Detection of infiltrating mast cells using a modified toluidine blue staining

Mast cells are part of the immune system and characteristically contain histamine- and heparin-rich basophilic granules. While these cells are usually associated with allergy and anaphylaxis, they also promote wound healing and angiogenesis and confer protection against pathogens. The presence of these cells is sometimes indicative of a poor prognosis, especially in skin cancer, pancreatic cancer, and lymphoma. Toluidine blue staining of acid-fast granules is an established method for the identification and quantification of mast cells. Generating detailed information on the location of mast cells within tissues is problematic using this technique and often requires serial sections from adjacent tissue to be separately stained with hematoxylin and eosin (H&E). Staining serial sections is not always possible, particularly if the sample is very small or rare. In such cases, a method of simultaneously identifying and localizing mast cells in a tissue would be advantageous. Toluidine blue and H&E are not commonly combined because H&E includes repetitive washes in water, which may affect the efficacy of the aqueous-soluble toluidine blue. We have developed and tested a novel staining technique that integrates toluidine blue between hematoxylin and eosin in one simple procedure. This protocol works on both frozen and formalin-fixed, paraffin-embedded tissue and readily allows for the identification of purple-stained mast cells against a clean H&E background. This facilitates a more accurate localization and proper counting of mast cells in normal and affected tissu

Pharmacologically antagonizing the CXCR4-CXCL12 chemokine pathway with AMD3100 inhibits sunlight-induced skin cancer

One way sunlight causes skin cancer is by suppressing anti-tumor immunity. A major mechanism involves altering mast cell migration via the C-X-C motif chemokine receptor 4–C-X-C motif chemokine ligand 12 (CXCR4-CXCL12) chemokine pathway. We have discovered that pharmacologically blocking this pathway with the CXCR4 antagonist AMD3100 prevents both UV radiation-induced immune suppression and skin cancer. The majority of control mice receiving UV-only developed histopathologically confirmed squamous cell carcinomas. In contrast, skin tumor incidence and burden was significantly lower in AMD3100-treated mice. Perhaps most striking was that AMD3100 completely prevented the outgrowth of latent tumors that occurred once UV irradiation ceased. AMD3100 protection from UV immunosuppression and skin cancer was associated with reduced mast cell infiltration into the skin, draining lymph nodes, and the tumor itself. Thus a major target of CXCR4 antagonism was the mast cell. Our results indicate that interfering with UV-induced CXCL12 by antagonizing CXCR4 significantly inhibits skin tumor development by blocking UV-induced effects on mast cells. Hence, the CXCR4-CXCL12 chemokine pathway is a novel therapeutic target in the prevention of UV-induced skin cancer

Inhibition of GSK-3 by Tideglusib suppresses activated macrophages and inflammatory responses in lipopolysaccharide-stimulated RAW 264.7 cell line

Glycogen synthase kinase-3 (GSK-3) is an important immune regulator that controls inflammation via inhibition of its protein kinase activities. Persistent inflammatory responses through the activation of immune cells and excessive production of immune mediators may cause tissue destruction and implicated in the development of chronic inflammatory diseases. The objective of this study was to examine the role of Tideglusib, a GSK-3 inhibitor, in inflammatory responses elicited through macrophage activation by investigating the expression of cell surface biomarkers and inflammatory molecule levels. Method: The effects of GSK-3 inhibition by Tideglusib on the expression of CD11b and CD40 and secretion of pro-inflammatory cytokines in the lipopolysaccharide (LPS)-activated macrophage-derived RAW 264.7 cells were determined by flow cytometry, while the presence of nitric oxide (NO) was determined by Griess assay. Results: Stimulation of RAW 264.7 cells with LPS increased substantial levels of CD11b and CD40 expressions, and secretion of NO, TNF-α, and MCP-1. However, the expression of these molecules was suppressed through inhibition of GSK-3. Conclusion: These findings suggest the significant role of Tideglusib to limit the upregulation of immune responses in activated macrophages, and as a potential anti-inflammatory drug for the intervention and treatment of inflammatory diseases

A wake-up call: Covid-19 and its impact on reforming biosciences education towards resiliency and sustainability

COVID-19’s global pandemic has had a significant impact on bioscience education, which has switched to online learning. Every entity within the higher education ecosystem, whether technical, pedagogical, or social, has faced a number of challenges as a result of this. Regardless, biosciences education stakeholders have been fast to implement innovative strategies to maintain high standards and quality of biosciences online teaching and learning. This paper focuses on the biosciences education transition toward developing resiliency, as well as the technology resources and approaches that have been deployed in the current context to change biosciences education to be robust in the face of the COVID-19 upheaval. Finally, significant insights into ‘resilience sustainability’ approaches that may be employed in relation to the digitisation of biosciences education in a concerted effort to promote resiliency, adaptability and sustainability in biosciences education are presente

A persistent antimicrobial resistance pattern and limited methicillin-resistance-associated genotype in a short-term Staphylococcus aureus carriage isolated from a student population

