Microbial communities living in the oceans are major drivers of global biogeochemical cycles. With nutrients limited across vast swathes of the ocean, marine microbes eke out a living under constant assault from predatory viruses. Viral concentrations exceed those of their bacterial prey by an order of magnitude in surface water, making these obligate parasites the most abundant biological entities in the ocean. Like the pirates of the 17th and 18th centuries that hounded ships plying major trade and exploration routes, viruses have evolved mechanisms to hijack microbial cells and repurpose their cargo and indeed the vessels themselves to maximise viral propagation. Phenotypic reconfiguration of the host is often achieved through Auxiliary Metabolic Genes – genes originally derived from host genomes but maintained and adapted in viral genomes to redirect energy and substrates towards viral synthesis. In this review, we critically evaluate the literature describing the mechanisms used by bacteriophages to reconfigure host metabolism and to plunder intracellular resources to optimise viral production. We also highlight the mechanisms used when, in challenging environments, a ‘batten down the hatches’ strategy supersedes that of ‘plunder and pillage’. Here, the infecting virus increases host fitness through phenotypic augmentation in order to ride out the metaphorical storm, with a concomitant impact on host substrate uptake and metabolism, and ultimately, their interactions with their wider microbial community. Thus, the traditional view of the virus-host relationship as predator and prey does not fully characterise the variety or significance of the interactions observed. Recent advances in viral metagenomics have provided a tantalising glimpse of novel mechanisms of viral metabolic reprogramming in global oceans. Incorporation of these new findings into global biogeochemical models requires experimental evidence from model systems and major improvements in our ability to accurately predict protein function from sequence data. The Creator would appear as endowed with a passion for stars, on the one hand, and for beetles on the other’ [28 infections occurring per day in the oceans [Based on their extraordinary abundance and diversity, J.B.S. Haldane once quipped that ‘e other’ . In compe other’ . Assumine other’ . With 10e oceans , these be oceans  and the e oceans , 6. Relee oceans –9. Thus,Lysis and release of cellular material as dissolved organic matter is only the final step of a complex host-virus interaction where the invading pathogen can manipulate host metabolism and alter its phenotype to favour viral replication at the expense of host function. Once a virus has overcome host defences, lytic viral infection typically involves a shut-down of host metabolism, followed by degradation of macromolecules and scavenging of intracellular resources. Virally encoded ‘Auxiliary Metabolic Genes’ (AMGs) are expressed during infection to augment and redirect energy and resources towards viral production –13. ThesEscherichia coli, with 3 distinct phases of transcription of viral genes: (1) defence neutralisation and host metabolic rewiring; (2) synthesis of viral components; and (3) viral assembly [Cellulophaga baltica. Infection with a specialist phage was initiated with expression of 15 genes putatively involved in overcoming host defences. Of these, one was an anti-restriction defence gene thought to provide viral defence against host degradation of viral DNA. The remaining 14 genes had unknown function but shared a promoter with the anti-restriction defence and were located in the same genomic neighbourhood, inferring a complementary functional role [Synechococcus hosts showed that within the first 30 min following infection, almost all transcription is associated with phage encoded genes and within the first hour of infection ~ 80% of cellular DNA has been scavenged for resources [First, we will consider the classic view of viral predation, where the virus replicates by lytic infection and redirects host resources for the purpose of viral production that ends in cell lysis and release of viral progeny , many of these viral genes encode for central metabolic proteins and are conserved across different marine phage lineages, signifying their importance to viral success . Two difnrdA/nrdB) using NADPH as a terminal electron donor [psbA and 50% containing both psbA and psbD [2 fixation [psbA gene suggested that the increase in energy available to the virus for replication is negated by the increased cost of encoding an extra gene. However, under high-light conditions, the virus gains an advantage by replacing photosystem II machinery damaged by light stress [Prochlorococcus-infecting cyanophage (P-TIM68) was isolated that encodes viral genes for photosystem II as well as a cassette for photosystem I (psaABCDEFJK) - the first known example of a phage to manipulate both photosystems during infection. Predictions of enhanced cyclic electron flow around photosystem I were also confirmed [2 fixation and towards nucleotide biosynthesis for phage replication [Virus-directed augmentation and redirection of host energy and resources to increase viral progeny production has been observed in both marine heterotrophs and phototrophs, with the latter the focus of much of the seminal research in this area. The lytic cyanophage Syn9 encodes photosystem II core protein D1 (which enables transfer of electrons from water to plastoquinone), as well as genes catalysing the synthesis of deoxyribonucleotides , nitrate (N) and iron (Fe) –40. Therutrients . Using med hosts . The phoiversity . Howeveren hosts , 46.amt) and ammonia monooxygenases (amoC), as well as genes associated with sulfur reduction (dsr) and oxidation (sox) [amoC genes encoded by viruses infecting Thaumarchaeota form a distinct phylogenetic clade, they can also comprise up to half of the total abundance of amoC in some metagenomic datasets [nar) have also been found in viromes from deep-sea hydrothermal vents [dsrC were observed during SUP05 blooms. Investigators concluded that phage-encoded DsrC is likely to function in the sulfur cycling of infected SUP05, and may modulate vital electron transfer reactions. Evidence indicating increased viral infection of SUP05 with increasing depth, and decreasing O2 levels, underlines the potential significance of this finding [Viral manipulation of cellular nitrogen and sulfur levels during infection is perhaps less well documented in cultured host-virus systems than phosphate regulation, but recent research is providing evidence that it is no less important. Phage genomes have been found to encode numerous proteins to manipulate concentrations of 2-oxoglutarate, which in turn, regulates cellular N-limitation response via the promotor NtcA . Beyond on (sox) . Screenidatasets . Nitrateal vents . Evidencal vents , 50, 51.al vents  and peak finding .speD, cysK/M, metK, dapC) and carbohydrates  [talC, zwf, gnd) to divert carbon towards the pentose phosphate pathway by converting glyceraldehyde-3P to fructose-6P, which is subsequently converted to reducing power and the synthesis of dNTPs for phage replication [manA) and a glycogen synthase (glgA), with the latter identified in all examined viral metagenomes [glgA was used to trigger a starvation response in the host in order to push carbon through non-glycolytic pathways that promote dNTP biosynthesis. Manipulation of succinate through the glyoxylate shunt is thought to facilitate energy production at the expense of amino acid synthesis, particularly in nutrient limited, deep ocean samples. Intracellular carbon is also redirected during viral infection for energy via virally-encoded genes for the Entner-Doudoroff pathway, the TCA cycle and fatty acid metabolism [Virally-encoded genes associated with carbon uptake have also been identified in both model experimental systems and viral metagenomes, including those involved in uptake and metabolism of amino acids  , 53. Cyalication . Viral mPseudomonas aeruginosa PAO1 and compared responses across host-virus infections between one host and six different phages [So how does expression of viral AMGs during infection influence cells at the fundamental level of metabolite synthesis and utilisation? The influence of viral hijacking at the metabolite level lacks a representative study in marine systems, but De Smet and colleagues performed an exemplary study in t phages . Followit phages .Cellulophaga baltica [Synechococcus and Prochlorococcus include many generalists, some capable of infecting both genera [Synechococcus-dominated to Prochlorococcus-dominated communities as nutrient availability waxes and wanes over seasonal, depth and diel gradients [Efficiency in host hijacking is thus not universal, and tends to be higher in infections between specialist phages and their preferred host (which are the focus of many host-phage model systems). Generalist phages that infect multiple hosts tend to have less efficient infections, fail to completely suppress host translation and transcription, have longer latent periods and decoupled translation and transcription , 13. Theh genera , 55. Theradients –58. Furtradients .6 cells per mL [From the perspective of viral fitness, the benefit of driving host energy and metabolites towards viral production during lytic infection only confers an advantage if viral progeny released following a lytic event can successfully infect new, actively growing hosts. Imagine a band of naïve pirates in the doldrums of a seldom-travelled ocean gyre. They seize a ship, kill the crew and strip its cargo with alarming efficiency, before scuppering the vessel and sailing off in rowboats. With no land in sight, nor new vessel to capture, their career as pirates would be short and they would soon succumb to the tropical sun, deprived of life-giving resources. In contrast, a savvy band of pirates would instead remain aboard the seized vessel, secure the mainmast during storms that may arise, ration the rum and steer her towards more profitable waters. Many marine viruses display a similar ‘batten down the hatches’ strategy by foregoing a lytic life cycle and instead increasing the fitness of the infected cell phenotype through metabolic manipulation Fig. b. Here, s per mL . A signis per mL . These ls per mL .E. coli [Vibrio cholerae [Vibrio harveyi [, Staphylococcus aureus [Listeria monocytogenes [Pseudoalteromonas strain from Arctic sea ice containing a filamentous phage, they noticed it had lower growth rates and cell density, and lower tolerance of NaCl and reactive oxygen species than when it was cured of the infection. Transcriptional analysis showed downregulation of succinyl-CoA synthetase and succinate dehydrogenase indicating virally-mediated suppression of central carbon metabolism. However, presence of the phage increased host motility. They postulated that the presence of the phage increased host fitness during the nutrient-limited polar winters by slowing down host metabolism and increasing its capacity to find new nutrient sources in the heterogeneous structure of the sea ice in which it lived [E. coli phage λ. This lysogen maintains integration in the host genome via a phage-encoded repressor known as cI. cI also represses the host gene pckA, which encodes a protein for the conversion of oxaloacetate to phosphoenolpyruvate. Thus, lysogeny results in a decoupling of central carbon metabolism from cellular synthesis, reducing host growth rate and potentially conferring a selective advantage in hosts within nutrient-poor environments [Anabena spp. and Nostoc spp. appear to regulate nitrogen-fixation through active