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Background . The study of red bone marrow in preclinical studies is an important component of the study program of the drug. Objective . To compare the results obtained in parallel studies of the material from the same animal by two different methods for rats, rabbits and mice (cytological examination of the material obtained from the femur and histological examination of the material obtained from the sternum). Design and methods . Bone marrow obtained from intact animals was studied. Cytological preparation was stained according to Romanowski–Giemsa, histological preparation was prepared in the usual way and stained with hematoxylin-eosin. Stained preparations were examined by microscope. Results . As a result of this study was sets of myelograms with leuko-erythroblast ratio and neutrophil maturation index. Conclusion . A number of conclusions were made: when conducting preclinical studies, the most appropriate can be considered a bone marrow sampling from the femur; the fence can be carried out both for cytological examination, and for histological; taking into account the high variability of indicators in preclinical studies of myelograms, it is necessary to have control groups of (intact) animals of the same species that did not receive the study drug; Myelograms in rabbits turned out to be the least varied, both according to the results of our own studies (with histological and cytological studies), and when compared with literature data, which allows us to recognize rabbits as the best object for studying the composition and function of red bone marrow; when comparing the literature data, significant patterns in the number of cells at different age periods were not observed in the studied animal species, and, therefore, in preclinical studies using KKM analysis, animals of any age can be used.

Neutrophils play a crucial role in establishing inflammation in response to an infection or injury, but their production rates, as well as blood and tissue residence times, remain poorly characterized under these conditions. Herein, using a biomaterial implant model to establish inflammation followed byin vivotracking of newly formed neutrophils, we determine neutrophil kinetics under inflammatory conditions. To obtain quantifiable information from our experimental observations, we develop an ordinary differential equation-based mathematical model to extract kinetic parameters. Our data show that in the presence of inflammation resulting in emergency granulopoiesis-like conditions, neutrophil maturation time in the bone marrow and half-life in the blood reduces by about 40%, compared to non-inflammatory conditions. Additionally, neutrophil residence time at the inflammatory site increases by two-fold. Together, these data improve our understanding of neutrophil kinetics under inflammatory conditions, which could pave the way for therapies that focus on modulating in vivo neutrophil dynamics.

SUMMARY This protocol describes a rapid and efficient feeder-, serum-, and xeno-free method for neutrophil generation from hiPSCs using ETV2 modified mRNA (mmRNA), which directs hematoendothelial programming of hiPSCs. Hematoendothelial progenitors were cultured with GM-CSF, FGF-2, and UM171 to expand myelomonocytic progenitors, followed by treatment with G-CSF and retinoic acid agonist Am580 to induce neutrophil maturation. This protocol is suitable for generating functional neutrophils from iPSCs to interrogate the role of genes in a neutrophil development and function. For complete details on the use and execution of this protocol, please refer to Brok-Volchanskaya et al. (2019)., Graphical Abstract This protocol describes a rapid and efficient feeder-, serum-, and xeno-free method for neutrophil generation from hiPSCs using ETV2 modified mRNA (mmRNA), which directs hematoendothelial programming of hiPSCs. Hematoendothelial progenitors were cultured with GM-CSF, FGF-2, and UM171 to expand myelomonocytic progenitors, followed by treatment with G-CSF and retinoic acid agonist Am580 to induce neutrophil maturation. This protocol is suitable for generating functional neutrophils from iPSCs to interrogate the role of genes in a neutrophil development and function.

Human bone-marrow-on-a-chip improves drug studies of hematopoietic cell function and identifies a neutrophil maturation defect in a genetic disorder.

Neutrophil serine proteases (NSPs), such as neutrophil elastase (NE), are activated by dipeptidyl peptidase 1 (DPP1) during neutrophil maturation. High NSP levels can be detrimental, particularly in lung tissue, and inhibition of NSPs is therefore an interesting therapeutic opportunity in multiple lung diseases, including chronic obstructive pulmonary disease (COPD) and bronchiectasis. We conducted a randomized, placebo-controlled, first-in-human study to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of single and multiple oral doses of the DPP1 inhibitor AZD7986 in healthy subjects. Pharmacokinetic and pharmacodynamic data were analyzed using nonlinear mixed effects modeling and showed that AZD7986 inhibits whole blood NE activity in an exposure-dependent, indirect manner-consistent with in vitro and preclinical predictions. Several dose-dependent, possibly DPP1-related, nonserious skin findings were observed, but these were not considered to prevent further clinical development. Overall, the study results provided confidence to progress AZD7986 to phase II and supported selection of a clinically relevant dose.

