Plastics are synthetic materials made from organic polymers that are ubiquitous in daily living and are especially important in the healthcare setting. However, recent advances have revealed the pervasive nature of microplastics, which are formed by degradation of existing plastic products. Although the impact on human health has yet to be fully characterised, there is increasing evidence that microplastics can trigger inflammatory damage, microbial dysbiosis, and oxidative stress in humans. Although there are limited studies investigating their effect on the ocular surface, studies of microplastics on other organs provide some insights. The prevalence of plastic waste has also triggered public outcry, culminating in the development of legislation aimed at reducing microplastics in commercial products. We present a review outlining the possible sources of microplastics leading to ocular exposure, and analyse the possible mechanisms of ocular surface damage. Finally, we examine the utility and consequences of current legislation surrounding microplastic regulation. Plastics are synthetic or semi-synthetic polymers. Their use is ubiquitous in daily living and particularly in the healthcare setting ranging from packaging to equipment manufacturing. Despite its utility, there have been concerns over the adverse health effects of plastics.All plastics undergo degradation into smaller particles, termed “microplastics” and “nanoplastics”. The term “microplastic” was first coined in 2004 to describe microscopic plastic particles in marine sediments . These eMicroplastics have a high surface-area-to-volume ratio and bioaccessibility due to their small size, allowing them to exert effects on human health at a cellular level . ExposurThis review outlines possible sources of ocular surface exposure to microplastics, the impact on the ocular surface, and proposed mechanisms of damage. Current quantification methods for microplastics and nanoplastics, along with their limitations, will also be discussed. Lastly, this review provides a summary of existing regulations governing the manufacturing and monitoring of microplastics.Possible environmental sources include exposure to microplastic-contaminated fluids or air . The mosShedding of microplastics from household items has also been reported. There is evidence of microplastics released from plastic food containers, disposable cups, and plastic tea filter bags ,17,18. CMicroplastics have also been retrieved from the surgical environment and are thought to arise from the abundant use of plastics in the healthcare setting . Given wIn vitro studies showed both human cornea and conjunctival epithelial cell lines could take up polystyrene microplastic particles with microplastics accumulating around the cell nuclei. These particles were cytotoxic, with decreased cell viability and proliferation markers identified . To explOcular surface fluorescein staining was evaluated weekly and increased staining was observed in the test group but not in the control or normal group. Interestingly, sporadic punctate staining was seen in the group of mice receiving administration of normal saline. There was no mention of how the normal saline was stored (presumably in a plastic bottle); neither was testing of the normal saline solution for microplastics described beforehand. Tear film secretion was investigated weekly with a phenol red thread test. A reduction in tear secretion was identified and tear secretion reduced over time in the two treatment groups.Stereo-fluorescence microscopy further demonstrated accumulation of microplastic particles in the lower conjunctival sac that increased over time. Analysis of ex vivo tissues at the end of the study showed reduced size and density of goblet cells of the lower lid compared to the control group. Proliferation-related markers  were also downregulated in the treatment groups compared to controls.Compared to the normal and control groups, there was an irregular arrangement of lacrimal gland acini in both treatment groups. Inflammatory cells between acini and upregulation of inflammatory factors and cytokines  in a time-dependent fashion were also reported. There were higher rates of apoptosis identified in mice receiving the suspension containing 50 nm compared with 2 μm microplastic particles .Exposure of the murine ocular surface to particulate matter 2.5 (PM2.5) environmental pollutants, which can contain microplastics, causes reduced tear volume, tear film break-up time, and destruction of corneal epithelial microvilli and corneal desmosomes . IncreasAlthough limited studies have been performed on the ocular surface, much work has been performed in other organs which may predict the impact of microplastics on the ocular surface. Tissue and cell damage may be caused by: (1) inflammation and oxidative damage, (2) microbial dysbiosis, and (3) toxicological effects from additives and sequestrated compounds.While the role of inflammation in patients with dry eye disease is well-established, our understanding of how it fits into pathological mechanisms remains controversial. For instance, key differences in how inflammation arises and perpetuates in patients with dry eye disease remain unknown . RegardlOxidative stress can cause inflammation and dry eye disease . Dry eyeMytilus spp., Caenorhabditis elegans, Artemia parthenogenetica) increased intestinal expression of glutathione S-transferase 4 and lipid peroxidation, and reduced catalase and glutathione reductase, suggesting that oxidative damage is a key mechanism in microplastic-induced epithelial damage [Similar oxidative damage can be induced by microplastic exposure. Microplastic uptake in the intestinal system is governed via microvilli endocytosis and cilia movement, which transfers these particles into digestive tubules . Knowledl damage ,33,34.Danio rerio, Poecilia reticulata, Girella laevifrons, Larimichthys crocea) increased intestinal levels of TNF-α, IFN-γ, IL-1α, IL1-β, and IL-6 [Oral ingestion of microplastics in aquatic vertebrates  has been associated with lower levels of diseases ,56.Parabacteroides in the meconium (ethylene-vinyl acetate) and Bacteroides in the placenta (polyethylene), suggesting that specific microplastics may impact upon the viability of various microbiota.A similar dysbiotic impact of microplastics has also been observed in non-gastrointestinal