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In this episode we look at the work of Professor Robert C Allen, who over the past four decades, has invented techniques for quantifying the activities of specialised immune cells – the phagocytic leukocytes and macrophages. Allen has investigated how neutrophil respiratory burst metabolism transforms oxygen to a microbe-killing agent, which can be detected by the associated light emission. Building on this, he is now developing an assay to measure the function of the immune system using the tiniest drop of blood.
Oxygen is essential to the survival of many organisms on Earth, including humans. In our bodies, however, oxygen also acts as a powerful defence weapon against microbial invaders. In the blood, red cells known as erythrocytes carry oxygen molecules throughout the body as required for metabolism. The white cells, known as neutrophils, respond to microbe infections by migrating to the site of infection, contacting and ingesting the microbe (a process known as ‘phagocytosing’), and then converting oxygen to reactants responsible for combustive microbicidal oxygenations. Neutrophil oxygenating activity is fast and focused on killing microbial pathogens.
Through a series of elegant studies, Professor Robert C Allen has shown that the key to understanding the role of oxygen within the complex biochemical machinery of our immune system is linked to the unique electronic structure of the oxygen molecule. Neutrophils change the spin multiplicity state of the oxygen molecule by modifying the distribution of the frontier orbitals electrons. In neutrophils, the chemically inert triplet multiplicity oxygen molecule found in our atmosphere is converted to the metastable electronically excited singlet multiplicity oxygen molecule which readily reacts with the singlet multiplicity biomolecules of the microbe. This results in the combustive oxygenation reactions responsible for microbial killing. Allen’s central premise is that reactions of singlet oxygen with the singlet multiplicity molecules of microbes are ‘spin allowed’ and highly exergonic. Such oxygenation reactions can yield electronically excited singlet multiplicity carbonyl products that relax to ground state by light emission, or ‘chemiluminescence’.
In the conventional chemical process of burning, energy is applied to a singlet multiplicity fuel molecule causing homolytic bond cleavage yielding two radical doublet products. The doublet radicals produced can participate in doublet-triplet reactions with ground state triple oxygen yielding a doublet product capable of reacting with additional triple oxygen in a process of radical propagation.
Instead of radicalizing the microbial substrate molecule to facilitate reactivity via doublet-triplet reaction with propagation, neutrophils de-radicalize paramagnetic triple oxygen to create diamagnetic singlet oxygen – thus allowing its participation in singlet-singlettes reaction with the singlet molecules of microbes. Spin must be conserved for combustion of any type, either burning or neutrophil dioxygenation. Combustions have exergonicities sufficient for light emission.
Chemiluminescence allows non-destructive measurement of phagocyte microbicidal oxygenation reactions. The sensitivity and specificity of luminescence measurements of phagocyte redox metabolism and haloperoxidase activity are greatly increased by using chemiluminigenic probes, that is, organic molecules susceptible to dioxygenations yielding electronically excited singlet carbonyl functions and photon emission. In this way, lucigenin luminescence requires reductive dioxygenation and allows high sensitivity and specific measurement of phagocyte NADPH oxidase activity. Luminol luminescence requires simple dioxygenation. Luminol permits high-sensitivity luminescence measurement of neutrophil haloperoxidase action, but it is not haloperoxidase specific. Luminol can measure oxidase activities in macrophages lacking haloperoxidase and in haloperoxidase-deficient heterophile leukocytes. In an influential paper published in 1986, Allen demonstrated the use of the chemiluminigenic probes lucigenin and luminol for high sensitivity and differential quantification of phagocyte oxidase and myeloperoxidase activities, respectively.
Allen’s method provides information on immune system dynamics that can be applied to investigating how the body responds to pathogens under a variety of conditions and environments. Intriguingly, these are not limited to life on Earth. In recent work carried out on the International Space Station, Allen’s approach has been used to monitor the reactions occurring in respiratory burst conditions in mammalian macrophages, a type of tissue phagocyte that play an important role in the immune system by engulfing and digesting pathogens such as microbes, cancer cells, and foreign substances. This study has shown that macrophages adapt very quickly, literally within seconds, to microgravity after an initial inhibitory phase of the respiration burst reactions. This is an important finding, which sheds light onto the long-standing question of why many astronauts can spend protracted periods of time in space with no apparent adverse effects on their health, despite the severe stress mammalian cells experience in microgravity conditions.
The human immune system is an intricately complex network, characterized by the dynamic interplay of genes, proteins, cells, and tissues. It is a vast and sophisticated architecture, billions of times larger than the human genome. This complexity is further accentuated by individual variations among people and by the continual modulation influence of factors such as age, genetics, and environmental conditions. This intricate system serves as the foundation for crucial health interventions, encompassing vaccines and state-of-the-art immunotherapies.
Advanced computing capabilities combined with the classification statistic method of discriminant analysis facilitate investigation of immune system functions. Chemiluminigenic probing allows measurement and differentiation of resting and stimulated neutrophil oxidase and haloperoxidase functions and permits estimation of the ratio of circulating (or COR) to maximal primed neutrophil opsonin receptor (or MOR) expression. The COR-to-MOR ratio gauges the state of inflammation in the body. Metric acquisitions require only tiny (sub-microliter) quantities of blood or tissue fluids. Discriminant analysis of temporally acquired data provides information applicable to research and infectious disease clinical management. Such temporal analysis is also relevant to monitoring of bone marrow myelopoiesis.
Allen and collaborators have demonstrated the utility of composite chemiluminigenic probing with discriminant analysis. Recent efforts have been directed to procedural and technical simplification regarding minimal blood specimen collection, rapid dilution, and direct luminescence measurement using a 96-well microplate format. Blood or fluid undergoes a thousand-fold dilution with buffered medium that reconstitutes divalent cations and obviates the effect of anticoagulant.
Less than a microliter of specimen is directly contacted with appropriate chemiluminigenic probes, with or without immune primers, and with or without chemical or phagocytic stimuli. Light emission is measured by an automated Berthold microplate luminometer and the composite data is collected and used for discriminant analysis. Testing is robust, yields reproducible results, and can be adapted to point-of-care testing using a hand-held or portable luminometer.
Professor Allen’s focus is now on the creation of a point-of-care test tool for the immune system and make it available for general use.
As such, he has shown that neutrophil number and function can be rapidly assessed in real-time using a sub-microliter volume of blood or body fluids by dilution and luminometry measurement. Such measurements are applicable to the clinical laboratory environment or any venue where a point-of-care luminescence detection device can operate.
That’s all for this episode – thanks for listening. Links to the original research can be found in the show notes for this episode. And, as always, stay subscribed to Research Pod for more of the latest science.
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