Wignot Research Projects

1. Wignot Research Projects • Inhibition and Activation of Pancreatic Enzymes (Lipase & Protease) • IgA Stimulation by Various Stimuli (ProbioticYogurt, Lemon Aroma) • Removal of Trimethylamine from Industrial Exhaust (“Fish Smell Project”)

2. Lipase Inhibition Proposed Mechanism for HAART Induced Diarrhea in HIV T.Wignot

3. Introduction -Pancreatic Lipase • pH optimum • Human = pH 8.0 • Porcine = pH 9.0 • Requirements for activity of pancrelipase • oil/water interface • bile salts (micelle) • colipase • Procolipase is activated by trypsin • Trypsinogen is activated by enterokinase

4. Lipase-Bile Salt (micelle)-Colipase Pignol D, Ayvazian L, Kerfelec B, Timmins P, Crenon I, Hermoso J, Fontecilla-Camps JC, Chapus C. Critical role of micelles in pancreatic lipase activation revealed by small angle neutron scattering. J BiolChem 275 (6): 4220-4224 (2000).

5. Lipase-Bile Salt (micelle)-Colipase Pignol D, Ayvazian L, Kerfelec B, Timmins P, Crenon I, Hermoso J, Fontecilla-Camps JC, Chapus C. Critical role of micelles in pancreatic lipase activation revealed by small angle neutron scattering. J BiolChem 275 (6): 4220-4224 (2000).

6. Lipase Reaction

7. Possible Mechanism for PI Induced Diarrhea • Undigested fat can cause diarrhea • Protease Inhibitors directly inhibit: • Pancreatic lipase • Direct inhibition of enzyme • Interferes with binding of colipase • Interferes with binding to bile salt micelle • Disrupts oil/water interface

8. Aims of Phase I Research Program • Test the inhibitory activity of HAART drugs on: • pancreatic lipase • Determine the reactivating effect of colipase on drug inhibited lipase reaction mixtures

9. Phase I Results • Inhibition of Pancrelipase • Nucleoside Reverse Transcriptase Inhibitors • No significant inhibition with any NRTI drug • Non-Nucleoside Reverse Transcriptase Inhibitors • % inhibition at physiological concentration • Sustiva® 26% • Rescripta® 18%

10. Phase I Results cont. • Inhibition of Pancrelipase • Protease Inhibitors • % inhibition at physiological concentration • Agenerase ®solution 100% • Agenerase ®capsule 99% • Fortovase ® 74% • Norvir ® 73% • Viracept ® 72% • HAART Cocktails • No difference in inhibition of pancrelipase by cocktails containing PI compared to single PI

11. Phase I Results cont. • Inhibition of Pancrelipase • Protease Inhibitors • Addition of pancrelipase to overcome inhibition • Colipase reactivation

12. Recovery of Pancrelipase Activity After Inhibition by Protease Inhibitors • Point A • Time 0-enzyme added to substrate • Point B • 5 minutes-protease inhibitor added to reaction mixture • Point C • 12 minutes-additional enzyme added to reaction mixture

13. Specific Drug Results

14. Reactivation of Pancrelipase by Colipase • A: No Colipase • Pancrelipase + protease inhibitor • B : With Colipase • Pancrelipase +protease inhibitor +colipase

15. Specific Drug Results

16. Phase I Conclusions • Agenerase, Fortovase, Norvir, and Viracept inhibit pancrelipase at physiological concentration • The inhibition can be overcome by addition of pancrelipase • The addition of excess colipase can reverse the inhibition of Agenerase • Drug cocktails containing PI inhibit pancrelipase to the same extent as individual PI

17. Future Studies • Determine lipase inhibition by • Excipient D-a-tocopherylpolyethylene glycol 1000 succinate (TPGS) • Antibiotics that have diarrhea side effects • Purify pancreatic lipase and colipase

18. IgA Stimulation • Examine changes in levels of Immunoglobulin A (IgA) when a subject is exposed to various external stimuli • Probiotic Yogurt • Green tea • Lemon Aroma

19. Many studies on Stress & IgA • High levels of stress correlated with LOWER IgA levels • Low levels of stress correlated with HIGHER levels of IgA • Probiotics • Microorganisms that improve health • Some products claim to “boost” immune response

20. Antibody found in serum and external body secretions Humoral immunity – produced by B cells (specifically plasma cells) One of the five antibody isotypes (IgA, IgD, IgE, IgG and IgM) Body’s initial defensive counterattack against antigen invasion What is IgA? Image from: http://img.tfd.com/vet/thumbs/gr222.jpg http://www.medterms.com/script/main/art.asp?articlekey=21061prepared by J. Woloski

21. Objective • To examine levels of Immunoglobulin A (IgA) • In saliva (human oral fluid) before and after eating a single 4-oz container of yogurt. • Measure baseline IgA (before consumption) • Measure IgA 30 minutes after consumption • Short term response • Measure IgA after eating a single container of yogurt daily for a one week period • Long term response

22. Capture or Sandwich ELISA • Antibody Capture • Ab (orange) bound to surface and standard OR sample (saliva) containing Ab (green) is added • Wash • Add labeled Ab (blue with star) • wash • Add substrate • Positive test (presence of Ab) would result in color formation

23. Tetramethybenzidine (TMB) Substrate Reaction

24. Plate reading via microtiter plate reader • Set for 450 nm for TMB

25. Conclusions • Yogurt Study • Over a 30-minute interval, no statistically significant differences observed in salivary IgA concentrations in any of the groups • After a week’s time, no statistically significant differences observed in salivary IgA concentrations in any of the groups • Green Tea Study • A decrease in IgA was observed for the green tea and iced tea 30 and 60 min after consumption • Lemon Aroma • Inconclusive

26. Additional and Future Work • Test a new stimulus and analyze its effects on IgA • Repeat lemon aroma • Probiotc supplements • Red wine

27. “Fish Smell” Project • Catawissa PA is the home of a fishmeal processing plant • One of the by-products of the production process is trimethylamine • Dead fish smell

28. Capture gaseous trimethylamine Dowex® resin Scope of Project

29. Future Work • Quantitate • Develop process that can be used at plant