Fuentes, Ana-Lucia

In this study we compared the effects of the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS), flagellin, the synthetic bacterial triacylated lipopeptide Pam3-Cys-Ser-Lys4 (Pam3CSK4), Polyinosinic:polycytidylic acid (Poly I:C), and macrophage-activating lipopeptide (MALP-2), which are TLR4, TLR5, TLR1/2, TLR3, and TLR2/6 agonists, respectively, on cell morphology and phagocytosis of zymosan particles, derived from Saccharomyces cerevisiae, and rich in fungal PAMPs including beta-glucan, mannose, and chitin. LPS, Pam3CSK4, and MALP-2 induced an activated macrophage phenotype and enhanced zymosan phagocytosis. In contrast, flagellin and Poly I:C, respectively, had little effect on cell morphology and phagocytosis. We examined the role of scavenger receptor A (SR-A) on zymosan phagocytosis. Cells cultured in medium alone expressed SR-A, and LPS induced further expression of the receptor. We also observed inhibitory effects of scavenger receptor antagonists fucoidan, dextran sulphate, and Polyinosinic (Poly I), respectively, on zymosan phagocytosis of cells in medium alone and those pre-treated with LPS. We conclude that exposure to specific TLR ligands impacts both cellular morphology and phagocytic capacity, and that scavenger receptors contribute to zymosan ingestion as well as LPS-induced augmentation of phagocytosis.--Publisher.

In recent years, information on the diversity and abundance of microorganisms associated with the human body has significantly expanded. Recent research on the microbiota reveals its importance in the maintenance of normal host physiology and particular dysbioses have been associated with disturbances of gastrointestinal and metabolic health, brain function, and immunological responses. College students in health sciences and related programs, to our knowledge, receive little information regarding the microbiota and its importance in health. Adequate descriptions of the microbiota are only recently starting to appear in undergraduate human anatomy and physiology textbooks and most curricula fail to address the topic of microbiota with the attention that it deserves. Given the growing amount of evidence demonstrating the vital role of the microbiota in body function, it is crucial to educate students about the microbiota in undergraduate human anatomy and physiology courses for health practitioners. In this review, we present examples of recent findings illustrating the importance of the microbiota, and discuss how we may incorporate the microbiota into the teaching of human anatomy and physiology courses

Phagocytosis is a fundamental aspect of innate resistance against microbes, including fungi. In this study we investigated the significance of beta-glucan on the surfaces of zymosan particles, derived from Saccharomyces cerevisiae, during phagocytosis by RAW 264.7 macrophages. Phagocytosis was assessed in vitro by macrophage exposure to zymosan particles followed by cell staining and light microscopy. Macrophage ingestion of zymosan was dependent on cellular recognition of the particles' beta-glucans since laminarin, a soluble beta-glucan, inhibited phagocytosis in a concentration dependent manner when added to cell cultures. In contrast, the presence of another carbohydrate, mannan, had no effect on zymosan phagocytosis by cells. In addition we showed that LPS and dexamethasone had opposing effects on phagocytosis of zymosan. LPS significantly augmented ingestion while in contrast dexamethasone, like laminarin, suppressed it. The LPS-enhanced ingestion of zymosan was insensitive to the presence of laminarin in cell cultures, however dexamethasone partially ameliorated the effects of LPS on phagocytosis. Our findings confirm beta-glucan as an important ligand identified by macrophages and required for zymosan phagocytosis in naïve cells, but not in cells previously exposed to LPS.--Publisher.

The chapter, "Karyotype analysis of murine macrophages for undergraduate students" was written by the listed authors including Lynette B. Sigola (Douglas College Faculty). We have developed a karyotype analysis laboratory involving photomicroscopy which is suitable for use by students in biology courses at the undergraduate level. The laboratory activity uses the murine macrophage RAW 264.7 cell line maintained in vitro. In this laboratory activity, students benefit from an introduction to conditions required for cell culture (media, growth conditions, sterile technique), and use an inverted microscope to observe live cells in culture. We describe the use of colchicine to arrest dividing cells in metaphase when chromosomes are most condensed. Cells are then harvested, treated in a hypotonic solution, fixed, and then dropped onto glass microscope slides. The slides are then stained and mounted and sealed. Photomicrographs are taken for analysis. We discuss the significance of cell preparation for microscopic analysis in engaging students as active learners.

The chapter, "Study of cell form and function through microscopy: Macrophages in action for undergraduate students" was written by the listed authors including Lynette B. Sigola (Douglas College Faculty). Microscopy at the undergraduate level can be pursued over a wide range of activities, from a relatively passive experience where students use microscopy to look at prepared slides to record their findings in the form of sketch diagrams, to inquiry-based labs, where students learn to appreciate many dimensions of observation and discovery through microscopy. The laboratory activity we describe here, stemmed from our research on mechanisms of fungal recognition and phagocytosis of a murine macrophage cell line. Phagocytosis of zymosan particles (cell wall derivatives of the yeast Saccharomyces cerevisiae) by macrophages can be easily quantified, using a compound microscope, by counting the number of cells ingesting zymosan as well as the number of particles ingested by each particular cell. Phagocytosis can be assessed after different pre-treatments of both particles and macrophages, involving students in qualitative and quantitative data analysis. Furthermore, this research-inspired microscopy laboratory activity exposes undergraduates to many aspects of research, ranging from technical and logistical skills to ethical aspects of scientific inquiry.

Background: Fungal and bacterial coinfections are common in surgical settings; however, little is known about the effects of polymicrobial interactions on the cellular mechanisms involved in innate immune recognition and phagocytosis. <p>Materials and methods: Zymosan particles, cell wall derivatives of the yeast Saccharomyces cerevisiae, are used to model fungal interactions with host immune cells since they display carbohydrates, including beta-glucan, that are characteristic of fungal pathogens. Using in vitro cell culture, RAW 264.7 macrophages were challenged with zymosan, and phagocytosis determined via light microscopy. The effects of different concentrations of lipopolysaccharide (LPS) on zymosan phagocytosis were assessed. In addition, the transfer of supernatant from LPS-treated cells to naive cells, the effects of soluble carbohydrates laminarin, mannan, or galactomannan, and the impact of complement receptor 3 (CR3) inhibition on phagocytosis were also determined. <p>Results: LPS enhanced phagocytosis of zymosan in a dose-dependent manner. Transfer of supernatants from LPS-primed cells to naı¨ve cells had no effect on phagocytosis. Laminarin inhibited zymosan phagocytosis in naı¨ve cells but not in LPS-primed cells. Neither mannan, galactomannan, nor CR3 inhibition had a significant effect on ingestion of unopsonized zymosan in naı¨ve or LPS-treated cells. <p>Conclusions: Zymosan recognition by naı¨ve cells is inhibited by laminarin, but not mannan, galactomannan, or CR3 inhibition. LPS enhancement of phagocytosis is laminarin insensitive and not mediated by supernatant factors or zymosan engagement by the mannose or CR3 receptors. Our data suggest alternative mechanisms of zymosan recognition in the presence and absence of LPS.