systems. A study analysing the microplastic and microbiome composition in human placenta and newborn meconium samples retrieved 16 different types of microplastics from all samples . ResultsBacillus, Tatumella, and Lactobacillus species while soft contact lens users had lower abundance of Delftia and more Elizabethkingia than non-contact lens wearers [Contact lens wear is associated with ocular surface microbial dysbiosis with a shift towards periorbital skin biota . Additio wearers . These d wearers . No studMicroplastics and their degradation products may harbour toxic chemicals arising from either additives during the manufacturing process, or chemicals absorbed by plastics from the environment . PlasticAdditives are chemicals incorporated into plastics during production to augment their properties, such as colour, transparency, and durability. There are numerous additives of concern that have the potential to damage human tissues. Phthalates are esters of phthalic acid  used to produce polyvinyl chloride (PVC). Epidemiological studies have identified phthalates as key culprits of suppressed reproductive hormones, altered thyroid function, and the development of obesity and metabolic syndrome ,66,67,68Heavy metal additives incorporated into plastics to imbue specific properties can also potentiate diseases. Cadmium, mercury, and arsenic can induce carcinogenesis, while copper and cobalt can induce the formation of ROS ,78,79,80Pseudomonas aeruginosa, Staphylococcus aureus, and Fusarium solani showed that preincubation of solutions with contact lenses led to a decrease in effectiveness against the bacterial strains [Plastics sequester other toxic chemicals and heavy metals from their surroundings ,84,85. OUnfortunately, our understanding of the interactions between plastics and various chemicals remains limited, and the vast majority of chemicals isolated from plastic compounds remain unidentified and unstudied . CurrentDespite ongoing progress, this field remains relatively new and challenges in accurate identification and quantification of microplastics exist. This section describes common protocols for sample processing and analysis, with an emphasis on predictability, reproducibility, and accuracy of methods.Prevention of environmental contamination during sample preparation and analysis is important. The proposed methodology to reduce plastic contamination includes utilising non-plastic apparatus such as glass or steel devices, deep cleaning of work stations prior to experiments with plastic-free ethanol and Milli-Q water, wearing natural fibre clothing, minimising movement of personnel in and out of the laboratory, and keeping a database of plastics that may come into contact with the samples ,90. ExpePretreatment aims to remove contaminants which may confound subsequent analyses. To separate inorganic contaminants from microplastics, samples are first filtered and subsequently added to solutions  as vehicles to separate microplastics from the original sample via density differences ,94,95. AAcid-base solutions such as hydrogen peroxide and potassium hydroxide are used to remove organic materials . A majorFollowing pretreatment, further analysis to quantify and characterise microplastics is performed, with common methods summarised in Visual identification of microplastics can be conducted with visual, light, or digital microscopes. This method is preliminary, operator dependent, subjective, and associated with high rates of misidentification and the inability to detect very small sizes  ,102. HenThe stereomicroscope allows three-dimensional visualisation of microplastic particles by allowing observation of the sample from two different angles. Although this is limited by a lower magnification compared to conventional light microscopy, it provides a better visual characterisation of the surface structure and morphology of microplastic particles. Unfortunately, the usage of this technique is limited by the quality of samples—samples with impurities which cannot be chemically digested and samples with thick, dense sediment often makes visualisation difficult. Previous studies have shown a 20–70% identification rate of transparent particles using stereomicroscopy when validated against other techniques ,104.Fluorescence microscopy, such as staining the sample with Nile Red reagent, can highlight microplastics and assess the count and nature of microplastics . This meTransmission electron microscopy (TEM) is one of the most commonly utilised techniques in characterising nanoparticles as it provides chemical information and imagery at atomic resolutions . HoweverScanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy (EDS) can analyse the chemical composition of particles . SEM-EDSAtomic force microscopes (AFM) operate on the principle of surface sensing using a sharp tip of a rigid conductive material fixed to a cantilever . A recenFourier-transform infrared (FTIR) spectroscopy involves the detection of unique spectral signals released from molecules after excitation with infrared irradiation. Comparison of recorded signals against a spectral library of known plastic materials provides information regarding its composition. The theoretical limit of FTIR spectroscopy is 10 μm due to the diffraction limit of light; however, there is approximately a 35% underestimation of the number of particles even at sizes of 20 μm . An addiAs an alternative to FTIR, laser direct infrared (LDIR) imaging analyses microplastic particles faster than conventional FTIR spectroscopic methods. It is an infrared (IR) spectrometer utilising a fast-tunable quantum-cascade laser (QCL) as a light source which is coupled to a rapidly scanning imaging system. The instrument was originally designed for the pharmaceutical analysis of tablets, laminates, tissues, and fibres, but can also be used to analyse microplastics. Similar to an FTIR, the imaging system provides information on particle enumeration, size, and morphology, while the polymer type can be identified by the spectrometer .Raman spectroscopy is another vibrational spectroscopy technique which analyses the Raman shift of microplastic particles after irradiation with a monochromatic light source . SimilarFTIR and Raman spectroscopy utilise established custom and open-source spectral libraries such as the Spectral Libraries of Plastic Particles (SLoPP) and μATR-FTIR Spectral Libraries of Plastic Particles (FLOPP) to identify microplastics ,122. TheIn contrast to FTIR and Raman spectroscopy, pyrolysis or thermal desorption gas chromatography-mass spectrometry (Pyr-GC-MS) is an analytical method which involves the thermal degradation of large particles into fingerprint chromatograms, known as pyrograms, to assess their chemical composition. Products of pyrolysis are separated using gas chromatography and analysed with mass spectrometry to identify synthetic polymers which make up the original microplastic particles . The advAlthough a variety of preparatory and analytical methods are available to characterise microplastics, there is still no consensus on a standardised protocol, partly due to the limitations of current methodologies. Differences in sample preparation and analytical methodologies may limit reproducibility and comparison between studies.Legislation has been driven by environmental and human health concerns. Several countries, including the United States and European Union, have legislated to combat growing microplastic and nanoplastic contamination in the environment ,130,131.Enforceability of regulations is restricted by limitations in current analytical techniques that may be unable to accurately detect and fully characterise all polymeric particles, which may be present in minute amounts. In fact, conventionally used analytical techniques may be unable to accurately identify the lower limits of particles (1 nm) outlined in the ECHA definition .The lack of consensus on the definition of microplastics further confounds the usefulness of regulations. The United States Microbead-Free Waters Act of 2015 has been criticised for the limited scope of the included definition because it does not cover microbeads added to certain types of cosmetics (“leave-ons’’) and secondary microplastics produced by degradation of larger plastics, which make up the majority of microplastics found in the environment. Conversely, the original 2019 ECHA definition of microplastics was criticised as overly stringent and included all forms of polymers, including polymers functionally important to the manufacturing industry. Polymers such as derivatised celluloses, which are frequently used in the pharmaceutical sector for medication production, have also been included. Increasingly specialised drugs, such as those targeted at penetrating the blood–brain barrier, often capitalise upon the unique properties of these polymers. In many cases, there are no suitable alternatives that possess properties of the original polymer . LimitinIt is important to note that the largest source of microplastics still arises from secondary degradation of existing plastic products and waste after natural environmental exposure . Large-sPlastics are abundant materials that are relatively inexpensive to manufacture. Numerous industries, including healthcare, remain dependent on plastics across a wide range of applications. Eye care, with its widespread use of topical ophthalmic formulations, surgical equipment, contact lenses, and syringes for intraocular delivery of therapeutics is no different . There i


We describe the rationale for and design of an innovative, nested, tripartite prospective observational cohort study examining whether relative estrogen insufficiency-induced inflammation amplifies HIV-induced inflammation to cause end organ damage and worsen age-related co-morbidities affecting the neuro-hypothalamic-pituitary-adrenal axis (Brain), skeletal (Bone), and cardiovascular (Heart/vessels) organ systems (BBH Study).The BBH parent study is the Multicenter AIDS Cohort/Women’s Interagency HIV Study Combined Cohort Study (MWCCS) with participants drawn from the Atlanta MWCCS site. BBH will enroll a single cohort of n = 120 women living with HIV and n = 60 HIV-negative women, equally distributed by menopausal status. The innovative multipart nested study design of BBH, which draws on data collected by the parent study, efficiently leverages resources for maximum research impact and requires extensive oversight and management in addition to careful implementation. The presence of strong infrastructure minimized BBH study disruptions due to changes in the parent study and the COVID-19 pandemic.BBH is poised to provide insight into sex and HIV associations with the neuro-hypothalamic-pituitary-adrenal axis, skeletal, and cardiovascular systems despite several major, unexpected challenges. Funded in 2018 U54AG062334), the Specialized Centers of Research Excellence on Sex Differences (SCORE) at Emory University focuses on the HIV-host pathogen interaction as a model for probing the influence of sex as a biologic variable (SABV) on the pathology and pathogenesis of infectious diseases. Globally, over 50% of the 37.7 million people living with HIV are women AG062334,, skeletaThe Emory SCORE is investigating the effect of relative estrogen insufficiency on the HIV-host interaction in women via the Brain, Bone, Heart (BBH) study, a nested, tripartite sub-study of the Multicenter AIDS Cohort Study (MACS)/Women’s Interagency HIV (WIHS) Study Combined Cohort (MWCCS). BBH is examining the degree to which, if any, estrogen insufficiency-induced inflammation converges with HIV-induced inflammation to cause end organ damage and worsen age-related co-morbidities affecting the neuro-hypothalamic-pituitary-adrenal (neuro-HPA) axis (Brain), skeletal (Bone), and cardiovascular (Heart/vascular) organ systems. Understanding mechanisms underlying ongoing inflammation in virologically suppressed women living with HIV could ultimately lead to novel preventative and therapeutic interventions to limit inflammation and subsequent end organ damage in the women. This manuscript describes the rationale behind BBH, outlines the BBH protocol, and describes protocol adjustments to meet two serious study disruptions: a change in the scope and activities of the parent study and the COVID-19 pandemic.Important sex differences exist in the immune response to antigens, including infectious diseases, vaccines, and self-antigens, and estrogen has been shown to exert a potent effect on both the innate and the adaptive immune systems , 20. CliHIV establishes a chronic and latent infection that is not eliminated by host immune defenses nor completely eradicated by ART. Extensive immune system