBINARS@QIAGEN.COM - 74 - Sample & Assay Technologies