The Effect of Beta Glucan on Neutrophil Chemotaxis: Role of PI3Kinase

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The Effect of Beta Glucan on Neutrophil
Chemotaxis Role of PI-3-Kinase Leon Edward
Cushenberry, Jr. McNair Scholars
Program University of California, Davis
Jonathan Reichner, Ph.D. Liz Lavigne, Ph.D.
Department of Surgical Research Rhode Island
Hospital Brown University
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BACKGROUND - NEUTROPHILS

• A part of the innate immune system
• Most abundant leukocyte
• Originates in bone marrow
• Polymorphonuclear granulocytes
• Relatively short lifespan
• Acute inflammation

Figures taken from Junqueira Basic Histology
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EXTRAVASATION

• Steps
• Rolling
• Activation by chemical stimuli
• Arrest and adhesion to vessel wall
• Transmigration into tissue

Adapted from www.helsinki.fi/ml/
biotiet/biokemia/lag/Fig9.html
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CHEMOTAXIS

• Chemically induced directed movement
• Based on a gradient
• Migration to site of injury or infection
• Chemotactic factors (i.e. C5a, fMLP, IL-8) serve
  as distress signals.

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BETA GLUCAN

• Glucose polymer present in yeast, fungi, plants,
  some bacteria
• ß(1?3) backbone that gives bioactivity
• Documented enhancement of neutrophil chemotaxis
  to C5a and IL-8

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PHOSPHOINOSITIDE-3-KINASE (PI3K)

• Cytosolic cell signaling protein
• Heterodimeric protein
• There are 4 isoforms ?, ?, ?, and ?
• Class 1A p110 ?, ?, and ?
• Class 1B p110 ?
• All PI3K isoforms are expressed in neutrophils

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PHOSPHOINOSITIDE-3-KINASE (PI3K)

• Activates PI3K cell signaling pathway leading to
  proliferation, apoptosis, cell motility, and
  adhesion
• Responsible for directed but not random migration
  of neutrophils

Figure taken from Sandhu, et al.
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RESEARCH QUESTION

• Is the PI3K cell signaling pathway involved in
• neutrophil chemotactic migration to fMLP in the
• presence of Beta Glucan?
• Hypothesis
• The Beta Glucan effect on neutrophil
  chemotaxis will be attenuated with inhibition of
  PI3K?.
• Specific Aims
• Test neutrophil chemotactic migration to fMLP in
  the presence of glucan.
• Test the effect of PI3K inhibition on neutrophil
  chemotactic migration to fMLP in glucan.

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METHODS

• In vitro study
• Neutrophil Preparation
• Human blood
• Centrifugation steps to isolate neutrophils
• Beta Glucan
• Added to the agar solution and coating of the
  slide

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METHODS

• Under Agarose Migration Assay

• Slides are coated with fibronectin and agar
• Three wells are punched
• Cells migrate under agar layer
• 2 hour incubation

PBS
Cells
Chemoattractant (fMLP )
ß glucan
-ß glucan
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MIGRATION ASSAY
Cell count area
fMLP
PBS
Random migration
Migrating cells viewed under inverted microscope
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RESULTS
cells migrating
beta glucan
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RESULTS
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CONCLUSIONSHypothetical Model
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SUMMARY

• Beta glucan enhances migration to fMLP.
• Beta glucan interaction with PI3K is
  inconclusive.
• Limitations
• Human study
• Chemoattractant concentrations

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ACKNOWLEDGEMENTS

• Jonathan Reichner, Ph.D. - Brown University
• Liz Lavigne, Ph.D. - Brown University
• Members of the Reichner Lab
• University of California, Davis McNair Scholars
  Program
• The Leadership Alliance at Brown University

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The Effect of Beta Glucan on Neutrophil
Chemotaxis Role of PI-3-Kinase Leon Edward
Cushenberry, Jr. McNair Scholars
Program University of California, Davis
Jonathan Reichner, Ph.D. Liz Lavigne, Ph.D.
Department of Surgical Research Rhode Island
Hospital Brown University