damage occurs affecting both cellular and humoral immune responses and leads to severe T cell (both CD4+ and CD8+) depletion and B cell exhaustion, predisposing patients to secondary and opportunistic infections. Commonly, untreated HIV infection involves persistent viral replication leading to a gradual loss of CD4+ T cells, and increased immune activation affecting all major immune system cell populations, driving chronic inflammation . HIV denEmerging data suggest that the modifying effects of sex on HIV associated age-related co-morbidities is particularly pronounced in certain end organs including the central nervous, skeletal, and cardiovascular systems. Exposure to stress and trauma that leads to posttraumatic stress disorders and other stress-related psychiatric conditions are highly prevalent among WLH –11, 27, The identification of causal pathways underlying the influence of sex on the pathology and pathogenesis of chronic HIV and other infections will enable the development of interventions and therapies that take these factors into account. New insights into sex-based effects will play a critical role in the development of more individualized treatment concepts in infectious disease that take into account host factors as well as pathogen diversity and susceptibility. Given the inherent over representation of men in clinical trials and the limited analysis of existing clinical trial data according to sex, focused research on women as an important and underrepresented group in infectious diseases research is even more critical given the global challenges of historic and emerging infectious diseases.To examine the causal pathways underlying the influence of sex on the pathology and pathogenesis of chronic HIV infection, BBH is investigating, within the same cohort of women three interlinked Projects: 1) Brain, which hypothesizes that estrogen deficiency promotes inflammation in WLH that is exacerbated by trauma exposure and trauma-related psychophysiological hyperarousal; 2) Bone, which hypothesizes that HIV-induced immunodeficiency and ART-induced inflammation, could exacerbate estrogen deficiency bone loss in older WLH; and 3) Heart/vascular, which hypothesizes that HIV-related inflammation in women worsens endogenous reparative/regenerative processes in the setting of estrogen deficiency and that these have a combined impact on the presence and progression of sub-clinical coronary and carotid artery atherosclerosis. The specific aims of each BBH Project are listed in A unique feature of BBH is that it was designed as a prospective WIHS sub-study and all research participants for the tripartite study were originally intended to be drawn from the Atlanta (Emory University) WIHS site. WIHS was a prospective cohort study of WLH or women at risk for acquiring HIV. In addition to Atlanta, WIHS participants were enrolled at 9 other sites nationally. Every six months WIHS participants underwent a physical and gynecological exam, provided specimens for laboratory analysis, and updated information about their current behaviors, socio-demographics, medications, and physical health status. Additional information about WIHS and MWCC, including cohort descriptions, is described elsewhere –44.Recruitment for BBH started in April 2019 with a (revised) targeted conclusion date of September 2022. During the second year of participant recruitment into BBH, WIHS and MACS, a prospective cohort study of HIV infection among gay and bisexual men, were merged by the NIH to form MWCCS. The transition from a WIHS sub-study to a MWCCS sub-study significantly affected BBH due to revisions to the parent study’s design and implementation, recruitment and retention procedures, revision of the data collection forms that altered both the content and frequency of information and specimens collected from participants, and a reduction from semiannual to annual study visits by participants. Details about the implications of these changes are described below.Target enrollment for BBH is n = 120 WLH and n = 60 women at risk for HIV, equally divided by menopausal status .Inclusion/exclusion criteria are described in Each aim of the three BBH Projects were separately powered see  using thWritten informed consent is obtained from all BBH participants. The parent WIHS protocol was initially approved by the Institutional Review Board (IRB) at each WIHS site, including Emory University and, in addition, the BBH protocol was approved by the Emory University IRB and the Grady Health System Research Oversight Committee. After WIHS and MACS merged, the new MWCCS study protocol was approved for all sites by the Johns Hopkins University IRB and prior WIHS and MACS participants were re-recruited and re-consented into MWCCS. Due to challenges with a single IRB for a multi-site study, the MWCCS reverted to individual site IRB approval and informed consent was again obtained from all participants. This slowed BBH recruitment as former WIHS study subjects had to wait for MWCCS recruitment begin and then be re-consented into MWCCS before being consented into BBH.The protocol for Project 2: Bone Aim 2 (mouse models) was approved by the Emory University Institutional Animal Care and Use Committee (IACUC). See Prior to study initiation, each project conducted an extensive literature to determine potential confounders. A comprehensive evaluation of proposed elements  for data collection from each BBH Project was undertaken to identify inter-Project and WIHS parent study redundancies that would increase participant burden. Questions identified as being common to more than one Project were removed from individual Project forms and asked in a single collective BBH intake form. All BBH forms were assessed for readability of the wording of each question and individual response option, understandability, and consistency, as well as interdependence.The intense review process was undertaken a second time after WIHS merged into MWCCS in order to ensure that all required BBH data would still be collected by the MWCCS parent study at the same frequency, using the same methods, and the same wording.Prior to the MWCCS transition, initial eligibility information from WIHS participants was available for assessment. Use of this information expedited preliminary screening and reduced wasted effort by narrowing recruitment to only those potential