Granulocyte Colony — Stimulating Factor Multiplies Normal Blood ROS Generation at Less than 1 μ g / l

Background: The neutrophils (PMN) are our main blood cells to combat fungi, bacteria, and fibrin. For normal function, an activated PMN generates a certain concentration of reactive oxygen species (ROS). If the generated blood ROS concentration is too low, then fungi, bacteria or fibrin might threaten the life of the patient, and it could be of great medical interest to stimulate PMN by physiologic drugs. Granulocyte-Colony Stimulating Factor (G-CSF) is a cell hormone that increases the cell number of PMN and that stimulates the individual PMN. The blood ROS generation assay (BRGA) is an innovative physiologic test to monitor the ROS generation of PMN in blood. Here the ROS generating action of G-CSF on normal PMN is quantified. Material and Methods: 40 μl 0 10.3 ng/ml (final conc.) G-CSF (in 5% human albumin) in black Brand® 781608 high quality polystyrene F-microwells was incubated in triplicate with 125 μl Hanks’ balanced salt solution (HBSS; modified without phenol red) and 10 μl normal citrated blood. Immediately (BRGA) or after 60 min (BRGA-60-) 10 μl 5 mM luminol sodium salt in 0.9% NaCl and 10 μl 0 or 36 μg/ml zymosan A in 0.9% NaCl was added. The photons were counted within 0 318 min (37 ̊C) in a photons-multiplying microtiter plate luminometer. At about 0.5 t-maxn (0.5 fold the time to normal maximum) the approx. SC200 of G-CSF was determined. Results and Discussion: The approx. SC200 of G-CSF on normal blood ROS generation was 0.2 μg/l (=20 IU/ml). In clinical situations where an increased blood ROS generation is pharmacologically required, few micrograms of G-CSF could be a sufficient dosage for an adult patient. The BRGA helps to find out the correct stimulating G-CSF dosage for each individual. An enhanced PMN function could favor a better clinical outcome in situations of wanted increase of the innate immunology or in cellular fibrinolysis. G-CSF plasma concentrations of 0.1 1 μg/l might favor singlet oxygen generation without immunosuppression or cell fragmentHow to cite this paper: Stief, T. (2018) Granulocyte Colony—Stimulating Factor Multiplies Normal Blood ROS Generation at Less than 1 μg/l. Optics and Photonics Journal, 8, 1-10. https://doi.org/10.4236/opj.2018.81001 Received: October 24, 2017 Accepted: January 6, 2018 Published: January 9, 2018 Copyright © 2018 by author and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access

SC200 of G-CSF on normal blood ROS generation was 0.2 µg/l (=20 IU/ml).In clinical situations where an increased blood ROS generation is pharmacologically required, few micrograms of G-CSF could be a sufficient dosage for an adult patient.The BRGA helps to find out the correct stimulating G-CSF dosage for each individual.An enhanced PMN function could favor a better clinical outcome in situations of wanted increase of the innate immunology or in cellular fibrinolysis.G-CSF plasma concentrations of 0.1 -1 µg/l might favor singlet oxygen generation without immunosuppression or cell fragment-

Results
In albumin samples, the BRGA maximum of 2389 RLU/s was reached after 124 min.In NaCl samples, the maximum of 1694 RLU/s was reached after 137 min.
At 318 min, the blood ROS generation was 51% or 37% of the maximum, respectively (Figure 1).µl Hanks' balanced salt solution (HBSS; modified without phenol red) and 10 µl normal citrated blood.10 µl 5 mM luminol sodium salt in 0.9% NaCl and 10 µl 36 µg/ml zymosan A in 0.9% NaCl were added.The photons were counted within 0 -318 min (37˚C) in a photons-multiplying microtiter plate luminometer (LUmo).In albumin samples the maximum of 2389 RLU/s was reached after 124 min, in NaCl samples the maximum of 1694 RLU/s was reached after 137 min.At 318 min the blood ROS generation was 51% or 37% of the maximum, respectively.The experiment was repeated twice, the standard deviations were <10%.In albumin samples, the BRGA-60-maximum of 6502 RLU/s was reached after 84 min.In NaCl samples, the maximum of 6254 RLU/s was reached after 84 min, too.At 264 min, the blood ROS generation was 43% or 22% of the maximum, respectively (Figure 2).This means that a protein-poor environment facilitates the down-regulation of the ROS generation.

