G-CSF Infusion for Stem Cell Mobilization Transiently Increases Serum Cell-Free DNA and Protease Concentrations.

G-CSF for stem cell mobilization increases circulating levels of myeloid cells at different stages of maturation. Polymorphonuclear cells (PMNs) are also mobilized in high numbers. It was previously reported that G-CSF primes PMNs toward the release of neutrophils extracellular traps (NETs). Since NETs are often involved in thrombotic events, we hypothesized that high G-CSF blood concentrations could enhance PMN priming toward NET formation in healthy hematopoietic stem cell donors, predisposing them to thrombotic events. However, we found that G-CSF does not prime PMNs toward NETs formation, but increases the serum concentration of cell-free DNA, proteases like neutrophils elastase and myeloperoxidase, and reactive oxygen species. This could possibly create an environment disposed to induce thrombotic events in the presence of additional predisposing factors.

Placental Protein 13 (Galectin-13) Polarizes Neutrophils Toward an Immune Regulatory Phenotype.

Termed as galectin-13, placental protein 13 (PP13) is exclusively expressed in the placenta of anthropoid primates. Research on PP13 in normal and pathologic pregnancies show alteration of PP13 concentrations in pregnancy affected by preeclampsia or gestational diabetes. Galectins are also described as potent immunomodulators, and PP13 regulates T cell function in the placenta. Therefore, this study aims to investigate the effects of PP13 on neutrophils; a cell type often ignored in pregnancy, but present in the uterus and placenta from the early stages of pregnancy. Since neutrophil function is dysregulated during pathologic pregnancies, a link between PP13 and neutrophil activity is possible. We determined that PP13 reduces the apoptosis rate in neutrophils. Also, PP13 increases the expression of PD-L1 and production of HGF, TNF-α, reactive oxygen species (ROS), and MMP-9 in these cells. This phenotype resembles one observed in permissive tumor neutrophils; able to sustain tissue and vessel growth, and inhibit T cell activation. At the same time, PP13 does not alter all neutrophil functions, i.e., extrusion of neutrophil extracellular traps, degranulation, phagocytosis, and ROS production following bacterial exposure. PP13 seems to play an essential role in regulating the activity of neutrophils in the placenta by polarizing them toward a placental-growth-permissive phenotype.

Computational Methodologies for the in vitro and in situ Quantification of Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) are a neutrophil defensive mechanism where chromatin is expelled together with antimicrobial proteins in response to a number of stimuli. Even though beneficial in many cases, their dysfunction has been implicated in many diseases, such as rheumatoid arthritis and cancer. Accurate quantification of NETs is of utmost importance for correctly studying their role in various diseases, especially when considering them as therapeutic targets. Unfortunately, NET quantification has a number of limitations. However, recent developments in computational methodologies for quantifying NETs have vastly improved the ability to study NETs. Methods range from using ImageJ to user friendly applications and to more sophisticated machine-learning approaches. These various methods are reviewed and discussed in this review.

Constructing artificial urinary conduits: current capabilities and future potential.

INTRODUCTION: Intestinal segments are currently used in reconstructive urology to create urinary diversion after cystectomy. Ileal conduit (IC) is the dominant type of urinary diversion. Nevertheless, IC is not an ideal solution as the procedure still requires entero-enterostomy to restore the bowel continuity. This step is a source of relevant complications that might prolong recovery time. Fabrication of artificial urinary conduit is a tempting idea to introduce an alternative form of urinary diversion which might improve cystectomy outcomes. AREAS COVERED: The aim of this review is to discuss available research data about artificial urinary conduit and identify major challenges for future studies. EXPERT OPINION: Fabrication of artificial urinary conduit is in range of current tissue engineering technology but there are still many challenges to overcome. There is an urgent need for studies to be conducted on large animal models with long follow up to expose the limitation of experimental strategies and to gather data for translational research.

Excessive Neutrophil Activity in Gestational Diabetes Mellitus: Could It Contribute to the Development of Preeclampsia?

Gestational diabetes mellitus is a transient form of glucose intolerance occurring during pregnancy. Pregnancies affected by gestational diabetes mellitus are at risk for the development of preeclampsia, a severe life threatening condition, associated with significant feto-maternal morbidity and mortality. It is a risk factor for long-term health in women and their offspring. Pregnancy has been shown to be associated with a subliminal degree of neutrophil activation and tightly regulated generation of neutrophil extracellular traps (NETs). This response is excessive in cases with preeclampsia, leading to the presence of large numbers of NETs in affected placentae. We have recently observed that circulatory neutrophils in cases with gestational diabetes mellitus similarly exhibit an excessive pro-NETotic phenotype, and pronounced placental presence, as detected by expression of neutrophil elastase. Furthermore, exogenous neutrophil elastase liberated by degranulating neutrophils was demonstrated to alter trophoblast physiology and glucose metabolism by interfering with key signal transduction components. In this review we examine whether additional evidence exists suggesting that altered neutrophil activity in gestational diabetes mellitus may contribute to the development of preeclampsia.