participants known to meet initial BBH eligibility criteria. Potentially eligible Atlanta WIHS participants were notified of the BBH sub-study during their WIHS study visit and, if interested in study participation, were scheduled for a BBH screening visit during which final eligibility was assessed and informed consent obtained.During the COVID-19 pandemic, there was an 11-month institutional research pause at Emory University, during which time WIHS transitioned to MWCSS. No recruitment occurred during the pause. Once research activities resumed, the efficacy of prescreening using information previously collected in WIHS became limited as it was no longer current enough for BBH pre-screening purposes and new MWCSS pre-screening information was not yet available. BBH adapted to these changes by redoubling recruitment efforts to make up for increasing screen-fails.To streamline data collection, minimize costs, and reduce participant burden, BBH utilizes as much data as possible collected by the parent study: social and demographic characteristics, medication use and adherence, health behaviors, health history, blood chemistry and lipid panels. New BBH data collection is limited to outcomes assessments, important Project-specific covariates, and specific medical history questions.Entry, one- and two- year follow-up visits were scheduled to take place within 6 months of a corresponding semiannual WIHS visit in order to minimize the time delay between parent study data collection (WIHS visit) and the sub-study data collection (BBH visit). After the MWCCS transition, the parent protocol changed to only require one annual visit from participants. Consequently, MWCCS data are now potentially widely separated in time (up to one year) to the corresponding BBH visit. In response, BBH investigators reevaluated the chronological windows for optimal data collection and the protocol was revised to ensure adequate timeliness of previously designated parent-study data elements.BBH collects Project-specific data at study entry, one- and two-years post-study entry see . The entDue to the complexity of participating in three separate but linked research Projects across multiple physical locations lasting variable lengths of time, BBH protocols require participants to complete all necessary Project components for each time point within 90 days after completion of the first component. Information about BBH data collection is summarized in After enrolling BBH, every effort is made to schedule participants for data collection in a way that minimizes patient burden while maximizing data collected across Projects. Scheduling is based on a complex synthesis of a participant’s availability, their willingness to undergo more than one procedure in a day, the time needed to travel to the fixed location of scanning equipment, time needed to complete each procedure, and equipment/personnel availability. As a result, a given participant may have from 1 to 7 separate scheduled appointments to complete their entire entry visit. To facilitate this, BBH staff developed and implemented a database that is used for scheduling and tracking to ensure that each participant completes all study components in the right order within each Project-specific time window over the course of BBH Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


A stroke occurs when there is a lack of blood flow to the brain. Stroke injures the brain and can have devastating outcomes depending on the size and location of the brain tissue affected. Currently, there are only a limited number of treatment options for stroke. Extracellular vesicles are small vesicles secreted by cells. Importantly, extracellular vesicles have specific markers indicating the cell they were released from and can pass from the brain into the blood. For these reasons, assessing extracellular vesicles in the blood may create a window into changes occurring in the brain. Assessing changes in extracellular vesicles in the blood during stroke may produce new insight into the cellular changes in the brain causing injury during stroke. This in turn may generate potential targets for the development of future treatments. We summarize what is known about changes in brain-cell-specific extracellular vesicles during stroke and stress the importance of continuing to study these changes.Stoke is a prevalent and devastating neurologic condition with limited options for therapeutic management. Since brain tissue is rarely accessible clinically, peripheral biomarkers for the central nervous system’s (CNS’s) cellular response to stroke may prove critical for increasing our understanding of stroke pathology and elucidating novel therapeutic targets. Extracellular vesicles (EVs) are cell-derived, membrane-enclosed vesicles secreted by all cell types within the CNS that can freely pass the blood-brain barrier (BBB) and contain unique markers and content linked to their cell of origin. These unique qualities make brain-derived EVs novel candidates for non-invasive blood-based biomarkers of both cell specificity and cell physiological state during the progression of stroke and recovery. While studies are continuously emerging that are assessing the therapeutic potential of EVs and profiling EV cargo, a vast minority of these studies link EV content to specific cell types. A better understanding of cell-specific EV release during the acute, subacute, and chronic stages of stroke is needed to further elucidate the cellular processes responsible for stroke pathophysiology. Herein, we outline what is known about EV release from distinct cell types of the CNS during stroke and the potential of these EVs as peripheral biomarkers for cellular function in the CNS during stroke.  