Ceramide-induced integrated stress response overcomes Bcl-2 inhibitor resistance in acute myeloid leukemia.

Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

From the bench to the lectern: teaching immunology in higher education as a rewarding career opportunity.

This article covers the career pathway of a research-trained scientist to an immunology educator in a university setting.

Intracellular immunoglobulin A (icIgA) in protective immunity and vaccines.

Virus neutralization at respiratory mucosal surfaces is important in the prevention of infection. Mucosal immunity is mediated mainly by extracellular secretory immunoglobulin A (sIgA) and its role has been well studied. However, the protective role of intracellular specific IgA (icIgA) is less well defined. Initially, in vitro studies using epithelial cell lines with surface expressed polymeric immunoglobulin receptor (pIgR) in transwell culture chambers have shown that icIgA can neutralize influenza, parainfluenza, HIV, rotavirus and measles viruses. This effect appears to involve an interaction between polymeric immunoglobulin A (pIgA) and viral particles within an intracellular compartment, since IgA is transported across the polarized cell. Co-localization of specific icIgA with influenza virus in patients' (virus culture positive) respiratory epithelial cells using well-characterized antisera was initially reported in 2018. This review provides a summary of in vitro studies with icIgA on colocalization and neutralization of the above five viruses. Two other highly significant respiratory infectious agents with severe global impacts viz. SARS-2 virus (CoViD pandemic) and the intracellular bacterium-Mycobacterium tuberculosis-are discussed. Further studies will provide more detailed understanding of the mechanisms and kinetics of icIgA neutralization in relation to viral entry and early replication steps with a specific focus on mucosal infections. This will inform the design of more effective vaccines against infectious agents transmitted via the mucosal route.

Role of saturated and unsaturated fatty acids on dicarbonyl-albumin derived advanced glycation end products in vitro.

Glycation is a non-enzymatic reaction that occurs between the free amino group of proteins and reducing sugars and/or lipids, leading to the formation of advanced glycation end products (AGEs). The reaction also produces reactive oxygen species that have detrimental effects on cellular and extracellular proteins. Aminoguanidine is a known inhibitor of AGEs, and some fatty acids are known to have a beneficial role in vivo by reducing inflammation and oxidative stress. However, the role of fatty acids on AGE formation has not been thoroughly reported. We investigated the role of a range of fatty acids in the formation of AGEs and their reactive intermediates using an in vitro BSA-dicarbonyl model. The model assessed a time-dependent (0-72 h) and dicarbonyl concentration (0-2 mM) -dependent studies for the optimal formation of AGEs. A 72 h time point was found to be optimal for the reaction of BSA with either methylglyoxal (MGO) or glyoxal (GO) to generate AGE-BSA complexes. When arachidonic, eicosapentaenoic or docosahexaenoic acids were included in the reaction, a significant decrease in protein-bound fluorescent AGEs was seen compared to the respective controls. In contrast, saturated and 18 carbon polyunsaturated fatty acids showed no significant activity. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed saturated fatty acids significantly decreased the production of Nε-carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL) from GO and MGO models, respectively, whilst increasing methylglyoxal-derived hydroimidazolone (MG-H1). In contrast, arachidonic, eicosapentaenoic and docosahexaenoic acids did not significantly change either CEL or MG-H1 compared to no treatment controls whilst significantly reducing CML levels.

Folic acid deficiency increases sensitivity to DNA damage by glucose and methylglyoxal.

