Lipopolysaccharide-mediated enhancement of zymosan phagocytosis by RAW 264.7 macrophages is independent of opsonins, laminarin, mannan, and complement receptor 3.

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. 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 naïve cells, the effects of soluble carbohydrates laminarin, mannan, or galactomannan, and the impact of complement receptor 3 (CR3) inhibition on phagocytosis were also determined. RESULTS: LPS enhanced phagocytosis of zymosan in a dose-dependent manner. Transfer of supernatants from LPS-primed cells to naïve cells had no effect on phagocytosis. Laminarin inhibited zymosan phagocytosis in naïve cells but not in LPS-primed cells. Neither mannan, galactomannan, nor CR3 inhibition had a significant effect on ingestion of unopsonized zymosan in naïve or LPS-treated cells. CONCLUSIONS: Zymosan recognition by naïve 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.

Immunoproteomic discovery of Mycobacterium bovis antigens, including the surface lipoprotein Mpt83 as a T cell antigen useful for vaccine development.

Tuberculosis (TB) is one of the leading causes of death from infectious diseases, killing approximately 1.3 million people worldwide in 2022 alone. The current vaccine for TB contains a live attenuated bacterium, Mycobacterium bovis BCG (Bacille Calmette-Guérin). The BCG vaccine is highly effective in preventing severe forms of childhood TB but does not protect against latent infection or disease in older age groups. A new or improved BCG vaccine for prevention of pulmonary TB is urgently needed. In this study, we infected murine bone marrow derived dendritic cells from C57BL/6 mice with M. bovis BCG followed by elution and identification of BCG-derived MHC class I and class II-bound peptides using tandem mass spectrometry. We identified 1436 MHC-bound peptides of which 94 were derived from BCG. Fifty-five peptides were derived from MHC class I molecules and 39 from class II molecules. We tested the 94 peptides for their immunogenicity using IFN- γ ELISPOT assay with splenocytes purified from BCG immunized mice and 10 showed positive responses. Seven peptides were derived from MHC II and three from MHC class I. In particular, MHC class II binding peptides derived from the mycobacterial surface lipoprotein Mpt83 were highly antigenic. Further evaluations of these immunogenic BCG peptides may identify proteins useful as new TB vaccine candidates.

Challenges in training the ideal Doctor for Africa: lessons learned from Zimbabwe.

BACKGROUND: Limited competencies among doctors and reduced numbers from medical migration in Africa could be corrected through innovative curricula and retention of trained manpower. The University of Zimbabwe Medical School simultaneously decided to increase the quality and quantity of doctors to address shortages. AIMS: To evaluate the outcome of innovative medical education at the University of Zimbabwe Medical School. METHOD: A structured questionnaire was administered to a broad group of staff and student representatives. In addition, a desk review of academic documents and policies and procedures was carried out. RESULTS: Early patient contact and community attachment which were introduced to the traditional curriculum remained but other teaching methodologies were not sustained with traditional didactic training still taking centre stage with limited staff development and retention. Whilst the annual student enrolment increased from less than 80 to 200 per year the vacancy rate of academic staff increased to 50%. CONCLUSION: Innovative curricula were partially implemented. The annual student intake increased but the staff complement declined. There is an urgent need to monitor and evaluate outcomes of medical education in Africa to arrest further decline in the quality of health care services.

Effects of Toll-like receptor ligands on RAW 264.7 macrophage morphology and zymosan phagocytosis.

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.

Rifampicin and dexamethasone have similar effects on macrophage phagocytosis of zymosan, but differ in their effects on nitrite and TNF-alpha production.

The antibiotic rifampicin is used extensively in the treatment of mycobacterial and other infections. It has previously been suggested that rifampicin binds to and activates the glucocorticoid receptor potentially leading to pharmacological glucocorticoid-like effects such as host immunosuppression (Calleja et al.). This study compares the effects of rifampicin with the synthetic glucocorticoid dexamethasone, on macrophage phagocytosis of zymosan particles and production of nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) by splenocytes or macrophages. Rifampicin and dexamethasone, partially suppressed zymosan phagocytosis by macrophages, respectively, and both effects were ameliorated by the glucocorticoid receptor antagonist RU486. In other experiments, rifampicin had no effects on NO responses; however, dexamethasone inhibited NO in an RU486-sensitive manner. At high doses, rifampicin moderately suppressed TNF-alpha while dexamethasone inhibited it in a dose-dependent manner, which was ameliorated by the presence of RU486. These findings suggest that rifampicin has differential immunomodulatory effects on these innate immune mechanisms.

Laminarin, a soluble beta-glucan, inhibits macrophage phagocytosis of zymosan but has no effect on lipopolysaccharide mediated augmentation of phagocytosis.

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.