Stroke is major contributor to disability and is the second leading cause of death worldwide . Acute iHemorrhagic strokes account for the remaining 13% of strokes and occur as a result of vascular rupture in the brain usually due to aneurysm or arteriovenous malformation . HemorrhCharacterizing the changes in cellular function that occur during stroke may not only help us better understand the distinct subtypes of stroke in both the acute and chronic stages but may elucidate novel targets for future treatments. More so than any other organ, brain tissue is rarely available to access clinically. For this reason, blood-based biomarkers offer unique potential in CNS injury to understand the processes or pathophysiologic state of the brain tissue. The ability to infer cellular activity in the brain in recovering stroke patients would produce novel insight into stroke pathophysiology and could be key to discovering targets for future treatments. A variety of mechanisms exacerbate cellular injury post-stroke. Within minutes following the obstruction of blood flow to the brain, a drastic shift in ionic homeostasis causes spreading depolarizations (SDs) to occur ,14,15. SThe clinical outcome of stroke largely depends on the size and location of infarct, although sex and genetic factors can also play a role . The NatAs previously discussed, under normal conditions, the brain is secured by the BBB. Although stroke results in disruption to this BBB, it holds true that the ideal biomarker is one that is freely able to pass through the BBB and elicit a picture of the physiologic state of the CNS. Additionally, the ideal biomarker would be of CNS origin and, moreover, would correlate with distinct cell types in order to offer a window into the physiologic processes occurring in the brain during stroke . Such knThe term extracellular vesicles (EVs) refers to a broad family of cell-derived, membrane-bound vesicles . SecreteExosomes, also known as intraluminal vessels, are the smallest category of EVs, ranging from 30–150 nm in diameter ,34. ExosMicrovesicles (MVs), also known as ectosomes, are larger irregularly shaped vesicles, ranging from 50–1000 nm in diameter ,34. In cWhile both exosomes and MVs were originally thought to simply be a method of cellular waste removal, it has since been found that they play a key role in many biological processes. Their role in cell communication, regulation of microenvironments, and specificity to their parent and target cells have attracted significant scientific attention. EVs, particularly exosomes, have been studied extensively in the context of cancer for their potential role in metastasis, tumor microenvironment formation, and immune modulation ,40,41,42EVs’ small size and heterogeneous nature is the primary challenge for both isolation and characterization. In 2018, the International Society for Extracellular Vesicles provided updated guidelines on “Minimal information for studies of extracellular vesicles” (MISEV), which in summary follow these four guiding principles: (1) Describe the EV source using quantitative means; (2) demonstrate the presence of intact lipid bilayers and characterize purity; (3) utilize a combination of EV characterization techniques, preferably one optical and one biophysical/biochemical; and (4) when assessing the biological function of EVs, utilize a robust and rigorous experimental design ,47. The first step in this process typically involves the isolation of EVs from biological fluid. Common methods to achieve this include ultracentrifugation, precipitation, size exclusion chromatography, or density gradients. Tissue can also be used as an EV source. When isolating EVs from tissue, the additional step of tissue homogenization must be first carried out using dissociation buffers and physical shear stress to release the EVs trapped in the extracellular matrix ,49. AfteQuantification is another important step in EV characterization. Many conventional approaches such as light microscopy and flow cytometry lack the requisite resolution for EV quantification. Newer technologies with higher resolution combating this limitation include scanning, transmission and cryo-electron microscopy, atomic force and super-resolution microscopy, dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). Nanoflow cytometry represents a variation of flow cytometry that allows for multiparameter, high-throughput analysis of EVs and is optimized for the detection of EVs smaller than 1000 nm in diameter . NanofloEV cargo analysis also holds great interest for the development of EV biomarkers. Methods of EV cargo analysis include mass spectrometry, nucleic acid sequencing, sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Northern blots (for RNA), and Western blots (for protein) ,49,51,57In addition to disruption of the BBB, the cellular changes and inflammatory mechanisms of injury following stroke also induce the release of EVs from the cells within the CNS and infiltrating cells from the periphery. EVs have been isolated from neurons, endothelial cells, microglia, astrocytes, oligodendrocytes, neural progenitor cells, erythrocytes, granulocytes, and leukocytes ,65,66,67Collectively, EVs have the potential to serve as biomarkers indicative of changes in cellular function during stroke. EVs have been isolated from brain tissue but are also found in all biofluids, including cerebrospinal fluid (CSF), urine, and blood . Common In addition to serving as biomarkers for changes in cellular function in the acute stages of stroke, EVs may also provide novel insight into changes occurring in the more chronic stages of stroke. For example, neuronal EVs have been suggested to transport proteins and miRNAs involved in synaptic plasticity while microglia-derived EVs were proven to stimulate synaptic regeneration and remyelination via membrane lipids . The uniFollowing acute ischemic stroke, the increased risk of cognitive impairment and dementia are major clinical concerns and require further investigation to understand their onset. Post-stroke EVs may play an integral role in damage resolution but may also create a neuronal environment susceptible to deterioration. Several publications have demonstrated the presence of pathological protein aggregates in EV cargo as disease-spreading vehicles in neurodegenerative diseases . Thus, tFollowing ischemic insult, endothelial cells undergo significant swelling and inflammatory stress, promoting the release of EVs ,85. Impo+, CD105+, and CD41a- (platelet marker)) relative to age-matched controls [+ EVs ) being most strongly correlated with infarct volume and endoglin+ EVs  correlating with long-term clinical outcome. They demonstrated the ability to differentiate severe from minor AIS; however, they were not able to distinguish minor stroke from controls. A separate study assessing EV counts in TIA and AIS relative to healthy controls found a marked increase in CD146+, CD62E+, and annexin V+ EVs in both TIA and stroke within 48 h of the event, with a