Type 2 diabetes (T2D) is associated with elevated frequencies of micronuclei (MNi) and other DNA damage biomarkers. Interestingly, individuals with T2D are more likely to be deficient in micronutrients (folic acid, pyridoxal-phosphate, cobalamin) that play key roles in one-carbon metabolism and maintaining genomic integrity. Furthermore, it has recently been shown that deficiencies in these nutrients, in particular folic acid leaves cells susceptible to glucose-induced DNA damage. Therefore, we sought to investigate if the B lymphoblastoid WIL2-NS cell line cultured under folic acid-deficient conditions was more sensitive to DNA damage induced by glucose, or the reactive glycolytic byproduct methylglyoxal (MGO) and subsequent advanced glycation endproduct formation. Here, we show that only WIL2-NS cultured under folic acid-deficient conditions (23 nmol/l) experience an increase in MNi frequency when exposed to high concentrations of glucose (45 mmol/l) or MGO (100 µmol/l). Furthermore, we showed aminoguanidine, a well-validated MGO and free radical scavenger was able to prevent further MNi formation in folic acid-deficient cells exposed to high glucose, which may be due to a reduction in MGO-induced oxidative stress. Interestingly, we also observed an increase in MGO and other dicarbonyl stress biomarkers in folic acid-deficient cells, irrespective of glucose concentrations. Overall, our evidence shows that folic acid-deficient WIL2-NS cells are more susceptible to glucose and/or MGO-induced MNi formation. These results suggest that individuals with T2D experiencing hyperglycemia and folic acid deficiency may be at higher risk of chromosomal instability.

Methylglyoxal Impairs Sister Chromatid Separation in Lymphocytes.

The accurate segregation of sister chromatids is complex, and errors that arise throughout this process can drive chromosomal instability and tumorigenesis. We recently showed that methylglyoxal (MGO), a glycolytic by-product, can cause chromosome missegregation events in lymphocytes. However, the underlying mechanisms of this were not explored. Therefore, in this study, we utilised shotgun proteomics to identify MGO-modified proteins, and label-free quantitation to measure changes in protein abundance following exposure to MGO. We identified numerous mitotic proteins that were modified by MGO, including those involved in the separation and cohesion of sister chromatids. Furthermore, the protein abundance of Securin, an inhibitor of sister chromatid separation, was increased following treatment with MGO. Cytological examination of chromosome spreads showed MGO prevented sister chromatid separation, which was associated with the formation of complex nuclear anomalies. Therefore, results from this study suggest MGO may drive chromosomal instability by preventing sister chromatid separation.

Proteomic Analysis of Methylglyoxal Modifications Reveals Susceptibility of Glycolytic Enzymes to Dicarbonyl Stress.

Methylglyoxal (MGO) is a highly reactive cellular metabolite that glycates lysine and arginine residues to form post-translational modifications known as advanced glycation end products. Because of their low abundance and low stoichiometry, few studies have reported their occurrence and site-specific locations in proteins. Proteomic analysis of WIL2-NS B lymphoblastoid cells in the absence and presence of exogenous MGO was conducted to investigate the extent of MGO modifications. We found over 500 MGO modified proteins, revealing an over-representation of these modifications on many glycolytic enzymes, as well as ribosomal and spliceosome proteins. Moreover, MGO modifications were observed on the active site residues of glycolytic enzymes that could alter their activity. We similarly observed modification of glycolytic enzymes across several epithelial cell lines and peripheral blood lymphocytes, with modification of fructose bisphosphate aldolase being observed in all samples. These results indicate that glycolytic proteins could be particularly prone to the formation of MGO adducts.

Methylglyoxal induces chromosomal instability and mitotic dysfunction in lymphocytes.

Type 2 diabetes is associated with elevated levels of DNA damage, in particular micronuclei (MNi) which are formed by acentric chromosome fragments caused by double-stranded DNA breaks (DSBs), or whole chromosomes which fail to segregate during mitosis. We investigated if methylglyoxal (MGO), a reactive dicarbonyl known to be elevated in type 2 diabetes is capable of increasing chromosomal instability and DNA damage as measured by the cytokinesis block micronucleus cytome (CBMNcyt) assay in B-lymphoblastoid WIL2-NS cells and primary peripheral blood lymphocytes (PBL). We also investigated the level of various dicarbonyl stress biomarkers, including extracellular and intracellular MGO, protein and MGO modifications of DNA. WIL2-NS cells exposed to either MGO or a glyoxalase 1 inhibitor showed increases in MNi and nuclear buds, which were associated with an increase in intracellular MGO. DNA damage in the form of MNi and nucleoplasmic bridges were observed in primary PBL exposed to 10 µM MGO, suggesting low concentrations of MGO may be genotoxic. Furthermore, we showed, using fluorescent in situ hybridisation, that the majority of MNi caused by MGO in WIL2-NS cells were caused by whole chromosome loss events, rather than DSBs. Our data suggest that MGO, a reactive metabolite elevated in type 2 diabetes and other pathologies, can affect genomic integrity by impairing chromosome segregation during mitosis.