lysogeny. Here, prophages interrupting N-fixation genes  are excised from the genome during N-limitation, re-activating the genes  . This in turn inhibits RNA transcription and promotes transcription of the global stress response regulator RpoS, shifting the cell into stationary phase . Increaspulation . Similar viruses .E. coli cells that enter stationary phase [E. coli and thus repress production of MazF [mazG is common in viral genomes from marine environments and has been reported in viruses infecting both heterotrophs [mazEF expression increases host resistance to phages, it is conceivable that infecting viruses can use MazG to manipulate ppGpp concentrations and thus maintain the susceptibility of hosts within a population to subsequent infection by viral progeny. In pirate terms, viruses have evolved the capacity to sail under a false flag, disabling host alarm systems until it is too late. Further experimental evidence of the use of MazG and its effects on marine populations is required to explore whether this phenomenon is observed in nature.Given its dual role as both a regulator of the stringent response and of MazEF-regulated resistance and cell death, increasing cellular levels of ppGpp pose a clear threat to viral replication. Many viruses are dependent on host RNA transcription for replication, and viral replication has been shown to be drastically reduced or suspended in infected ry phase . Cell de of MazF . Thus inrotrophs , 85 and rotrophs , 86. Therotrophs . In addirotrophs  . Given rotrophs , and thaThe current understanding of how viruses hijack host metabolism during infection is the result of both culturing experiments in model systems; and culture-independent techniques such as viral metagenomics. Culture-dependant techniques have significant advantages: Model systems enable us to study the relationship between host and viruses in controlled conditions; viral replication cycles and critical parameters  can be defined, and the functionality of viral genes may be determined in vitro. In culture, predator-prey interactions can be isolated from those occurring in complex microbial communities. This reduction in complexity allows for investigation into how transcriptomes, proteomes and metabolites are altered during infection and thus enable a systems-biology approach to understanding complex metabolic cascades and regulation (e.g. , 45, 90.For systems outside of cultured representatives, viral metagenomic studies to date have provided major insights into to viral taxonomic and functional distribution and diversity , 47, 53.This approach has successfully to identify new viruses and novel AMGs , 17, 96.It is worth noting however, that no matter how sophisticated sequencing methods become, perhaps the greatest barrier to understanding how marine viruses influence cellular metabolism during infection lies in our extremely limited capacity to identify the function of viral genes, in both cultured isolates and genomes constructed from environmental DNA. Whilst machine learning approaches are rising to meet this challenge , 99, oneTreasure Island and J.M. Barrie’s Peter Pan. Piecemeal accounts and a lack of historical records has enabled myth and legend to supersede the violent and unpleasant reality written in Charles Johnson’s A General History of the Robberies and Murders of the Most Notorious Pyrates [The contemporary image of pirates is typically one of swashbuckling, romantic characters of Robert Louis Stevenson’s  Pyrates . Similar Pyrates , 109 and


A large body of research has documented disparities in health and access to care for lesbian, gay, and bisexual (LGB) people in the United States. Less research has examined how the level of legal protection afforded to LGB people (the state policy environment) affects health disparities for sexual minorities. This study used data on 14,687 sexual minority adults and 490,071 heterosexual adults from the 2014–2016 Behavioral Risk Factor Surveillance System to document differences in health. Unadjusted state-specific prevalence estimates and multivariable logistic regression models were used to compare poor/fair self-rated health by gender, sexual minority status, and state policy environments (comprehensive versus limited protections for LGB people). We found disparities in self-rated health between sexual minority adults and heterosexual adults in most states. On average, sexual minority men in states with limited protections and sexual minority women in states with either comprehensive or limited protections were more likely to report poor/fair self-rated health compared to their heterosexual counterparts. This study adds new findings on the association between state policy environments and self-rated health for sexual minorities and suggests differences in this relationship by gender. The associations and impacts of state-specific policies affecting LGB populations may vary by gender, as well as other intersectional identities. Numerous studies have documented wide disparities in health status for sexual minorities, or gay, lesbian, bisexual (LGB), and other nonheterosexual populations ,4,5,6,7.A growing body of research has also shown that the state policy environment for sexual minorities, defined as the legal protections afforded, is related to health. Rooted in social ecological theories of health , public This study therefore builds on previous research and extends the analysis of self-rated health disparities by gender and sexual minority status. We hypothesize that living in a state with comprehensive legal protections for sexual minorities is associated with narrower disparities in self-rated health for lesbian, gay, and bisexual individuals. To test this hypothesis, we used recently collected data from a large, population-based, and multistate survey to compare self-rated health by state policy environment (comprehensive versus limited protections for LGB people). While previous studies have found that self-rated health for sexual minorities was generally better in states with legal protections, early studies often lacked a comparison group of heterosexual individuals to examine disparities in self-rated health. This study examines (1) how disparities in self-rated health vary across the United States and (2) whether disparities in self-rated health are modified by comprehensive legal protections for sexual minorities.This study analyzed recently collected data from the 2014–2016 Behavioral Risk Factor Surveillance System (BRFSS). The BRFSS is a nationally representative and telephone-based survey of civilian, noninstitutionalized adults aged 18 years and older in the United States. Conducted since 1984 by the Centers for Disease Control and Prevention (CDC) in coordination with state health departments, the BRFSS now collects health information for approximately 450,000 randomly selected adults each year. The core questionnaire ascertains key demographic and socioeconomic characteristics, medical diagnoses, health behaviors, and access to medical care. States can participate in optional CDC-designed modules or add their own specific questions to their statewide BRFSS surveys. The median response rate across all states was 47.1% in 2016 (which ranged from 30.7% in Louisiana to 65.0% in Wyoming) .The core demographic questionnaire in the BRFSS currently does not ask respondents about their sexual orientation, but some states have previously added sexual orientation questions to their individual BRFSS surveys ,24,25. Bn = 1945), respondents who did not know the answer (n = 5117), respondents who refused to answer (n = 8961), and respondents missing information on self-rated health (n = 1521). Previous research on nonresponse to a related sexual orientation question in state-level BRFSS surveys reported higher prevalence of nonresponse among racial and ethnic minorities [In the sexual orientation and gender identity module, respondents were asked which of the following categories best represents how they identify themselves: straight; lesbian or gay; or bisexual. While the BRFSS does include information on transgender status, we chose to focus this study on the association between state policy environments and self-rated health for sexual minorities. Our final sample included 14,687 sexual minority adults aged 18 years and older and 490,071 heterosexual adults aged 18 years and older. Our analysis excluded respondents indicating their sexual orientation as something else  by sexual minority status for all 31 states ascertaining sexual orientation. We restricted state-specific estimates to states with 50 or more sexual minority respondents to ensure a large enough sample size . Then, we compared poor/fair self-rated health by sexual minority status and state policy environments. Individuals from all 31 states were classified into two categories based on the level of legal protections granted to sexual minorities in 2014  ,30.svy command to reflect the complex survey design of the BRFSS. This study was deemed exempt by the Institutional Review Board because data were obtained from publicly available and secondary sources.Unadjusted and adjusted logistic regression models were conducted to compare poor/fair health by sexual minority status, first for all adults and then stratified by state policy environments . All analyses were conducted separately by gender. Our fully adjusted models controlled for age, race/ethnicity, relationship status, the presence of a child in the household, educational attainment, employment status, household income, chronic disease diagnoses, health insurance status, having a usual source of care, state of residence, and survey year. Results from the logistic regression models are presented as odds ratios (ORs) with 95% confidence intervals (CIs). All analyses were conducted in Stata version 14 using survey weights and the This study used a well-validated, population-based, and multistate sample of sexual minority and heterosexual adults in the United States to document disparities in self-rated health by state policy environment. We tested whether living in a state with comprehensive legal protections for sexual minorities  was associated with narrower differences in self-rated health by sexual minority status. As hypothesized, we found narrower and nonsignificant differences in self-rated health for sexual minority men in states with comprehensive protections, but sexual minority men in states with limited protections were more likely to report poor/fair self-rated health compared with heterosexual men. Unexpectedly, we did not find a similar relationship for sexual minority women, who were more likely to report poor/fair self-rated health regardless of the state policy environment.This study is consistent with previous research that has documented the association between better self-rated health among same-sex couples , sexual Our research also demonstrates the importance of state-level data for monitoring health disparities among sexual minorities. Having more comprehensive sexual orientation data at the state and municipal levels can inform targeted interventions for improving health outcomes and health behaviors in sexual minorities. Additional data can also help identify groups within the sexual minority population at greatest risk of worse health. For instance, while not examined directly in this study, sexual minority women experienced different trends and disparities in self-rated health compared with sexual minority men. On average, sexual minority women were more likely to report poor/fair self-rated health (20.7%) compared with sexual minority men (17.4%). Relatedly, sometimes disparities in self-rated health