Oxidative burst

Neutrophils (& monocytes) produce hydrogen peroxide (through superoxide production) to kill phagocytosed bacteria. 

2O₂ + NADPH —> 2O₂^•– + NADP⁺ + H⁺

This reaction is catalaysed by NADPH oxidase. A series of reactions then generates hydrogen peroxide from this superoxide radical, and finally hypochlorous acid.

In vitro, this reaction can be visualised by Dihydrorhodamine (DHR), which is oxidised by H2O2 from normal, stimulated neurophils to give fluorescence (in the FITC channel). EDTA/ heparin blood must be used within 2 hours of collection, in room temparature all the time.

Gating neutrophils, lymphocytes and monocytes from whole blood

Before and after stimulation; note presence of DHR peak ('oxidative burst') in neutrophils and monocytes, and not in lymphocytes

The lack of superoxide produces a (weirdly named) disease.

Chronic granulomatous disease

Well, this has got (almost) nothing to do with granulomas (maybe a little, indrectly). This is a defect in several components of NADPH oxidase, resulting in inability to produce hydrogen peroxide. An X linked form (75%) & an autosomal recessive form (25%) occur.

Agent

XR: Mutation in phagocyte NAPH-O complex due to defect in gene for gp91phox.

AR: Defects in genes p47,67,22 phox or RAC2

It manifests as recurrent pyogenic infection with catalase +ve organisms.

Pathology

Three scenarios:

1. Normal neutrophils accumulate H2O2 in the phagosome containing ingested bacterium → MPO (myeloperoxidase) is delivered to the phagosome by degranulation, → H2O2 acts as a substrate for MPO to oxidize halide to hypochlorous acid and chloramines, which then kill the microbes. The quantity of hydrogen peroxide produced by normal neutrophils is sufficient to exceed the capacity of catalase, a hydrogen peroxide-catabolizing enzyme of many aerobic microorganisms.

2. When catalase +ve organisms such as E. coli gain entry into the CGD neutrophils, they are not exposed to hydrogen peroxide because the neutrophils do not produce it, and the hydrogen peroxide generated by microbes themselves is destroyed by their own catalase. Thus catalase-positive microbes, such as E. coli, can survive within the phagosome of the CGD neutrophil.

3. When CGD neutrophils ingest catalase negative organisms such streptococci or pneumococci, the organisms generate enough hydrogen peroxide to result in a microbicidal effect; i.e. they are killed by their own hydrogen peroxide.

(Ref: Williams, Hematology)

Thus there is a preponderane by recurrent infections by Catalse positive organisms.

CGD; note the lack of oxidative burst in stimulated neutrophils

Foxp3

Titration of FoxP3 (tagged with PE). Note the gradual and slow population shift over increasing doses of antibody.  (UU - unstimulated unstained, US - unstimulated stained, SS - stimulated & stained)

As the dose of antibody is inreased, the entire helper T cell population is taking up the stain (which is unwanted behavior)

There is no drop in median PE expression with doses upto 10 microL. This means there might be yet more antibody targets left to bind

In this case, 2.5 miroL dose which gives good positive population (2.86%) without staining the negative population. So we select this dose.

Antibody clone: 236A/E7, FoxP3 - PE

Transforming growth factor beta 1

Titration of anti TGFB1 antibody

Without stimulation

Titration plots for increasing doses of anti-TGFB1. Note the vertical line drawn at unstained (UU) and successive population shifts (non specific binding?). The ideal dose in this case must be close to 0.625 microL.

Doses ranging from 0.625 to 10 microL; note that entire cell population is moving

Plotting median FITC versus dose shows a continuously increasing trend. so no help from there!

With antigenic (MTb) stimulation

Does not make much of a difference

Thus, still undecided on the dose.

(Antibody clone TW4-9E7 bound to Alexa fluor 488, detected on FITC channel; cells - healthy peripheral blood mononuclear cells).