Design, implementation, and assessment of an interactive simulation to teach undergraduate immunology students hemolytic disease of the newborn.

Hemolytic disease of the newborn (HDN) is a potentially fatal condition caused by a Rhesus (Rh) antigen incompatibility between a mother and fetus. As a result, determining the Rh status of expectant parents is a routine clinical assessment. Both the physiological and immunological basis of this condition are taught to undergraduate students. At the University of South Australia, some undergraduate immunology students find this topic challenging. The author designed, implemented, and assessed the impact of an interactive simulation to facilitate student learning of HDN. The students were actively engaged in determining the blood grouping and Rh status of an expectant mother and father and then determining the possibility of developing HDN. The simulation was found to take only 15 min to complete yet led to a significant increase in student performance in an end of semester exam question. Student perceived understanding was found to significantly improve following the introduction of the simulation, even though the content had been covered in a formal lecture. Student feedback was highly positive of this learning approach. In conclusion, short, interactive simulations can be used effectively to enhance student learning of challenging concepts.

Kelch-like protein 5-mediated ubiquitination of lysine 183 promotes proteasomal degradation of sphingosine kinase 1.

Sphingosine kinase 1 (SK1) is a signalling enzyme that catalyses the phosphorylation of sphingosine to generate the bioactive lipid sphingosine 1-phosphate (S1P). A number of SK1 inhibitors and chemotherapeutics can induce the degradation of SK1, with the loss of this pro-survival enzyme shown to significantly contribute to the anti-cancer properties of these agents. Here we define the mechanistic basis for this degradation of SK1 in response to SK1 inhibitors, chemotherapeutics, and in natural protein turnover. Using an inducible SK1 expression system that enables the degradation of pre-formed SK1 to be assessed independent of transcriptional or translational effects, we found that SK1 was degraded primarily by the proteasome since several proteasome inhibitors blocked SK1 degradation, while lysosome, cathepsin B or pan caspase inhibitors had no effect. Importantly, we demonstrate that this proteasomal degradation of SK1 was enabled by its ubiquitination at Lys183 that appears facilitated by SK1 inhibitor-induced conformational changes in the structure of SK1 around this residue. Furthermore, using yeast two-hybrid screening, we identified Kelch-like protein 5 (KLHL5) as an important protein adaptor linking SK1 to the cullin 3 (Cul3) ubiquitin ligase complex. Notably, knockdown of KLHL5 or Cul3, use of a cullin inhibitor or a dominant-negative Cul3 all attenuated SK1 degradation. Collectively this data demonstrates the KLHL5/Cul3-based E3 ubiquitin ligase complex is important for regulation of SK1 protein stability via Lys183 ubiquitination, in response to SK1 inhibitors, chemotherapy and for normal SK1 protein turnover.

Manipulating leukocyte populations to mimic immune disease states: a novel active approach to teaching flow cytometry to undergraduate immunology students.

Flow cytometry detects and measures the physical and chemical characteristics of cells or particles. In medical laboratories, flow cytometers are used to quantify changes in cell populations associated with disease states, such as AIDS. While a powerful technique, it is challenging to teach the principles of flow cytometry to undergraduate students. One approach is to have students process and analyze a patient sample. However, this is not possible when the patient has an infectious disease. Here we report a two-stage approach to address this challenge. Magnetic beads were used to manipulate leukocytes cell populations in healthy blood to mimic the phenotype of eight immune disease conditions. The cells were then stained against cell surface markers for cell populations and analyzed by flow cytometry. The second stage focused on teaching flow cytometry over 2 wk. Week 1 involved a lecture, followed by a laboratory session where students learned how to stain a blood sample. In week 2, students worked in a computer pool to analyze the previously generated data and determine the immunological status of a control and patient sample. Using this approach, all students achieved 100% correct diagnosis of both control and patient samples. Student feedback via a questionnaire was overwhelmingly positive, and student perceived knowledge of flow cytometry increased after the session significantly. We effectively mimicked several disease states, eliminating the need to source patient samples, yet still teaching undergraduate students the principles of flow cytometry.