between heterosexual individuals and sexual minorities were wider for women than men in the same state . Future research should use qualitative or mixed-methods study designs to better understand how health is understood or interpreted for sexual minorities and whether definitions of `excellent’ health vary by sexual orientation and within the sexual minority population. Other research should continue to use alternative data  and different settings  to confirm or negate these findings.Our findings should be interpreted with caution, as several limitations were present throughout this study, such as reporting bias, missing data, small sample sizes, and noncausal research designs. First, all responses in the BRFSS—including sociodemographic characteristics and health status—were self-reported, which may suffer from recall and response bias. Relatedly, responses to the sexual orientation question may be threatened by selection bias. The sample of sexual minorities examined in this study only included adults in noninstitutionalized settings (with landline or cell phones) who were comfortable disclosing their sexual orientation to BRFSS surveyors. Homeless adults and adults in nursing homes, incarceration facilities, and institutionalized medical facilities were missing from this study. Also missing were adults not comfortable disclosing their sexual minority status, which may bias our estimates to the extent that vulnerable subpopulations were included among nonrespondents.Meanwhile, adults in states not using the sexual orientation module in their BRFSS survey were also missing from this analysis. Because our sample only included data from 31 states, our results may not be generalizable to the rest of the country or to the entire sexual minority population. States in the southeastern U.S. were especially underrepresented, which includes numerous states with limited legal protections for LGBT people. Differences in self-rated health may be wider than what we detected here. We are hopeful that additional states will participate in the sexual orientation and gender identity module or that questions on sexual orientation and gender identity will be added to the core BRFSS questionnaire in the near future.Small sample sizes also restricted our analysis to sexual minority status. We were unable to separate our analysis by specific sexual orientation . Relatedly, the BRFSS module only asks a single question about sexual orientation identity and does not measure other dimensions of sexual orientation, such as sexual behavior or sexual attraction. Our analysis precludes individuals who are sexually active with or attracted to people of the same sex but do not identify as lesbian, gay, or bisexual. Additionally, our study only focused on health disparities by sexual minority status and did not include comparisons by transgender status or gender identity—both because of the limited sample size of transgender respondents and because gender identity is a separate and distinct concept from sexual orientation . We hopeFinally, this analysis is limited by its observational research design. The BRFSS is a cross-sectional health survey and the correlations identified in this analysis cannot definitively establish the causal pathways between sexual minority status, self-rated health, and state policy environments. Omitted and unmeasured variables—such as experiences of sexual-orientation-based discrimination and micro-level exposures to inclusivity or hostility in various settings —may explain the relationship between state policy environments and self-rated health for sexual minorities reported here. Future research should examine the impact of micro-level exposures to discrimination on self-rated health. Other research should use quasi-experimental designs or longitudinal data to test and establish causality in the relationship between legal protections and health outcomes for sexual minorities. Still, more research should build on this study and examine the relationship between legal protections and alternative dimensions of health, including mental health, activity limitations, and health behaviors.This study adds new evidence of the association between state policy environments and self-rated health disparities for sexual minorities using a population-based and multistate sample in the United States. While much focus has been placed on the impact of state health policy on health outcomes, this study emphasizes the importance of further understanding the relationships between non-health-related legal protections and the health of populations vulnerable to discrimination. Ultimately, the ability to elucidate the impact of public policies at the federal, state, and local levels will enable the development of better strategies to reduce disparities and improve the overall health of vulnerable populations, including sexual minorities.


Exercise alters human homeostasis by different means, with energy metabolism particularly affected by heavy and strenuous activities. From a substantial increase in an expenditure of cellular energy to an up-regulation of proteins involved in all aspects of energy turnover  is highly warranted to track down a feasible tissue-specific performance of this compound for the duration of exhaustive exercise. Theoretically, alterations in circulating GAA might help us to better understand a complex interplay between changes in circulation and bioenergetics among different organs during heavy exercise.Sotgia et al.  were amoOnce dropped after an exercise session, GAA levels seem to steadily return to pre-exercise values. Al Fazazi et al.  recentlyr = 0.79) . In particular, having in mind that GAA-synthesizing enzyme (L-arginine:glycine amidinotransferase) is stimulated by testosterone while the main GAA transporter (SLC6AB) is down-regulated by estrogen  being its only metabolic fate. It appears that GAA availability in the circulation could be modulated by physical exercise, perhaps due to an exercise-induced disbalance in tissue-specific GAA release and removal. Initial studies suggest that strenuous activities diminish circulating GAA levels in a magnitude-depended manner thus putting forward its use as a prospective biomarker of exhaustive exercise. Nevertheless, the exact mechanism of GAA utilization during heavy exercise needs to be further clarified and a more detailed exposure-response relationship  is required before recommending serum GAA as an accurate tool for general practice in exercise physiology and medicine. To achieve this, further carefully designed exercise intervention studies in both athletic and clinical settings are necessary to drive knowledge gain in this exciting area.SO drafted the manuscript. VS, LR, JB, PA, and AO performed a critical revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The exception was the inhibitor Dynasore and its more potent analogue Dyngo-4a, small molecules that target dynamin family GTPases, but also have off-target effects on the plasma membrane. Significantly, while Dynasore blocked stress-stimulated dye uptake at the ocular surface of ex vivo mouse eyes when treatment was performed at the same time as eyes were stressed, it had no effect when used after stress was applied and the ocular surface was already damaged. Thus, Dynasore could not be working by inhibiting endocytosis. Employing cytotoxicity and western blotting assays, we demonstrate an alternative mechanism, showing that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to oxidative stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into mechanisms of vital dye uptake and emphasize the importance of using a differentiated cell culture model for such studies. They also suggest that Dynasore and analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.“Vital” dyes such as fluorescein and rose bengal are used clinically to evaluate ocular surface health; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal cell damage stimulates fluorescein dye uptake. Since damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. Using an oxidative stress model, we show that damage to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates uptake of fluorescein and rose bengal dyes and also stimulates endocytosis. Importantly, dye uptake was blocked by co-treatment with three different endocytosis inhibitors. Damage to stratified and differentiated corneal epithelial cell cultures, which are a better model of the ocular surface, also stimulated dye uptake; however, endocytosis was  The wet ocular surface comprises the stratified squamous mucosal epithelia of the cornea/conjunctiva and the overlying tear film . These cDirectly exposed to the external environment, the ocular surface epithelia are subject to damaging agents and physical insults such as ultraviolet light, microorganisms and allergens, that cause ocular surface damage, barrier disruption and increased desquamation . Ocular Considering the widespread use of vital dyes, it is surprising that the mechanism of staining is still not well understood . Studiesin situ under epifluorescent illumination -1,3-benzene disulfonate; Thermo Fisher Scientific). Superoxide anions generated by NAD(P)H-dependent cellular oxidoreductase activity reduce MTT or WST to water-soluble formazans which absorb visible light . MTT dyeThe trypan blue exclusion assay is based on the principle that live cells possess intact cell membranes that exclude certain dyes, whereas dead cells do not exclude the dye . Cells wTo determine the presence of mucin-type glycoconjugates on cell surfaces, a binding assay was performed with jacalin, a lectin that specifically recognizes the T-antigen present on O-glycans , 43. Met°C with gentle shaking. Membranes were then incubated for 1 hour with secondary antibody–horseradish peroxidase conjugates  at a dilution of 1:10,000. Specific signals were developed for 1 min using the enhanced chemiluminescence (ECL) kit components 1 and 2 . Chemiluminescence was visualized by exposure of photographic film .Proteins from equal volume cell culture media samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). Membranes were probed with a primary antibody against LGALS3  overnight (at 1:200 dilution) at 4As a measure of cell lysis, cell culture medium was collected and an equal portion of the media was subjected to polymerase chain reaction (PCR) using primer sets for detection of ACTB gene fragments , as described previously .The goal of our first set of experiments was to characterize and validate our oxidative stress model in monolayer cultures of human corneal epithelial cells, referencing the two recent studies discussed in the Introduction , 21. RepAs visualized under epifluorescent illumination , fluoresTo characterize cell damage caused by oxidative stress, we probed for both early (ANXA5 binding assay) and late (TUNEL assay) stages of apoptosis. Phosphatidylserine exposed on the outer leaflet of the membrane surface leads to ANXA5 binding. The percentage of cells that bound ANXA5 was substantially increased by oxidative stress, as judged by visual inspection . This agNext, we attempted to determine whether oxidative stress would also stimulate hyperstaining in stratified and differentiated cell cultures . To asseNext, we investigated the possible role of endocytosis in oxidative stress-stimulated vital dye uptake by monolayer cell cultures. Representative results are shown in First, we determined whether endocytosis was stimulated by oxidative stress. Cells were stressed in the presence of Alexa Fluor 568-conjugated TF, internalization of which is a marker of the endocytic process. As predicted, oxidative stress-stimulated TF uptake