The Interaction Between Mycobacterium tuberculosis and Human Neutrophils.

Shalona Beeput, Willem Sturm

Abstract


Study Design

Specimens: The clinical isolates of Mycobacterium tuberculosis following will be used.

v 3 Beijing susceptible strains

v 3 New York - Beijing HN878 strains

v 2 KZN susceptible strains

v 3 KZN Multi-drug Resistant strains

v 3 KZN Extensively-drug Resistant (from surviving patients) strains

v 3 KZN Extensively-drug Resistant (from deceased patients) strains

v 3 Unique strains

v 3 F28 MDR strains

v 1 H37Rv Control strain

v 1 CDC1551 Control strain

Tests:

  • Nitroblue Tetrazolium (NBT) assay. This test measures the oxidative burst of the neutrophils on contact with pathogens. The production of intracellular superoxide anion and this will be used as an indication of the respiratory burst of neutrophils.
  • Ziehl-Neelsen staining technique. This stain will be used to determine whether or not phagocytosis of pathogens, by the neutrophils has occurred. This will be achieved by viewing samples under light microscopy after the samples have been stained.
  • Quantikine Myeloperoxidase (R&D Systems), Quantikine Human pro-Cathepsin B (R&D Systems), Human Lactoferrin (Kamiya Biomedical Company) and Quantikine Human neutrophil lipocalin-2 (R&D Systems) Immunoassay ELISA. This will be obtained commercially and applied to investigate the degranulation of neutrophils. Myeloperoxidase and Cathepsin B will be used as markers to measure the release the contents of primary granules and human Lactoferrin and human neutrophil Lipocalin will be used as markers to measure the release of the contents of secondary granules of neutrophils.
  • Transwell-24 well Permeable Supports Assays and the Under Agarose method. This will be preformed to investigate the chemotaxis of neutrophils. The Transwell assay will determine whether chemotaxis does occur or not. While the under agarose method will determine the distance migrated if chemotaxis does occur.