Measuring the Asian seabass (Lates calcarifer) neutrophil respiratory burst activity by the dihydrorhodamine-123 reduction flow cytometry assay in whole blood.

The neutrophil oxidative respiratory burst response is a key component of the innate immune system responsible for killing microbial pathogens. Since fish rely on the innate immune system for health, monitoring the respiratory burst activity may be an effective means of gauging fish health status. Here we report that the respiratory burst of Asian seabass neutrophils can be measured in whole blood by the dihydrorhodamine (DHR)-123 reduction assay and flow cytometry. Neutrophils responded to phorbol myristate acetate (PMA) in a concentration dependent manner with significant respiratory burst activity at 100-1000 nM. Other known neutrophil agonists, such as bacterial lipopolysaccharide, tumor necrosis factor, the tripeptide f-met-leu-phe and zymosan, did not induce a significant DHR reduction. Thus, the findings enable us to propose that the DHR-123 flow cytometry whole blood assay, incorporating PMA as a stimulator, would not only facilitate future studies into fish blood neutrophil research but provides a simple, rapid and reliable assay for gauging fish natural immunity status and health.

Polyandric acid A, a clerodane diterpenoid from the Australian medicinal plant Dodonaea polyandra, attenuates pro-inflammatory cytokine secretion in vitro and in vivo.

Dodonaea polyandra is a medicinal plant used traditionally by the Kuuku I'yu (Northern Kaanju) indigenous people of Cape York Peninsula, Australia. The most potent of the diterpenoids previously identified from this plant, polyandric acid A (1), has been examined for inhibition of pro-inflammatory cytokine production and other inflammatory mediators using well-established acute and chronic mouse ear edema models and in vitro cellular models. Topical application of 1 significantly inhibited interleukin-1ß production in mouse ear tissue in an acute model. In a chronic skin inflammation model, a marked reduction in ear thickness, associated with significant reduction in myeloperoxidase accumulation, was observed. Treatment of primary neonatal human keratinocytes with 1 followed by activation with phorbol ester/ionomycin showed a significant reduction in IL-6 secretion. The present study provides evidence that the anti-inflammatory properties of 1 are due to inhibition of pro-inflammatory cytokines associated with skin inflammation and may be useful in applications for skin inflammatory conditions including psoriasis and dermatitis.

Enhancing teaching effectiveness in biochemistry labs: Author reflections and improvement strategies.

This article details the outcome of a joint reflective approach undertaken by the authors to identify common difficulties experienced by 2nd-year undergraduate Biochemistry students in laboratory classes. Difficulties experienced in laboratories can affect the development of hand skills, an understanding of how to correctly operate laboratory equipment and the linkage between didactic content and their experimental demonstration. These difficulties covered were identified based on their common appearance across multiple cohorts and are grouped into five broad areas. The context of the laboratory exercises is detailed and the common difficulties experienced by students are outlined. The potential causes of these difficulties are then discussed along with the approaches and strategies that were implemented to help resolve future occurrences. The approach and resources developed to address these difficulties may help other Biochemistry educators who are facing similar experiences with their undergraduate students.

Identification and functional characterization of two novel mutations in the α-helical loop (residues 484-503) of CYBB/gp91(phox) resulting in the rare X91(+) variant of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is mainly caused by mutations in X-linked CYBB that encodes gp91. We have identified two novel mutations in CYBB resulting in the rare X91(+)-CGD variant, c.1500T>G (p.Asp500Glu) in two male siblings and c.1463C>A (p.Ala488Asp) in an unrelated male. Zymosan and/or PMA (Phorbol 12-myristate 13-acetate)-induced recruitment of p47(phox) and p67(phox) to the membrane fraction was normal for both mutants. Cell-free assays using recombinant wild-type and the mutant proteins revealed that these mutants were not activated by NADPH (nicotinamide adenine dinucleotide phosphate). Interestingly, the Ala488Asp mutant was activated by NADPH in the presence of glutathione. These data suggest that the mutations prevented NADPH from binding to gp91(phox) and the requirement of a negative charge at residue 500 in gp91(phox) for NADPH oxidase assembly, in contrast to a previously described Asp500Gly change. These mutations and the effect of glutathione provide a unique insight into disease pathogenesis and potential therapy in variant X91(+)-CGD.