by cells in monolayer culture . Co-treaNext, we determined whether endocytosis inhibitors blocked stress-stimulated vital dye uptake. Stress-stimulated fluorescein dye uptake was blocked by genistein, an inhibitor of caveolin-mediated endocytosis, and chlorpromazine, an inhibitor of clathrin-mediated endocytosis. It was also blocked by Dynasore, which is required for both forms of endocytosis .These results link between stress-stimulated endocytosis and vital dye uptake in monolayer cell cultures.In our next set of experiments, we examined the possible connection between endocytosis and vital dye uptake by stratified and differentiated cell cultures. These experiments were designed similarly to those described above, using monolayer cell cultures. Representative results are shown in First, we investigated whether endocytosis was stimulated by oxidative stress, as was done in the monolayer cell culture experiments described above. The results were quite different in stratified cell cultures. Oxidative stress did not stimulate TF uptake, in fact there appeared to be some inhibition .Next, we investigated the possible role of endocytosis in oxidative stress-stimulated vital dye uptake, as done for the monolayer cell culture experiments. Again, the result was quite different in stratified and differentiated cell cultures. Genistein and chlorpromazine had no effect on vital dye uptake , in thesThese results indicate that the link between vital dye uptake and endocytosis, observed in monolayer cells, does not hold for stratified and differentiated cells, which better model the ocular surface. The fact that Dynasore and its analogue Dyngo-4a inhibit vital dye uptake must thus occur through an alternative mechanism.As already noted, stratified cell cultures of corneal epithelial cells are a better model of the ocular surface than monolayer cultures. However, the stratified mounds of cells that develop in differentiation medium do not form a smooth and continuous layer like the corneal epithelium. The goal of the next set of experiments was to validate the cell culture findings made thus far to the actual ocular surface, and also to extend those findings. These experiments made use of the mouse eye organ culture model. Representative results are shown in ex vivo mouse eyes were placed in organ culture and left unstressed or subjected to oxidative stress for three hours. At the end of this time period, eyes were removed from the culture medium and the vital dye staining assay was performed immediately. Application of stress stimulated a significant increase in dye uptake at the ocular surface  was doubled (80 uM).To extend our investigation, we examined the effects of adding an endocytosis inhibitor  seconds . NeverthThese results provide further evidence to reject the hypothesis that stress stimulates vital dye uptake via endocytic vesicles.The goal of the next set of experiments, was to investigate an alternative hypothesis, that Dynasore protects the ocular surface against damaging oxidative stress, thus precluding vital dye uptake. Representative results are shown in First, we investigated protection of the cells themselves, using monolayer cultures. Applying the MTT/WST-1 metabolic assay, we found that absorbance was greatly increased by application of oxidative stress, indicative of cell damage. Treatment with Dynasore at the same time as stress was applied was protective, in particular for the WST-1 assay . ApplyinNext, we investigated protection of the glycocalyx, using stratified cell cultures with mucosal differentiation. Representative results are shown in A fluorescently-labelled lectin binding assay was used to assess damage to the mucins at the surface of differentiated cells. Jacalin is a lectin that binds to O-linked glycans found on membrane-associated mucins of the ocular surface , 43. Jacnd lane to 4th lane in set 1 and set 2). Levels of genomic DNA in the culture medium remained unaffected, consistent with an increase in LGALS3 shedding from the glycocalyx itself, rather than greater shedding of cells containing LGALS3 on their cell surface.While we were unable to detect any changes using the lectin assay, damage to the glycocalyx was evident when LGALS3 was examined by Western blotting. Oxidative stress caused an increase in LGALS3 in the cell culture medium consistent with an increase in shedding from the apical cell surface; treatment with Dynasore inhibited this increase , and a operties .How Dynasore and its analogue Dyngo-4a are so effective in protecting the ocular surface epithelial cells against oxidative stress remains an open and very intriguing question. These compounds were developed to specifically target dynamin family GTPases as an aid for the study of endocytosis ; howeverAfter we submitted this manuscript for review, we became aware of a new publication by the same team that conducted one of the studies that served as a reference here . Again, Dynasore has been proposed as a candidate therapeutic to treat diseases involving abnormal mitochondrial dynamics , 63, andHere we made the unexpected and novel discovery that Dynasore and its more potent analog Dyngo-4a protect ocular surface epithelial cells and their glycocalyx against damaging stress. These unexpected and novel findings provide greater insight into the mechanisms of vital dye uptake and point the direction for future study. Our results also suggest that Dynasore and its analogues might have therapeutic value in the treatment of ocular surface disease, and this use should be explored.


Transcatheter aortic valve implantation (TAVI) has emerged as an important alternative to surgical aortic valve repair (SAVR) for patients with severe aortic stenosis. This rapidly advancing field has produced new-generation devices being delivered with small delivery sheaths, embolic protection devices and improved retrieval features. Despite efforts to reduce the rate of thrombotic complications associated with TAVI, valve thrombosis and cerebral ischaemic events post-TAVI continue to be a significant issue. However, the antithrombotic treatments utilised to prevent these dreaded complications are based on weak evidence and are associated with high rates of bleeding, which in itself is associated with adverse clinical outcomes. Recently, experimental data has shed light on the unique mechanisms, particularly the complex haemodynamic changes at sites of TAVI, that underpin the development of post-TAVI thrombosis. These new insights regarding the drivers of TAVI-associated thrombosis, coupled with the ongoing development of novel antithrombotics which do not cause bleeding, hold the potential to deliver newer, safer therapeutic paradigms to prevent post-TAVI thrombotic and bleeding complications. This review highlights the major challenge of post-TAVI thrombosis and bleeding, and the significant issues surrounding current antithrombotic approaches. Moreover, a detailed discussion regarding the mechanisms of post-TAVI thrombosis is provided, in addition to an appraisal of current antithrombotic guidelines, past and ongoing clinical trials, and how novel therapeutics offer the hope of optimizing antithrombotic strategies and ultimately improving patient outcomes. Severe aortic stenosis (AS) represents a major global healthcare burden, with medical management being associated with a poor prognosis and high rates of mortality . CalcifiSince the first procedure in 2002, TAVI has not only offered a new approach to treating calcific aortic stenosis but has also provided the large cohort of patients deemed intermediate to high surgical risk access to life-saving treatment ,7. This The TAVI procedure involves guiding a prosthetic valve, typically via a transfemoral catheter, and implanting it into the existing aortic valve . With raAs a consequence, there have now been more than 500,000 TAVI procedures performed in over 70 countries and at over 1000 centres [In spite of these developments, the issue of ischaemic and bleeding events continues to impact the immediate and long-term morbidity and mortality of patients undergoing TAVI procedures .Based on the results from the Surgical Replacement and Transcatheter Aortic Valve Implantation (SURTAVI) trial, the Placement of Aortic Transcatheter Valves (PARTNER) 2 cohort and the US CoreValve studies ,12,13,15p < 0.0001) and that patients with major bleeding/life-threatening bleeding showed a 410% increase in mortality compared with patients without bleeding . In addition, the presence of atrial fibrillation was independently correlated with TAVI-associated bleeding  [A recent meta-analysis highlighted that post-TAVI bleeding was associated with a 323% increase in 30-day postoperative mortality (odds risk (OR): 4.23, 95% confidence interval (CI): 2.80–6.40; = 0.005) .In addition to the bleeding risk associated with antithrombotic therapy, it is important to note that scoring systems, such as the EuroSCORE II and the Society of Thoracic Surgeons (STS) risk score, can be utilised to stratify the mortality risk of patients undergoing TAVI. These scores incorporate a range of clinical variables such as age, renal impairment and New York Heart Association Functional Classification as part of a clinically validated risk assessment model to help predict outcomes in patients undergoing cardiac procedures ,18. p = 0.007) whilst low body mass index is linked with increased all-cause mortality [However, important factors that heavily influence mortality after percutaneous procedures, such as frailty and body mass index, are not included in these scoring systems . Highligortality ,21.Therefore, despite the adoption of these prediction scores, the ‘heart team’ plays a central role in determining suitable candidates for TAVI or SAVR. Thus, with advancements in TAVI systems and technical knowledge, the focus of heart teams has shifted from discussing the technicalities of the procedure to assessing the patient and the important prognostic variables not represented by the EuroSCOREII and STS scoring systems to ensure the selection of patients most likely to benefit from TAVI. These results emphasise the importance of appropriate antithrombotic therapy following a TAVI procedure, given the often high-risk group of patients that undergo TAVI. Moreover, given the high rates of bleeding observed post-TAVI, these data highlight the clinical need for antithrombotic strategies that are tailored towards minimizing bleeding risk.The mechanisms underlying thrombosis associated with TAVI are likely multifactorial. In this regard, a number of contributing factors potentially explaining the thrombotic risk associated with TAVI have been proposed. These include: (1) flow disturbances associated with prosthetic valve placement, (2) the introduction of a prothrombotic metallic frame, and (3) a co-existent prothrombotic tendency in an older, co-morbid population ,23,24. ERecent data indicates that flow stagnation in the neosinus is likely to be an important haemodynamic factor associated with a heightened thrombotic risk ,32,33,342 = 47%) and cerebrovascular events  accordingly [The placement of the metallic frame creates a further nidus for thrombus formation. Although not formally tested, it is likely that placement of the metallic frame results in endothelial damage that in the context of local perturbation of endothelial matrix components and haemodynamic disturbances creates a prothrombotic milieu. Akin to coronary stents, prior to the endothelialisation of the metallic frame, the exposed artificial surface acts as a substrate for platelet adhesion and activation, as well as the initiation of the contact activation coagulation system. Furthermore, it