trend of stroke showing more endothelial EVs than TIA [+ EVs in AIS but not TIA was noted. There were also increases in annexin V+ EVs along with EVs corresponding to platelets, granulocytes, and leukocytes but a decrease in erythrocyte-derived EVs in both AIS stroke and TIA. While this study did not confirm the cellular origin of annexin V+ EVs, a previous study that compared circulating EVs 48 h post AIS to high-cardiovascular-risk controls demonstrated an increase in annexin V+ EVs originating from endothelial cells (CD146+), neural progenitor cells , platelets (CD61+), erythrocytes (CD235ab+), and leucocytes (CD45+) [+, CD144+, and annexin V+) specifically carrying miRNA-155, a marked increase was observed both in the acute (<24 h) and subacute (24 h to 2 weeks) stages post AIS and was positively correlated to NIHSS score and infarct volume [Elevated levels of EVs post AIS were first reported in 2006 by Simak and colleagues, who identified an increase in endothelial EVs (PScontrols . Moreovethan TIA . Similar (CD45+) . In a mot volume . Alternat volume . However+ EVs in ICH patients compared to controls [+, CD106+, CD54+, or CD62E+) along with EVs of other origins [As previously described, AIS and ICH are known to have different mechanisms of cellular injury. Given the relationship between endothelial cells and vascular integrity, it stands to reason EVs released from endothelial cells may provide unique insights into ischemic vs. hemorrhagic insult in the brain. Previous studies have demonstrated an increase in annexin Vcontrols ,96,97, a origins ; this ma+ endothelial EVs are significantly associated with recent ischemic episodes, increased NIHSS scores, and infarct volumes in acute stroke patients [EVs have also been shown to demonstrate potential as indicators of cerebral small vessel disease, suggesting a utility for them in the detection of ongoing endothelial dysfunction occurring in the post-stroke scenario . Considepatients .Beyond stroke severity, endothelial EVs have been explored as biomarkers to monitor treatment efficacy following acute ischemic stroke. In a case-control study, E-selectin, P-selectin, and platelet-derived EVs were all significantly higher within 7 days of stroke onset. At 3 to 6 months after stroke, E-selectin and P-selectin fell significantly below controls while platelet-derived EVs remained elevated to reflect sustained platelet activation in the recovery phase . In concIonic perturbations, ATP depletion, SD, and other secondary mechanisms of injury, including excitotoxicity, ROS production, mitochondrial failure, microglial activation, pro-inflammatory cytokine release, and infiltrating immune cells from the periphery, all impact neuronal wellbeing . How neuInterestingly, a recent study proposed that neurons release EVs containing miRNA-98 as a “help me” signal during stroke, which in turn causes a decrease in microglial phagocytosis of stressed but still viable neurons in the penumbral region. Importantly, in rats subjected to MCAO, miRNA-98 expression was found to peak in the penumbra on the first day but rapidly declined 3 days post-stroke . SeparatA recent study found several EV miRNAs demonstrating differential expression in AIS vs. ICH. One of these miRNAs was miR-134, which has been predominantly found to be a neuronal miRNA. Critically, this study did not include a non-stroke control group, making true assessment of the diagnostic potential of these miRNA challenging . InteresDynamic neuronal EV changes are also associated with functional rehabilitation in patients with subacute stroke. Platelet-derived EVs were found to form the largest population post-stroke, followed by neuronal and endothelial-derived EVs . CriticaMicroglia are the resident immune cell of the CNS and play a complex role in stroke pathophysiology. Neuroinflammation stems from the activation of microglia within the brain and infiltration of leukocytes from the periphery due to BBB dysfunction ,111. Fol+/Iba1+) [The microglia-macrophage-specific protein CD11b is commonly used as a marker for microglial-derived EVs, and the more specific microglia transmembrane protein 119 (TMEM119) is also expressed on microglial EVs . CD13 an+/Iba1+) . Taken t+/Iba1+) . Moreove+/Iba1+) . In cont+/Iba1+) . It is clear that microglia play a dynamic role in both injurious and neuroprotective post-stroke processes. As reviewed by Taylor and Sansing, several studies taken together outline the timeline by which anti-inflammatory-type microglia initially predominate the ischemic core, followed by a shift to pro-inflammatory-type microglia over the course of 2 weeks . InteresAstrocytes are the most abundant cell within the CNS and play critical roles in BBB permeability and nutrient and waste metabolism . In AIS,Common protein markers for astrocyte-derived EVs include the astrocyte-specific proteins excitatory amino acid transporter1 (EAAT1/GLAST) and glial fibrillary acidic protein (GFAP) . AstrocyOligodendrocytes are the myelinating cells of the CNS. Common markers for oligodendrocyte-derived EVs include oligodendrocyte-specific proteins, oligodendrocyte myelin glycoprotein (OMG), and myelin basic protein (MBP) . OligodeAs demonstrated, EVs and their cargo are a promising means of peripherally assessing CNS cell function. Regarding stroke, miRNA represents a more highly studied component of EV cargo. Not mentioned in the above sections, there have been several other non-cell-specific key findings concerning miRNA fluctuations and stroke that have previously been outlined in recent reviews ,74. It iWhen thinking about the significance behind differential miRNA expression in stroke, cell origin also plays an important role. For instance, serum EV miRNA-9 and miRNA-124 were found to be significantly higher in AIS compared to controls . While tEV miRNAs are also actively involved in pathological changes seen in both stroke and neurodegenerative diseases , suggestSimilar points can also be made for differential EV-contained protein profiles. It is worth noting that the literature surrounding EV cargo post-stroke is heavily focused on the miRNA content and overall investigations of the protein content are relatively scarce. An overall increase in inflammatory proteins in circulating EVs in AIS patients has been found relative to controls. These findings included increased mRNA of TNF, CXCL-, CCL-2, and