Introducing First-Year Undergraduate Students the Fundamentals of Antibiotic Sensitivity Testing through a Combined Computer Simulation and Face-to-Face Laboratory Session.

Determining the antibiotic sensitivity of disease-causing microorganisms is a fundamental process in a clinical microbiology laboratory. With the continued use of antibiotics, the emergence of antibiotic resistance has become a significant health issue. However, the principles and laboratory testing to determine antibiotic sensitivity are generally not taught to first-year undergraduate students. This is partly due to the limited time to cover the fundamental biology of microorganisms and the mechanism of action of antibiotics in an introductory course. We overcame these limitations by teaching first-year students the fundamental principles of antibiotic sensitivity using an online data generator/simulation. Using the Kirby-Bauer disk diffusion test, students replicated the effects of antibiotic dose on bacterial growth and determined the antimicrobial susceptibility testing of their allocated bacterium. After 2-3 weeks, the antimicrobial sensitivity testing was replicated in an authentic face-to-face laboratory setting over 2 days. The impact of the intervention on student learning was assessed using a written laboratory report and a short questionnaire containing Likert and free-text questions. Student self-reported understanding of the content rose significantly, with nearly all students passing the written assessment. The approach was found to be enjoyable and interactive and facilitated authentic learning in first-year students. This cohort of students will continue to use more advanced versions of this simulation in future years, allowing for the long-term benefits of this approach to be assessed.

Using online simulations to teach biochemistry laboratory content during COVID-19.

At the University of South Australia (UniSA), Biochemistry is a second year undergraduate course. The student cohort is diverse, with students enrolled in courses with a laboratory focus, such as Laboratory Medicine, Medical Science, Nutrition and Food Science and Pharmaceutical Science. The course is taught in a traditional manner, with weekly lectures, fortnightly tutorials and three practical sessions. In response to the growing numbers of COVID-19 cases, in mid-March the University leadership moved to cease face-to-face teaching. By this time, 58 of 96 students had completed the first two (of three) face-to-face laboratory practicals. In response to this decision, teaching of all practical based content was moved online for all students. The first question was, how do we teach practical content online? And secondly, how do we teach hands-on skills? The first question was addressed using a suite of online simulations, progressively developed since 2013. Simulations are widely used and shown to be useful as teaching aids in STEM. A total of five simulations were introduced each covering key aspects of laboratory practice, including fundamental mathematical skills, reading, and setting a pipette, basic Biochemistry assays, protein quantification, and enzyme kinetics. The second issue of teaching hands on skills was addressed once restrictions were eased. Students were invited to attend the laboratory to learn the kinesthetic skills with instructor guidance. Both approaches used proved to be highly effective and can be readily adapted not only to teaching Biochemistry, but any aspect of science education.

Using a science simulation-based learning tool to develop students' active learning, self-confidence and critical thinking in academic writing.

Simulation is one teaching strategy that health educators use to promote active learning, however, evidence to support whether students utilising simulations develop skills in critical thinking and appraisal in their academic literacy, is lacking. This study explored the impact of embedding a desktop simulation about the hemolytic disease of the newborn (HDN) in a first-year, undergraduate nursing course on students' acquisition of knowledge, self-confidence, and development of critical thinking in academic writing. The study used a quasi-experimental, two-group, and pre-test-post-test design. After using the simulation, students reported i. a positive learning experience, ii. an increase in their self-confidence to critically appraise a case study, and iii. greater student success in two assessments for student's participating in the simulation compared to students who only used lecture notes as a study tool. The findings support the use of simulations to promote active learning, knowledge acquisition and opportunities to develop self-confidence and critical thinking in academic writing.

Cytoplasmic dynein regulates the subcellular localization of sphingosine kinase 2 to elicit tumor-suppressive functions in glioblastoma.