has recently been established that haemodynamic forces play a central role in mediating platelet activation ,37. Togeordingly . In addition to the emergence of AF and the local prothrombotic milieu that occurs in the setting of TAVI, periprocedural stroke can occur as a consequence of the dislodgment of calcified valvular components upon insertion of the prosthesis ,43,44. ERecent data has demonstrated that valve leaflet thrombosis is a common occurrence post-TAVI, with this complication being diagnosed in approximately 10–15% of patients by computed tomography angiography post-TAVI . InteresTo date, the available evidence suggests that leaflet thrombosis can affect aortic valve pressure gradients; however, the magnitude of this effect ranges from clinically insignificant leaflet restriction to symptomatic valve deterioration . In thisp = 0.912; for stroke- or transient ischaemic attack-free survival 98.5% vs. 96.8%, p = 0.331). In contrast, data from the MAUDE database highlights that leaflet thrombosis complicated by structural valve disease (SVD) (aortic stenosis or regurgitation) is associated with adverse outcomes including stroke, cardiogenic shock and death [Interestingly, recent data suggest that the development of subclinical leaflet thrombosis following TAVI is not associated with increased mortality or stroke . This obnd death . Indeed,With the inherent risk of stroke and leaflet thrombosis in the setting of TAVI, post-TAVI antithrombotic therapy is essential to minimise these risks. However, all currently clinically available antithrombotic therapy is invariably associated with a risk of bleeding . In the The current guidelines regarding the use of antithrombotic therapy post-TAVI are summarised in p = 0.50). A pattern, however, was noted in favour of fewer life-threatening and major bleeds in the ASA (aspirin) group compared with DAPT, questioning the value of clopidogrel in this context. Two recent meta-analyses conducted by Hu et al. and Ahmad et al. reached similar conclusions, suggesting that DAPT, when compared with SAPT following TAVI, displays non-superiority in reducing thrombotic events but involves added risk of major and life-threatening bleeds [Current recommendations comprise administering dual antiplatelet therapy (DAPT), with clopidogrel used for the first 3–6 months and aspirin maintained indefinitely, given there is no indication for anticoagulation . The theg bleeds ,63. HoweThe role of anticoagulation post-TAVI is vitally important but is still a highly contentious issue. This has come into focus with the observation that oral anticoagulation (OAC) may prevent subclinical leaflet thrombosis and valve deterioration . Indeed,Current guidelines recommend OAC ) in addition to aspirin for patients undergoing TAVI with concurrent AF. This practice has recently been supported by results from the FRANCE TAVI registry, where anticoagulation was correlated with decreased risk of bioprosthetic valve dysfunction after TAVI . HoweverRecently, the question as to whether NOACs may confer a lower bleeding risk compared to VKA in the setting of TAVI has begun to be addressed. A consistent finding from the large-scale trials evaluating NOACs for the prevention of arterial thromboembolism in AF and treatment of venous thromboembolism (VTE) has been the lower bleeding risk, especially intracranial bleeding, associated with NOACs in comparison to VKA ,72. ReceThe POPular-TAVI  trial is a multicentre open-label randomised trial currently investigating the hypothesis that monotherapy with aspirin or OAC is safer than dual therapy with clopidogrel for three months. Cohort A comprises patients on aspirin (up to 100 mg OD) versus aspirin plus clopidogrel 75 mg OD). Cohort B comprises patients on OAC (according to their indication) versus OAC plus clopidogrel. The primary outcome evaluated is freedom from non-procedure-related bleeding at one year and the trial will also assess for net clinical benefit [ mg OD. CThe ATLANTIS  trial is a Phase IIIb multicentre open-label randomised trial investigating the efficacy of apixaban following TAVI, compared with DAPT, for 12 months . The expThe ENVISAGE-TAVI  in Atrial Fibrillation) trial is a multicentre open-label randomised trial comparing edoxaban to VKA-based therapy in patients with an indication for OAC after TAVI for up to 36 months . The priThe AUREA  and AVATAR  trials are also smaller-scale multicentre randomised studies comparing VKA with DAPT and aspirin plus VKA therapy respectively for strokes and other complications ,79.The ongoing trials discussed will provide important new insights regarding how NOACs may fit in the antithrombotic paradigm post-TAVI. However, a number of important challenges remain. The bleeding risk associated with current antithrombotic regimes remains a significant concern. In this regard, there have been a number of new antithrombotic drugs developed in the last decade that hold the promise of inhibiting thrombosis without significantly impeding haemostasis ,81. ForeAnother aspect of TAVI-associated thrombosis that is yet to be tested is whether mechanistically, the metallic frame associated with the valve causes significant contact factor activation. The RE-ALIGN trial, which investigated dabigatran for the prevention of thrombosis in patients with mechanical heart valves, had to be ended prematurely due to the elevated risk of bleeding and thrombosis associated with dabigatran . SubsequAs discussed above, a major outstanding issue is identifying the patients most likely to benefit from more intense antithrombotic strategies post-TAVI. Our group has developed novel therapeutic and imaging agents based on a unique single-chain antibody that specifically binds to activated platelets . Thus, tIn addition to novel therapeutics, there remains much interest within the bioengineering field to design and develop new valves that may produce fewer haemodynamic disturbances and expose less thrombogenic material and as such, may result in a lower thrombosis risk . The introduction of TAVI represents one of the most successful innovations in cardiovascular medicine in the last two decades. This has afforded patients with severe aortic stenosis, especially the elderly and those with significant co-morbidities, access to treatment where surgery is often contraindicated. With significant improvements in mechanical durability and implantation technologies, TAVI-related thromboses and bleeding associated with antithrombotic therapy, are becoming the major determinants of the long-term outcome of TAVI. A growing recognition of the shortcomings of the current antithrombotic regimes used to prevent TAVI-associated thrombosis has led to several ongoing clinical trials, which will no doubt inform the development of new practice guidelines. Simultaneously, with a deepening understanding of the mechanisms causing the development of post-TAVI thrombosis and the recent advent of a range of novel antithrombotics that do not inhibit haemostasis, the stage is set for the development of safer and more effective TAVI-specific antithrombotic therapy.


The recent surge in incorporation of metallic and metal oxide nanomaterials into consumer products and their corresponding use in occupational settings have raised concerns over the potential for metals to induce size-specific adverse toxicological effects. Although nano-metals have been shown to induce greater lung injury and inflammation than their larger metal counterparts, their size-related effects on the immune system and allergic disease remain largely unknown. This knowledge gap is particularly concerning since metals are historically recognized as common inducers of allergic contact dermatitis, occupational asthma, and allergic adjuvancy. The investigation into the potential for adverse immune effects following exposure to metal nanomaterials is becoming an area of scientific interest since these characteristically lightweight materials are easily aerosolized and inhaled, and their small size may allow for penetration of the skin, which may promote unique size-specific immune effects with implications for allergic disease. Additionally, alterations in physicochemical properties of metals in the nano-scale greatly influence their interactions with components of biological systems, potentially leading to implications for inducing or exacerbating allergic disease. Although some research has been directed toward addressing these concerns, many aspects of metal nanomaterial-induced immune effects remain unclear. Overall, more scientific knowledge exists in regards to the potential for metal nanomaterials to exacerbate allergic disease than to their potential to induce allergic disease. Furthermore, effects of metal nanomaterial exposure on respiratory allergy have been more thoroughly-characterized than their potential influence on dermal allergy. Current knowledge regarding metal nanomaterials and their potential to induce/ exacerbate dermal and respiratory allergy are summarized in this review. In addition, an examination of several remaining knowledge gaps and considerations for future studies is provided. Over the past several decades, an extensive amount of scientific attention has been invested in the field of nanotechnology. Significant advances have been made in understanding the unique behaviors of matter with nano-scale dimensions. This progress has facilitated the capacity for manipulation of material properties to optimize their functional utility. Subsequently, nanomaterials have proven useful in diverse applications ranging from pharmaceutics and energetics to transportation and electronics. The exponential growth of the nanotechnology field has left few sectors unaffected by its momentum, as the global nanotechnology market has been valued at over $20 billion US . Although the resounding impact of these technological advancements has generated comparisons to the impact of the industrial revolution, the expansion of nanotechnology has also generated several notable concerns. In addition to the environmental, legal, ethical, and regulatory challenges imposed by the expanding presence of nanotechnology, the potential risk for adverse health effects following exposure to nanomaterials has also become a major concern.As a result, a unique discipline of toxicology has emerged to evaluate the potential health effects of nanomaterials. Nanotoxicology studies have consistently demonstrated that the unique properties of nanomaterials that render their industrial functionality also implicate unique interactions with biological systems. A general correlation between decreasing size and increased toxic potential has been observed for many nanomaterials . Howeverin vivo studies . In the United States, allergic diseases are the sixth leading cause of chronic illness with an annual cost exceeding $18 billion US (Centers for Disease Control and Prevention 2017). Although the development of allergy is dependent on a multitude of genetic, behavioral, and environmental factors, exposures to immunotoxic agents are a major underlying contributor to allergic diseases . ImmunotAs the nanotechnology market continues to expand and the global prevalence of allergic disease continues to increase, the knowledge gap regarding the immunotoxic potential of nanomaterials is becoming increasingly relevant. Specifically, the capacity for nanomaterials to cause or exacerbate allergic disease remains largely unknown, which is particularly concerning with respect to a specific class of nanomaterials. Metal-based nanomaterials  are one of the classes of nanomaterials being produced in the largest quantities. Noteworthy