Discussion
By contrast, in the BRGA, very low concentrations of G-CSF stimulate blood ROS generation.This could be of pharmacologic interest: in clinical situations where an increased blood ROS generation is pharmacologically required, few micrograms of G-CSF could be a sufficient dosage for an adult patient.The BRGA helps to find out the correct stimulating G-CSF dosage for each individual.
An enhanced PMN function could favor a better clinical outcome in situations of wanted increase of the innate immunology or in cellular fibrinolysis [9]- [17].
The normal plasma concentration of G-CSF is about 25 ± 20 pg/ml, and in acute infections, the G-CSF concentration can increase up to about 100 fold [18] [19] [20]; upon subcutaneous injection of 300 µg filgrastim, the G-CSF plasma concentration has increased about 1000 fold (blood half-life about 4 h), activating on neutrophils the CD11b/CD18 expression and the respiratory burst, on on endothelial cells the release of von Willebrand factor and F8, on hepatocytes the release of fibrinogen [21].There could be an enhanced generation of thrombin/systemically circulating micro-thrombi [14] [22].Thus, respective blood hemostasis, a G-CSF dosage of about 300 µg seems to be "too much of a good thing".The present work indicates that a G-CSF plasma concentration around 1 ng/ml (injection of about 3 µg G-CSF, i.e. 100 fold less than currently used) might favour the physiologic singlet oxygen generation (Figure 5) against pathogens without pathologic thrombin generation or immune suppressive side effects [23]- [47].The BRGA is a powerful tool to compare new analogues of G-CSF (e.g. the E. coli product filgrastim or the CHO product lenograstim).
Dose-finding studies are highly indicated to establish the range of beneficial G-CSF concentrations for each individual patient.

Figure 1 .
Figure 1.Blood ROS generation in presence of albumin or 0.9% NaCl in BRGA.40 µl 5% human albumin (Figure 1(a)) or 0.9% NaCl for control (Figure 1(b)) in black Brand® 781608 high quality polystyrene F-microwells were incubated in triplicate with 125 µl Hanks' balanced salt solution (HBSS; modified without phenol red) and 10 µl normal citrated blood.10 µl 5 mM luminol sodium salt in 0.9% NaCl and 10 µl 36 µg/ml zymosan A in 0.9% NaCl were added.The photons were counted within 0 -318 min (37˚C) in a photons-multiplying microtiter plate luminometer (LUmo).In albumin samples the maximum of 2389 RLU/s was reached after 124 min, in NaCl samples the maximum of 1694 RLU/s was reached after 137 min.At 318 min the blood ROS generation was 51% or 37% of the maximum, respectively.The experiment was repeated twice, the standard deviations were <10%.

Figure 2 .
Figure 2. Blood ROS generation in presence of albumin or 0.9% NaCl in BRGA-60-.40 µl 5% human albumin (Figure 2(a)) or 0.9% NaCl for control (Figure 2(b)) in black Brand® 781608 high quality polystyrene F-microwells were incubated in triplicate with 125 µl Hanks' balanced salt solution (HBSS; modified without phenol red) and 10 µl normal citrated blood.After 60 min 10 µl 5 mM luminol sodium salt in 0.9% NaCl and 10 µl 36 µg/ml zymosan A in 0.9% NaCl were added.The photons were counted within 0 -264 min (37˚C) in a photons-multiplying microtiter plate luminometer (LUmo).In albumin samples the maximum of 6502 RLU/s was reached after 84 min, in NaCl samples the maximum of 6254 RLU/s was reached after 84 min, too.At 264 min the blood ROS generation was 43% or 22% of the maximum, respectively.The experiment was repeated twice, the standard deviations were < 10%.
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