While the two mammalian sphingosine kinases, SK1 and SK2, both catalyze the generation of pro-survival sphingosine 1-phosphate (S1P), their roles vary dependent on their different subcellular localization. SK1 is generally found in the cytoplasm or at the plasma membrane where it can promote cell proliferation and survival. SK2 can be present at the plasma membrane where it appears to have a similar function to SK1, but can also be localized to the nucleus, endoplasmic reticulum or mitochondria where it mediates cell death. Although SK2 has been implicated in cancer initiation and progression, the mechanisms regulating SK2 subcellular localization are undefined. Here, we report that SK2 interacts with the intermediate chain subunits of the retrograde-directed transport motor complex, cytoplasmic dynein 1 (DYNC1I1 and -2), and we show that this interaction, particularly with DYNC1I1, facilitates the transport of SK2 away from the plasma membrane. DYNC1I1 is dramatically downregulated in patient samples of glioblastoma (GBM), where lower expression of DYNC1I1 correlates with poorer patient survival. Notably, low DYNC1I1 expression in GBM cells coincided with more SK2 localized to the plasma membrane, where it has been recently implicated in oncogenesis. Re-expression of DYNC1I1 reduced plasma membrane-localized SK2 and extracellular S1P formation, and decreased GBM tumor growth and tumor-associated angiogenesis in vivo. Consistent with this, chemical inhibition of SK2 reduced the viability of patient-derived GBM cells in vitro and decreased GBM tumor growth in vivo. Thus, these findings demonstrate a tumor-suppressive function of DYNC1I1, and uncover new mechanistic insights into SK2 regulation which may have implications in targeting this enzyme as a therapeutic strategy in GBM.

Colocalization of intracellular specific IgA (icIgA) with influenza virus in patients' nasopharyngeal aspirate cells.

Inhibition of viral replication by icIgA antibodies has only been observed with in vitro studies using epithelial cell lines in transwell cultures. This effect appears to involve an interaction between polymeric immunoglobulin A (pIgA) and viral particles within an intracellular compartment, since IgA is transported across polarized cells. Polyclonal guinea pig antisera against purified influenza A virus and mouse antisera prepared against Influenza A/H3N2 hemagglutinin (HA0) cleavage loop peptides, were used in confocal fluorescence microscopy to show specific staining of wild-type influenza H1N1 and H3N2 viruses in clinical specimens. The HA0 cleavage loop peptides used for intranasal immunization of mice were designed and synthesized from specific conserved regions of influenza A/H1N1 & A/H3N2 viruses. Anti-human secretory IgA antibodies were used to show co-localisation of influenza A virus and icIgA. The results showed specific immunofluorescent staining of influenza A/H3N2 (X31) (HA0 uncleaved)-infected MDCK cells and the presence of icIgA in respiratory exudate cells of infected patients. Both results confirm specific co-localisation and suggest interaction between influenza A virus and icIgA in patients' respiratory exudate cells. Importantly, antisera to the mouse anti-HA0 cleavage site were specific for wild-type virus in clinical specimens, indicating that the conserved region of HA0 was present in the uncleaved form. Similar staining and colocalization patterns between icIgA and virus were observed with polyclonal guinea pig antisera against influenza A virus. These are the first observations of co-localization of influenza A virus and intracellular IgA in clinical specimens. Role of icIgA: This report shows the co-localization of influenza A virus HA0 and icIgA antibodies in respiratory exudate cells of patients who were culture and viral RNA positive, suggesting that icIgA directed against the conserved HA0 site may have a privileged and unique opportunity to act on immature virus and thus prevent HA0 cleavage, maturation and subsequent cycles of viral replication. The precise mechanism by which icIgA mediates intracellular viral neutralization remains to be fully elucidated. SIGNIFICANCE: The above findings in clinical specimens would contribute strongly to our understanding of the mechanisms and kinetics of icIgA neutralization in relation to viral entry and early replication steps of mucosal viral infections. A rapid, objective and sensitive assay - by ex vivo enumeration of respiratory epithelial cells that have co-localized influenza virus and icIgA - would contribute to further mucosal immunity studies and inform the design of more effective vaccines against influenza and other viral infections transmitted via the mucosal route e.g. respiratory syncytial virus, rotavirus.