metal nanomaterials, their applications, and corresponding rates of production are shown in Metal nanomaterials are being increasingly incorporated into nano-enabled products and consumer goods, increasing the potential for exposures in the general public . AlthougSeveral specific concerns have emerged with respect to metal nanomaterials and their potential effects on allergic disease. First, the characteristic size profile of metal nanomaterials may confer enhanced potential for skin penetration and more efficient deposition in the lungs, circumventing one of the major barriers associated with limiting adverse immune effects induced by larger-sized metals. Secondly, the exposure threshold required for dermal and respiratory sensitization by metal nanomaterials may be lowered as a result of their unique chemistry. Lastly, the induction of inflammation and tissue injury caused by many metal nanomaterials may serve as an adjuvant, promoting the development of allergic disease to environmental allergens or exacerbating the severity of established allergic conditions. This review aims to summarize current scientific knowledge regarding these concepts. In addition to dermal and respiratory studies that examine specific metal nanomaterial effects, studies designed to delineate the role of physical and chemical properties in these effects are emphasized. Finally, considerations and knowledge gaps in the field are highlighted as potential directions for future research.Metals are a class of agents associated with expansively diverse immune effects including irritancy, autoimmunity, sensitization, and adjuvancy . The potThe most common metal-induced allergic disorder of the skin is ACD, a T-cell-mediated delayed-type hypersensitivity response. Dermal sensitization and the subsequent induction of ACD requires several key molecular and cellular events , which hThe preliminary requirement for skin sensitization is bioavailability of the sensitizing agent. Since a primary function of the skin is to serve as an effective barrier between the host and environment, the sensitizing potential of many antigens is limited by their capacity to evade this barrier . PassageThe next steps in the skin sensitization AOP involve the molecular initiating event of skin sensitization-antigen formation. The small size required for antigen passage through the stratum corneum is not conducive with cellular recognition . As a reIn addition to uptake of the hapten/carrier complex, activation of LC requires an additional antigen-nonspecific signal indicative of an elevated threat level. Many mediators capable of fulfilling this signal are released by non-immune cells including keratinocytes in response to injury . Presenc+ T-lymphocytes until recognition occurs by antigen-specific T-cell receptor (TCR). Given adequate costimulatory signals from the LC, T-lymphocytes undergo proliferation producing a pool of clonal antigen-specific effector T-cells. The T-cells enter the circulation, and following resolution of inflammation, a subset of these effector cells will survive and become memory T-cells, completing the process of sensitization.Once the LC reaches the lymph nodes, the processed hapten is presented via major histocompatibility Class I (MHC I) molecules to naïve CD8+ T-cells exhibit immediate cytotoxic effector functions. CD4+ T-helper (TH) of the TH1 phenotype have regulatory roles in ACD and produce high levels of the cytokines interleukin (IL)-2 and interferon (IFN)-γ, contributing to inflammatory cell recruitment  and respirable size range (< 10 μm) . Likewis+ T-lymphocytes along with costimulatory molecules, resulting in the preferential expansion of the TH−2 phenotype lymphocytes. These cells produce high levels of IL-4, IL-5, and IL-13, and stimulate isotype switching and allergen-specific IgE-production by B-cells. The Fc portion of secreted IgE is bound to FceRI receptors present on tissue-resident mast cell surfaces and circulating basophils, exposing the antigen-recognizing motif, completing the sensitization process (Since both low and high molecular weight (HMW) agents are capable of inducing asthma, the molecular initiating events of respiratory sensitization may differ accordingly. Similar to the skin, pulmonary immune responses following inhalation of metals can be nonspecific and self-limiting, or can result in the recruitment of the adaptive immune system. Lung-resident DC take up antigen, and given adequate second signals, the antigen is processed and DC migrate to the lung-draining lymph nodes. Here, the peptide is presented to naïve CD4 process .Upon subsequent encounters, the allergen is bound by allergen-specific IgE on the surface of mast cells and basophils. Binding induces crosslinking of receptors and the subsequent release of preformed mediators such as histamine, beginning the anaphylactic cascade responsible for the early asthmatic reaction experienced minutes after antigen encounter. Acute clinical manifestations of allergic asthma range from rhinitis and bronchoconstriction to anaphylactic shock. The late phase asthmatic response occurs 4–6 h later as a result of mast cell mediators and recruitment of inflammatory cells . ClinicaCompared to metal-induced ACD, metal-induced asthma occurs far less frequently. Cases tend to be isolated to individuals working in occupations involving metalwork where metal fumes, dust, or vapors are generated and inhaled . Nickel,In addition to their potential to induce sensitization, metals are also associated with the capacity to modulate allergic responses to nonmetal allergens. Contrasted with the consistent, sequential series of cellular processes involved in allergic sensitization and elicitation, adjuvant effects can emerge as a result of various mechanisms in various phases of allergic disorders .An example of metal adjuvant effects on the development of adaptive immune responses is best demonstrated by aluminum hydroxide, which is one of the most frequently used vaccine adjuvants. When administered with poorly-immunogenic antigens, aluminum hydroxide induces adequate stimulation of the innate immune system to generate antigen-specific immunological memory. Mechanisms of immunopotentiation associated with aluminum hydroxide include triggering release of alarmins, activation of inflammasomes (intracellular multi-protein complexes involved in innate immune responses), and DC activation; however, numerous other mechanisms including immune cell recruitment and activation, modulated cytokine production, and altered antigen delivery kinetics, can also enhance sensitization .Similarly, metals are also associated with adjuvant effects on established allergic conditions, as demonstrated by metal-rich ambient air pollution, which is known to exacerbate the severity of asthmatic responses to environmental allergens . AdjuvanMetals are known to induce many unique immune effects implicated in allergic disease. With respect to sensitization, the modulation of innate immune reactivity by some metals has been associated with their immuno-genicity. For example, some metal ions are known to produce functional mimicry of pathogen-associated molecular patterns (PAMP) . Gold io+ nickel-specific T-cell clones, which were shown to cross-react when presented with other transition metals including copper and palladium  and zinc oxide nanoparticles (ZnO NP) have led to their incorporation in sunscreens and cosmetics for their protective effects against ultraviolet radiation (UVR)  . Silver on (UVR) . Likewisl public . These nl public  are alsoThe potential for metal nanomaterials to penetrate the skin, induce dermal sensitization, and modulate skin allergy development/responses are the three main areas discussed in this section with respect to size and other physicochemical properties. In correspondence with the review of the literature, Adverse immune effects following dermal exposure to an agent are heavily dependent on the degree to which the skin protects from its entry into the body. Likewise, one mechanism by which dermal exposure to metal nanomaterials may lead to increased potential for adverse immune effects compared to larger-sized metals is by size-mediated evasion of skin barrier function. Although it seems logical that the small size of nanomaterials would inherently provide increased opportunity for absorption via the skin, there is currently no general consensus on the skin-penetrating capabilities of nanomaterials as a collective class of agents .in vivo and in vitro models. Iron-based nanoparticles (FeNP), gold nanoparticles (AuNP), palladium nanoparticles (PdNP), nickel nanoparticles (NiNP), AgNP, SiNP, and metal-based quantum dots (QD) have all been associated with penetration of the skin  can penetrate skin in various the skin . Moreovethe skin . Hydrophthe skin .2 NP and have not generated equally consistent findings. Numerous studies have demonstrated that the stratum corneum effectively restricts passage of TiO2 NP, irrespective of size shown to facilitate penetration of the skin by other metal nanomaterials. Repeated application of different forms of TiO2 NP did not lead to skin penetration in hairless rats, elevated levels of titanium in lymph nodes of minipigs, or penetration of human skin transplanted onto immunodeficient mice , 10 nm PdNP, 78 nm NiNP, and 80 nm CoNP  were worn by human subjects for 8 h a day for 5 consecutive days, following which levels of AgNP and aggregates in the skin were quantified. Compromised skin barrier was not associated with increases in AgNP skin accumulation; moreover, no differences in urine Ag levels were observed, indicating that atopic dermatitis did not impact the absorption of ions released from the textiles either .2 NP/ZnO NP-containing sunscreens. As noted by Discrepancies in findings regarding the importance of skin barrier integrity on metal nanomaterial skin penetration may be explained by several observations. The diverse degrees of epidermal barrier function disruption between studies represent a potential source of variation. Complete ablation of epidermal function is only observed in response to severe burns and lacerations; likewise, the diverse mechanisms of experimentally-induced disruptions of the stratum corneum should be compared cautiously. In addition, the pathogenesis of atopic dermatitis between experimentally-induced animal models and humans may explain some discordant findings. The severity of lesions between subjects of human studies is also subject to extreme variation, as well. Since chronic skin inflammation can result in epidermal thickening, enhanced barrier function is not uncommon in many skin disorders . Lastly,2 NP, and QD   . Accordinsitizer . Similartization .in vivo technique used to evaluate dermal sensitization potential that has been employed in the investigation of several metal-based nanomaterials. In one study, five UV-absorbing materials containing SiO2 NP, ZnO NP, and TiO2 NP were assessed. One out of 10 animals exhibited slight erythema following topical exposure to the ZnO NP and TiO2 NP-containing agents, leading to their classifications as mild skin sensitizers (The guinea pig maximization test (GPMT) is another sitizers . In anotsitizers . Similarsitizers . The GPMsitizers .