Neutrophil migration into the placenta: Good, bad or deadly?

Almost 2 decades have passed since the discovery that pregnancy is associated with a basal inflammatory state involving neutrophil activation, and that this is more overt in cases with preeclampsia, than in instances with sepsis. This pivotal observation paved the way for our report, made almost a decade ago, describing the first involvement of neutrophil extracellular traps (NETs) in a non-infectious human pathology, namely preeclampsia, where an abundance of these structures were detected directly in the placental intervillous space. Despite these remarkable findings, there remains a paucity of interest among reproductive biologists in further exploring the role or involvement of neutrophils in pregnancy and related pathologies. In this review we attempt to redress this deficit by highlighting novel recent findings including the discovery of a novel neutrophil subset in the decidua, the interaction of placental protein 13 (PP13) and neutrophils in modulating spiral artery modification, as well as the use of animal model systems to elucidate neutrophil function in implantation, gestation and parturition. These model systems have been particularly useful in identifying key components implicated in recurrent fetal loss, preeclampsia or new signaling molecules such as sphingolipids. Finally, the recent discovery that anti-phospolipid antibodies can trigger NETosis, supports our hypothesis that these structures may contribute to placental dysfunction in pertinent cases with recurrent fetal loss.

New Amniotic Membrane Based Biocomposite for Future Application in Reconstructive Urology.

OBJECTIVE: Due to the capacity of the amniotic membrane (Am) to support re-epithelisation and inhibit scar formation, Am has a potential to become a considerable asset for reconstructive urology i.e., reconstruction of ureters and urethrae. The application of Am in reconstructive urology is limited due to a poor mechanical characteristic. Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance, without affecting its unique bioactivity profile. This study evaluated biocomposite material composed of Am and nanofibers as a graft for urinary bladder augmentation in a rat model. MATERIAL AND METHODS: Sandwich-structured biocomposite material was constructed from frozen Am and covered on both sides with two-layered membranes prepared from electrospun poly-(L-lactide-co-E-caprolactone) (PLCL). Wistar rats underwent hemicystectomy and bladder augmentation with the biocomposite material. RESULTS: Immunohistohemical analysis (hematoxylin and eosin [H&E], anti-smoothelin and Masson's trichrome staining [TRI]) revealed effective regeneration of the urothelial and smooth muscle layers. Anti-smoothelin staining confirmed the presence of contractile smooth muscle within a new bladder wall. Sandwich-structured biocomposite graft material was designed to regenerate the urinary bladder wall, fulfilling the requirements for normal bladder tension, contraction, elasticity and compliance. Mechanical evaluation of regenerated bladder wall conducted based on Young's elastic modulus reflected changes in the histological remodeling of the augmented part of the bladder. The structure of the biocomposite material made it possible to deliver an intact Am to the area for regeneration. An unmodified Am surface supported regeneration of the urinary bladder wall and the PLCL membranes did not disturb the regeneration process. CONCLUSIONS: Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance without affecting its unique bioactivity profile.

Immunohistochemical localization of caffeine in young Camellia sinensis (L.) O. Kuntze (tea) leaves.

The anatomical localization of caffeine within young Camellia sinensis leaves was investigated using immunohistochemical methods and confocal scanning laser microscopy. Preliminary fixation experiments were conducted with young C. sinensis leaves to determine which fixation procedure retained caffeine the best as determined by high-performance liquid chromatography analysis. High pressure freezing, freeze substitution, and embedding in resin was deemed the best protocol as it retained most of the caffeine and allowed for the samples to be sectioned with ease. Immunohistochemical localization with primary anti-caffeine antibodies and conjugated secondary antibodies on leaf sections proved at the tissue level that caffeine was localized and accumulated within vascular bundles, mainly the precursor phloem. With the use of a pressure bomb, xylem sap was collected using a micro syringe. The xylem sap was analyzed by thin-layer chromatography and the presence of caffeine was determined. We hypothesize that caffeine is synthesized in the chloroplasts of photosynthetic cells and transported to vascular bundles where it acts as a chemical defense against various pathogens and predators. Complex formation of caffeine with chlorogenic acid is also discussed as this may also help explain caffeine's localization.