In this issue of Immunity, Riffelmacher et al. (2017) show that autophagy is necessary for the release of free fatty acids from intracellular stores within neutrophil precursor cells. This limits glycolysis, increases oxidative phosphorylation, and is essential for neutrophil maturation.

Background: Altered neutrophil maturation patterns have been reported useful for identification of myelodysplastic syndromes (MDS). Methods: Neutrophil maturation patterns based on CD11b, CD13, and CD16 were visually and numerically evaluated in 19 control, 23 MDS, 37 nondiagnostic for MDS (NDM) specimens, and 19 also processed 1 and 2 days subsequently. Results: In contrast to maturation patterns illustrated previously by others as “normal,” 84% of controls displayed diminished acquisition of CD16, imparting a contracted appearance. Such divergence from published “normal” patterns was usually mild-moderate, considered nonspecific, and associated with delayed processing: longer intervals between collection and processing (median 20.5 vs. 5.2 h), and following 1 and 2 days delay. Findings restricted to nonspecific contraction were found in 56% MDS and 78% NDM specimens. Evaluation for aberrant patterns was still performed with mild-moderate contraction present, but concern for over interpretation led to use of an equivocal-aberrant category. Nine cases had aberrant or equivocal-aberrant patterns (seven MDS, two NDM) with distinct visual alterations that differed from nonspecific contraction and had numerical evidence for a left shift: myeloblasts increased (67%) and least mature neutrophils (CD11b-/low, CD16-/low) increased (78%). Although evidence for a left shift was associated with MDS, it was also seen in NDM specimens with a synchronous left shift. Conclusions: Neutrophil maturation patterns that diverge from previously illustrated “normal” patterns, not specific for MDS, may be common in some settings. Laboratories seeking to implement FC evaluation for MDS must determine which findings have sufficient specificity for MDS within their own practice and patient population. © 2012 International Clinical Cytometry Society

The biological functions of avian cathelicidins are poorly defined. In mammals, cathelicidins have shown to possess potent broad-range antimicrobial activity as well as immunomodulatory activities. Therefore, we investigated the microbicidal activities and localization of Cathelicidin-2 in non-infected and Salmonella-challenged broiler chickens. Using immunohistochemistry, Cathelicidin-2 was shown to be abundantly present in heterophils, localized in the large rod-shaped granules, but absent in other peripheral blood cells and intestinal epithelial cells. Cathelicidin-2 synthesis was observed to be initiated at the early promyelocyte stage. Considerable infiltration of Cathelicidin-2 containing heterophils was observed in the jejunum of Salmonella enteritidis-challenged broilers within 8 h post-infection. Heterophils were shown to release mature Cathelicidin-2 peptide upon stimulation with Salmonella-derived LPS in a time-dependent way. Processing of the Cathelicidin-2 precursor was mediated by serine proteases with a divalent cation dependency. Cathelicidin-2 peptide showed potent bactericidal and fungicidal activity against all tested microorganisms, including chicken-specific Salmonella isolates. These results underscore the importance of avian heterophils as a first line of defence against invading pathogens and implicate that via heterophil-mediated release, cathelicidins may greatly contribute to avian innate immunity.

About 100 years ago, Eli Metchnikoff had already expressed the importance of phagocytic cells in host defense. Today everyone admits that neutrophils are playing the most important role. Neutrophils migrate near the invading microorganisms, ingest them, kill them and digest them. Number and quality of neutrophils must be adequate in order to fulfill its function. Abnormal neutrophil function can be thought of as intrinsic cell defects, defects due to mediators acting on the cell and abnormalities related to shifts in populations of normal cells. The recent reviews of abnormal processes of neutrophil maturation, margination, chemotaxis, phagocytosis and killing will be explained in this capter.

1. 1.|The post-nuclear particulate fractions of highly enriched mature and immature neutrophil preparations were dissolved in buffer containing sodium dodecyl sulfate and analyzed for protein and glycoprotein components by polyacrylamide gel electrophoresis. 2. 2.|The electrophoretic patterns of mature and immature neutrophils showed pronounced differences in both protein and glycoprotein bands. There were 12 prominent protein bands in the patterns of mature neutrophils which also stained for glycoproteins. With one exception, these bands were either greatly reduced in intensity or undetectable at corresponding positions in the patterns of immature cells. The patterns of immature neutrophils revealed three prominent glycoprotein bands, two of which were absent and the third was greatly reduced in intensity at corresponding position in the mature cell patterns. 3. 3.|The results indicate that a number of new glycoproteins appear in the later stages of neutrophil maturation, whereas other glycoproteins, present in immature cells, are either lost or greatly reduced in amount. These changes are presumably related to the development of specific cell functions that also appear in later stages of cell maturation.