+ T-cells and IL-17a, but not IFNγ. Moreover, ear swelling was observed in response to additional sizes of AgNP (50 and 100 nm) and Ag ions, suggesting that the immune response is not nanoparticle-specific. Further examination revealed that 3 nm NiNP was also capable of inducing sensitization in the model, whereas minimally-ionizable 10 nm AuNP and 10 nm SiNP were not  is an residues . While sresidues . Moreovetization .in vitro assay for determination of skin sensitizing potential involves evaluation of the keratinocyte response to test agents, since they are a source of numerous mediators that facilitate LC migration, antigen presentation, and T-cell activation during sensitization  on human APC. However, since the recommended cell lines for these assays (THP-1 and U937) are representative of general DC and not skin-specific DC, these studies will be discussed in the in vivo studies confirmed metal nanomaterials including QD are taken up by LC and subsequently transported to lymph nodes , AuNP, FeNP, and SiNP have all been shown to modify DC antigen cross-presentation capacity . The mececeptors . In this2 NP have been shown to cause lipid peroxidation, and these metal nanomaterials have also been associated with enhancing DC cross-presentation  increased susceptibility of mice to sensitization, as evidenced by a lower concentration of DNCB required to induce sensitization , ACD responses may be subject to modulation as a result of chemical modifications induced by interactions with metal nanomaterials. In their study, the topical application of nanomaterials was subject to removal prior to application of DNFB. As a result, the particle-specific modulation of allergic skin inflammation was not reflective of blocked adduct formation. Although metal nanomaterials exhibit characteristically increased surface reactivity and catalytic potential, their capacity to impact the chemical properties of skin sensitizing chemicals has not been extensively studied. However, a few studies have demonstrated the potential for such effects to impact both ACD sensitization and elicitation. AlNP and AuNP have been shown to act as non-protein carriers of haptens capable of facilitating the generation of hapten-specific adaptive immune responses in vivo . Similarin vivo .In addition to ACD, metal nanomaterial effects on IgE-mediated atopic dermatitis have also been examined. Atopic dermatitis is generally associated with protein allergens, which under normal circumstances are not capable of penetrating the skin . However2 NP, AgNP, and SiO2 NP during sensitization to house dust mite (HDM) in atopic dermatitis models has been associated with an amplification of TH2 responses. This effect was shown to be more pronounced with decreasing size with respect to AgNP and SiO2 NP, but not for TiO2 NP  sensitization resulted in decreased levels of IgG1 and IgG2 and suppression of TH1 and TH17 responses in mice .Knowledge regarding metal nanomaterial effects on skin sensitization is largely limited to TiO2 NP, and SiO2 NP, as a result of their incorporation into construction materials, sunscreen sprays, disinfectants, and cosmetic powders, which upon use can lead to their inhalation. Workers are at risk for inhalation exposure to these and other highly-produced metal-based nanomaterials  in their workplaces , that are potent mediators of bronchoconstriction  following pulmonary exposure . Nano-materials can evade clearance by this mechanism by numerous effects. First, since macrophages have been shown to selectively phagocytose nanomaterials according to size, charge, and surface modification, physicochemical properties may contribute to their persistence in the respiratory tract. Their clearance may also be compromised as a result of selective cytotoxic effects on pulmonary macrophages and subsequently fewer viable macrophages capable of neutralizing the nanoparticles. Pulmonary macrophage cytotoxicity has been associated with physicochemical properties including morphology, surface charge, and rate of dissolution . Metal nlearance . In addilearance .H2 responses in the lung and downregulate APC functions, cytotoxic effects may disrupt the maintenance of an immunological tolerant state  were associated with elevations in OVA-specific IgE, whereas insoluble SiO2 NP and TiO2 NP were not. Subsequent investigations confirmed the importance of metal ion release in the adjuvant effects on sensitization. The increase in OVA-specific IgE production associated with soluble NiO NP was not conserved in response to insoluble NiO microparticles in the same model , or surface area (1300 cm2). Both studies showed that serum OVA-specific IgE levels best correlated with particle number and surface area  simultaneous to OVA sensitization exacerbated pathological changes, inflammatory cell influx, and TH2 cytokine responses. These effects were specific to the unique surface chemistry of each type of SiO2 NP, but the most severe responses were associated with the nanoparticle with the highest surface area , mucus hypersecretion, and other measures of asthmatic responses - or eosinophil (TH2)-dominated inflammation  showed that functionalized AuNP were capable of binding fibrinogen independent of nanoparticle size, but certain sizes of AuNP induced conformational changes in the protein. Subsequent alterations in protein structure conferred its recognition by the Mac-1 receptor, subsequently activating NF-κB signaling in innate immune cells. Similarly, Bastus et al. (2009) demonstrated that while macrophages did not recognize AuNP or two biomedically-relevant peptides individually, their conjugation facilitated recognition by TLR-4 and the subsequent induction of pro-inflammatory cytokine production. Since these signaling pathways play critical roles in many of the adjuvant effects mentioned in previous sections, interactions between metal nanomaterials and host proteins can generate novel sources of immunogenicity that may promote allergic processes.in vitro has been shown to be impacted by the unique physicochemical properties of metal nanomaterials. The size-specific increase in surface energy of TiO2 NP was shown to promote associations between the metal and human serum albumin, resulting in increased bioavailability . 2 particle sizes (10–1430 nm); while all particles promoted activation marker expression, the 240 nm SiO2 particles induced the greatest degree of CD80 expression. Similar findings were reported by an investigation that assessed the potential for metal debris released from orthopedic implants to trigger immune activation. Both ~2 mm cobalt-chromium-molybdenum alloy particles and soluble metal ions induced elevations in THP-1 co-stimulatory molecule expression, suggesting that a wide range of metal particle sizes have the capacity to induce immune effects involved in allergic sensitization  has not yet adopted size-specific OEL values for TiO2 (NIOSH 2011).Accurate evaluation of nanomaterial physicochemical properties and their correlation to toxic effects has become increasingly relevant as emerging nanomaterials challenge the efficacy of traditional occupational exposure limits (OEL). The majority of respiratory occupational exposure limits do not discriminate for material size, so as new metal-based nanomaterials emerge, they are subject to the same mass-derived values enforced for other materials of the same elemental composition. This issue is proving problematic as nanotoxicity studies continue to demonstrate that mass may not be the best dose metric for prediction of pulmonary toxicity . MoreoveAlthough there is a growing amount of toxicological data demonstrating the vast potential for adverse immune effects following exposure to metal nanomaterials, advancements in understanding their interactions with biological systems have allowed for their unique characteristics to be harnessed for beneficial applications, as well. Numerous studies have demonstrated the potential utility of metal nanomaterials for novel vaccine adjuvants, drug delivery vehicles, diagnostic approaches, and immunotherapies. However, to optimize the use of metal-based nanomaterials for these and other advantageous purposes, a more complete understanding of their systemic immune effects, mechanisms of immunomodulation, and capacity to induce allergic sensitization is needed.


Stress stimuli are ubiquitous and women do not enjoy any exemptions. The physiologic “fight-or-flight” response may be deleterious to the female lower genital tract microbiome if the stress stimuli persist for longer than necessary. Persistent exposure to psychosocial stress and stimulation of the hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal-medullary (SAM) axes, and associated hormones are risk factors for several infections including genitourinary tract infections. Though this could be due to a dysregulated immune response, a cortisol-induced inhibition of vaginal glycogen deposition may be involved especially in the instance of vaginal infection. The estrogen-related increased vaginal glycogen and epithelial maturation are required for the maintenance of a healthy vaginal ecosystem (eubiosis). The ability of cortisol to disrupt this process as indicated in animal models is important in the pathogenesis of vaginal dysbiosis and the subsequent development of infection and inflammation. This phenomenon may be more crucial in pregnancy where a healthy Lactobacillus-dominated vaginal microbiota is sacrosanct, and there is local production of more corticotropin-releasing hormone (CRH) from the decidua, fetal membranes and placenta. To highlight the relationship between the stress hormone cortisol and the vaginal microbiomial architecture and function, the potential role of cortisol in the maintenance of vaginal health is examined. Stress is an organism's attempt to adequately respond to either internal or external threats or injuries. It refers to any physical or psychological challenge that threatens or has the potential of threatening the equilibrium of an organism's internal milieu (homeostasis) –3. Such Cortisol is a steroid (glucocorticoid) hormone produced by the zona fasciculata of the adrenal cortex within the adrenal gland. It is released by the adrenal glands as part of the fight-or-flight mechanism in response to stress or fear, but has been described as public health's foremost enemy . Its funThe release of cortisol is regulated by the HPA axis via the secretion of corticotropin-releasing hormone (CRH) by the paraventricular nucleus (PVN) of the hypothalamus, which stimulates corticotrophs in the anterior pituitary to secrete adrenocorticotropic hormone , which travels through the bloodstream to the adrenal cortex. ACTH stimulates the synthesis of cortisol and other glucocorticoids. Cortisol ultimately inhibits the HPA neuroendocrine response via a negative feedback mechanism to restore a steady state , 18. CorLactobacillus species dominance , BV, and preterm labor , were not included.With the use of words and phrases including (but not limited to) “stress and vaginal health,” “stress and bacterial vaginosis,” “stress and vaginal infection,” “stress and immune function,” “stress and vaginal Lactobacillus species. The most often identified species of Lactobacillus are L. crispatus, L. jensenii, L. gasseri, and L. iners  created by Lactobacilli amongst other protective mechanisms including production of lactic acid (~110 mM) (2O2), antimicrobial peptides and by competitive exclusion i.e., physically preventing the attachment of pathogens to vaginal epithelium  , 26, hydithelium , 27, 28.ithelium .Lactobacilli and other acid-producing microbes and a more alkaline pH  and other pathogens  patients argue otherwise showing only minimal association between prenatal stress and adverse pregnancy outcome  and chemokines IL-8, CCL5). Glucocorticoids also facilitates immunosuppression by inhibiting proliferation, migration and cytotoxicity of lymphocytes and leukocytes, and secretion of IL-2 and IFN-γ . On the . GlucocoLactobacillus have been demonstrated in a mouse model , this may not differ significantly