The aim of the present study was to assess and compare the antimicrobial susceptibility pattern against a panel of antibiotics and molecular and methicillin resistance-associated genotypes of 120 carriage S. aureus isolates previously isolated from a student population at two isolation events within a one-month interval. The antibiotic susceptibility of isolates was determined using the Kirby-Bauer disc-diffusion method (cefoxitin by Etest). The MRSA was screened using polymerase chain reaction for the presence of the mecA gene. The mecA-positive isolates were subjected to staphylococcal cassette chromosome (SCC) mec typing, multilocus sequence typing (MLST) and eBURST analysis. All isolates were characterized for the presence of the Panton–Valentine leukocidin (PVL) gene, an enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) pattern and the spa type. For the two occasions where S. aureus was isolated, the highest frequency of resistance was observed for penicillin (70% and 65%, respectively), with a lower rate against erythromycin and tetracycline (<12%). All isolates were susceptible to ciprofloxacin and gentamycin. As for methicillin resistance, eight isolates had minimum inhibitory concentrations (MIC) of resistant categories, but 10 isolates (8.33%) were positive for the mecA gene. The mecA-positive isolates belonged to SCCmec types I (n = 9) and V (n = 1). MLST was resolved for only three MRSAs, ST508 (n = 1), ST88 (n = 1) and ST96 (n = 1). The results of the eBURST analysis showed that the MRSA isolates analyzed in the present study were potentially related to MRSA identified in other countries. Approximately half of the persistent S. aureus carriers harbored S. aureus of a similar spa type in the respective individuals during both isolation events. A persistent antimicrobial pattern and limited distinct MRSAs were observed over the short study period. The latter frequently exhibited SCCmec type I, commonly associated with hospital-acquired (HA) characteristics, but further delineation is needed to justify the origins of these bacteria

Of Inclusion, Value, and Roman Elegy

Contemporary critiques and modern criticism of Roman elegiac poetry and its distinctly transgressive lifestyle align in casting its practitioner on the outside of Roman cultural values. It is a notion that has crystalized in the topos of erotic exclusion and the figure of the exclusus amator doomed to suffer it, which have become both the indelible image and the governing metaphor of the genre of Roman elegy as a whole. This project amplifies the conversation around the exclusion of elegy and its practitioners from insider status—from both the bedroom of the beloved and the theater of Roman cultural value—with a consideration of how elegy fashions its own inclusion. Through readings of the elegies of Propertius, Tibullus, and Ovid (the Amores), I consider how each harnesses the complementary topos of erotic inclusion to counter the cultural devaluation of the elegiac craft. I approach this issue through a study of two overarching themes, both anchored in the notion that the elegiac amator is categorically exclusus: the conception of love as lacking in substance and the conception of love as a waste of time, whose development I chart in two respective sections. Part I, In Venere Veritas, addresses the assessment that erotic love is a delusion that cannot survive an encounter with the beloved, tracing instances in elegy of the poet-lover shown in the company of his mistress and discussing the ways in which such episodes harness the state of inclusion to demonstrate the validity of passionate love—and the viability of elegiac poetics along with it. Part II, Operosus in Otio, turns to the charge that love and its poetry are idle and indolent. Against this accusation, I position the elegists’ own depictions of love as a form of personal industriousness through their representations of erotic activity in terms of lucubratio, the practice of working during the off-hours of the night, which challenges the devaluation of the erotic-poetic undertaking as an exercise incompatible with Roman mores

Antagonising the CXCL12 pathway with AMD3100 inhibits skin cancer

One way sunlight causes skin cancer is by suppressing the anti-tumor immune response. A major mechanism involves altering mast cell migration via the C-X-C motif chemokine receptor 4-C-X-C motif chemokine ligand 12 (CXCR4-CXCL12) chemokine pathway. I have discovered that pharmacologically blocking this pathway with the CXCR4 antagonist AMD3100 prevents both UV radiation-induced immune suppression and skin cancer. The majority of control mice receiving UV-alone developed histopathologically confirmed squamous cell carcinomas. In contrast, skin tumor incidence and burden was significantly lower in AMD3100-treated mice. Perhaps most striking was that AMD3100 completely prevented the outgrowth of latent tumors that occurred once UV irradiation ceased. AMD3100 protection from UV immunosuppression and skin cancer was associated with reduced mast cell infiltration into the skin, draining lymph nodes, and the tumor itself. Thus a major target of CXCR4 antagonism was the mast cell. The results presented in this thesis provide important evidence that the CXCR4-CXCL12 chemokine axis plays multiple roles in the development of UV-induced skin cancer. It makes a significant and unique contribution to our understanding of the effects of antagonising CXCR4 in chronic UV-induced skin damage, mast cell migration, UV- immune suppression and melanin production. These studies provide important pre-clinical proof of principle evidence that antagonising CXCR4 will be of therapeutic benefit to high risk, chronically sun-damaged patients