from the in vivo environment. This is supported by evidence of abundant supply of norepinephrine from nerve terminals innervating the cervicovaginal mucosa in humans and animals , reduction in neutrophil bactericidal potency and reduced abundance of commensal se model . Reducedmicrobes , most linfection . This ocnfection . Though  animals –91 and v animals . In addi animals . The commokines [; Figure d low pH .It also noteworthy that the incidence and prevalence of BV is influenced by other factors including frequent unprotected sexual activity with new or multiple partners –96, smokLactobacilli dominated vaginal microbiota rich in glycogen and lactic acid content observed in healthy pregnant women. However, because this experiments were conducted in rats with relatively high concentrations of cortisol (8.3 × 106 nmol/l) compared to circulating cortisol levels under maximum stress such as major surgery (726 and 1297 nmol/l)  , 108 or  nmol/l)  in human nmol/l)  is more Lactobacillus dominance. This recipe creates a conducive, less acidic ecosystem for infection by strict and facultative anaerobic bacteria, viruses, fungi, and protozoa -induced inflammation and more maternally-reported infant gastrointestinal symptoms and allergic reactions  for 6 weeks in later gestation showed activated HPA axis and increased cortisol levels , that was sufficient to cause altered intestinal microbiota characterized by decreased protective fsprings . Reducedre birth , 122. Thre birth . The patOptimal preventive/treatment approach to counteract the effect of stress on vthe aginal microenvironment should aim to alleviate or eliminate the stress stimuli and the associated infectious and inflammation agent(s). Though stress is almost inevitable, some life modifications can help an individual cope with stressors better. These adaptive measures include regular physical exercise, healthy eating, having adequate sleep and avoiding unhealthy habits like smoking, alcohol, and drug abuse. In addition to eliminating stressors and lifestyle modifications, appropriately administered antibiotics or antifungal agents can eliminate bacterial and fungal infections while inflammation can be targeted pharmacologically using anti-inflammatory agents. This is an important therapeutic goal because inflammation can persist even after the stress stimuli or infectious agents have been eliminated . AntibioLactobacillus species (L. crispatus), and prebiotics such as D-lactic acid, glycogen, oligosaccharide etc. that stimulate probiotic bacterial growth  when mice were exposed to mild prenatal stress  as observed in stress-induced vaginal colonization by anaerobic bacteria include phosphodiesterase (PDE) inhibitors, MAP kinase inhibitors, TNF biologics and NF-κB inhibitors . Some ofThe potential action of the stress hormone cortisol in the maintenance of vaginal health has been highlighted. Stress stimuli are ubiquitous and women do not enjoy any exemptions. The physiologic “fight-or-flight” response may be deleterious to their lower genital tract microbiome if the stress stimuli persist for longer than necessary. Persistent exposure to psychosocial stress and stimulation of the HPA and SAM axes, with a corresponding increase in cortisol and norepinephrine levels are associated with dysbiosis and increased susceptibility to several infections including genitourinary tract infections. Though this could be solely due to a dysregulated immune response, a cortisol-induced inhibition of vaginal glycogen deposition may be involved especially in the instance of vaginal infection. The estrogen-related increased vaginal glycogen deposition and epithelial maturation are requirements for the maintenance of vaginal eubiosis. The ability of cortisol to disrupt this process as shown in animal models is important in the pathogenesis of vaginal dysbiosis and subsequent development of infection and inflammation. If proven in humans, this phenomenon may be more crucial in pregnancy where a healthy Lactobacillus-dominated vaginal microbiome is sacrosanct, and there is local production of more CRH from gestational tissues including the decidua, fetal membranes (chorioamnion), and placenta. Because the current evidence of the repressive role of cortisol on vaginal glycogen deposition involve experiments in animals with high concentrations of cortisol compared to that observed during severe stress in humans, further work especially in humans is needed to elucidate the pathophysiologic association between cortisol levels and vaginal microbiomial architecture and function.EA conceived the research idea and conducted the literature search. EA and DA contributed to writing the manuscript. Both authors revised and approved the submission of the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tremendous advancements in syndromic surveillance strategies over the last two decades, and specifically from prior mass gatherings, have been incorporated into day-to-day healthcare analysis worldwide and have left a lasting indirect impact since their inception. Mass gatherings are a daily occurrence worldwide and provide a scenario ripe for public health aims and objectives utilising syndromic surveillance. Europe is less than a decade away from hosting a colossal worldwide gathering (2024 Summer Olympics) in likely a time when the global agreement is in flux. A call to arms is needed for additional surveillance strategies incorporating mobile application symptom checker data, telemedicine, social media and social data sensing. There remains a need for an optimal combination of real-time data sensing that captures the whole population, but to reach that goal we must incorporate new advancements into baseline epidemiologic data monitoring, otherwise we will be tracking real-time mass gathering events on top of inaccurate baseline epidemiologic data. Large mass gatherings pose a significant strain on the planning and resources of a regional event host. An influx of non-native visitors and efflux of native population combine to disrupt host baseline healthcare-related factors, including healthcare provider allocation, non-endemic seasonal and global disease variation, communication hurdles in response to a health crisis, and the strain on already limited physical resources. Preemptive event planning remains pivotal, but the real-time dynamic monitoring and response to new healthcare situations remain an imposing challenge.Syndromic surveillance is the real-time  collection, analysis, interpretation and dissemination of health-related data enabling early identification of the impact (or absence of impact) of potential human public health threats that require effective public health action . It reliSyndromic surveillance has been utilised in many mass gatherings to date. It has been utilised during major worldwide natural disasters  and pandemic outbreaks (2009 influenza) –4. BeforNations across Europe have built on each other's syndromic surveillance platforms. For example, the French Institute for Public Health Surveillance coordinated the Triple-S  to recognise potential data sources for syndromic surveillance across Europe .The 2012 London Olympic Games offered a prime opportunity to build on prior surveillance methods and incorporate new syndromic surveillance modalities such as GP out of hours and unscheduled care visits and daily emergency department (ED) symptom and diagnostic uploads . This quWith the many major mass gatherings looming and the 2024 Summer Olympic Games returning back to Europe (Paris), we must strive to align syndromic surveillance methodology with the evolving societal times and the on-demand technology-driven consumer. In 2013, the NHS performed a pilot analysis of online symptom checker data as an adjuvant to syndromic surveillance, given that roughly 90% of American and 75% of International Web users search for health information online [et al. analysing the potential diagnostic and triage capabilities of a multitude of online symptom checkers, with results varying vastly by diagnosis type, triage level and online symptom checker [Are online symptom checkers a valid and plausible addition to mass gathering strategies? A large audit study was performed in 2015 by Semigren  checker . Further checker . Critiqu checker . In addiFacebook, Snapchat, Instagram, Twitter, etc – may become a pivotal data source for real-time syndromic surveillance, and its reliance on clinical signs, epidemiologic trends, and proxy measures that create a provisional diagnosis (or ‘syndrome’). People are turning to less formal means to express the aforementioned measures, personal feelings, physical symptoms and geographic location. The current combination of hashtags and location pinning on social media allow easy means to track the current geographic impact of any subject. Online symptom checkers provide an organised modality to aid in syndromic surveillance but the future will reside in more non-healthcare specific social platforms to better capture, analyse, interpret and disseminate health-related data to enable prompt identification of potential human public health impacts. Questions do remain about missing surveillance data from those populations without access to electronic means or those technologically inexperienced.The evolving consumer wants access to supplemental information and decisional software at their fingertips, and incorporation of this demand into the syndromic surveillance algorithm for future mass gatherings must be made. The times of manual data entry and tracking consumer calls as a means for syndromic surveillance will continue to fade away. The future of syndromic surveillance for mass gatherings must incorporate the on-demand consumer, utilising such surveillance modalities such as telemedicine appointments or simply mobile search engine entries. Social media – be it et al. have proposed a research agenda for mass gatherings that encompasses a multidisciplinary evidence-based approach [et al. suggest, ‘such information is often incomplete, evolving and derived from an increasingly complex array of sources such as basic science researchers, epidemiologists, social and political scientists, and economists [New potential syndromic surveillance sources come with their own potential injustices, unlike prior systems, which are battle-tested and have been integrated into the syndromic surveillance foundation principles of legacy building and systemic sharing of information. Tam approach . Stresseonomists .’ RobustFacebook, Snapchat, Instagram, Twitter, etc. carry the ramifications of public vs. private data, and what constitutes proper sharing of one's personal data. As many of these new data sensing systems are for-profit based, legalities of governmental and epidemiologic data sensing will arise and create rich controversy. Regional and cultural variations may limit widespread use of such data sensing and hasten the fluid utilisation of such powerful epidemiologic data from emergent social media systems. The aforementioned intricacies may cloud the data for those monitoring the public health aspects of mass gatherings and alter epidemiologic baselines. In the end, the optimal incorporation of newer syndromic surveillance sources will rely on a stringent evidence-based research agenda, with continuous and preemptive multi-departmental testing and reliance on a systematic approach to determine the added value of such potential innovative modalities.Naïve syndromic surveillance systems have vast potential to adapt to the societal demands of the individual user-controlled, on-demand and informal surveillance modalities. Utilisation of real-time syndromic surveillance data from