IL-1β . AdditioStroke represents a prevalent and devastating condition in which significant benefit would come from a means of non-invasively gauging changes in CNS cell function. The characterization of EVs released from the brain represents a novel, peripheral window into the physiologic state of the brain and the cell-specific pathologic changes associated with stroke. While increasing studies are emerging that are examining the content of brain-derived EVs and their therapeutic potential, a vast minority of studies in this field correlate the EV content released during stroke to specific cell types within the CNS. To change this, there needs to be a detailed interrogation of cell-specific EV release and extravasation post-stroke. For instance, even common neuronal EV markers such as L1CAM are not restricted to CNS cells but are also expressed by melanocytes, immune cells, and certain populations of endothelial cells .Many studies have postulated a potential clinical role for EVs in stroke diagnosis and prognosis. For clinical application in acute stroke diagnosis, good EV biomarker candidates must be released in the hyperacute phase, be able to differentiate stroke subtypes, and be easily measurable with simple devices, which highly limits the complexity of targets . CriticaMoving forward, a focus on standardizing isolation and characterization methods along with advances in technology to allow for high-throughput analysis is requisite to one day see clinical application of EV biomarkers. The literature reviewed in this manuscript serves to generate a list of potential candidate EV biomarkers that may infer changes in cellular function in the CNS during stroke. To imply the clinical utility of these candidate biomarkers, future studies must first validate these biomarkers in a clinical setting with the appropriate controls, determine the effect of elapsed time on the biomarker, determine the optimal time for sampling, and finally compare these biomarkers to medical imaging/the current standard of diagnosis/prognosis. However, at this point in time, with currently available technology, we are able to profile the EV content over the course of stroke and relate it to cellular function. In doing so, EVs may serve as a form of a peripheral blood-based window into CNS cellular function. This will enhance our understanding of stroke progression and recovery and could reveal novel targets for future therapeutics. EVs themselves may also be used therapeutically for stroke in the future. Mesenchymal stem cells (MSCs) are multipotent stem cells primarily found in bone marrow that have been emerging as a promising means of enhancing post-stroke recovery and are being investigated in pre-clinical stroke models and clinical trials . MSCs arCollectively, a better understanding of the timing by which specific cell types within the CNS release EVs and how their cargo changes over time throughout the course of stroke presents an exciting opportunity to increase our knowledge of changes in cellular function manifesting stroke pathophysiology. A means of peripherally assessing CNS cellular function would allow for clinical sampling at distinct timepoints post-stroke in living stroke patients. Understanding the unique cellular events occurring in the recovering patient could reveal novel targets for future treatments.


Apoptosis plays an important role in development and in the maintenance of homeostasis. Apoptotic bodies (ApoBDs) are specifically generated from apoptotic cells and can contain a large variety of biological molecules, which are of great significance in intercellular communications and the regulation of phagocytes. Emerging evidence in recent years has shown that ApoBDs are essential for maintaining homeostasis, including systemic bone density and immune regulation as well as tissue regeneration. Moreover, studies have revealed the therapeutic effects of ApoBDs on systemic diseases, including cancer, atherosclerosis, diabetes, hepatic fibrosis, and wound healing, which can be used to treat potential targets. This review summarizes current research on the generation, application, and reconstruction of ApoBDs regarding their functions in cellular regulation and on systemic diseases, providing strong evidence and therapeutic strategies for further insights into related diseases. Apoptosis is a form of programmed process of cell death that occurs throughout life as a part of normal development, which includes distinct cell shrinkage, chromatin condensation, and plasma blebbing . Up to 1Extracellular vesicles (EVs) are recognized as key regulators of intercellular communications and can participate in multiple physiological and pathological processes, such as inflammation, immune responses, coagulation, tumorigenesis, and host–microbe interactions ,14,15,16All multicellular organisms have a pathway that strictly regulates and controls cell development, maintenance, and clearance. Controlling the number of cells is critically important to maintain the balance between cell proliferation and clearance . ApoptosApoptosis is mediated by the caspase protein family. Current studies have characterized the intrinsic and extrinsic apoptosis pathways in the body  . Cell death signals combine with their specific receptors to produce disc , which leads to recruitment and activation of caspase 8 and 10. Caspase 9 is activated by the intrinsic pathway and caspase 8 and 10 are activated by the extrinsic pathway to mediate caspase 3 activation. Activated caspase 3 can initiate apoptosis by cleaving more than 1300 cell substrates ,45,46. C+ and K+ gradients, the second stage is related with cytoskeleton organization and further K+ extrusion. In this second stage, both cytochrome C release and caspases seem to be involved and required for AVD. AVD is required for cell dismantling into ApoBDs [Vesicles are one of the earliest morphological changes that can be recognized in the process of apoptosis. The formation of membrane vesicles is the result of an increase of intracellular hydrostatic pressure after actin-mediated contraction . The repo ApoBDs . The inho ApoBDs . Although most apoptotic cells will appear as membrane vesicles, some cells will specifically form other types of membrane protrusions, such as microtubule spikes, beaded apoptotic structures, and so on ,55. For Phagocytes recognize the “find me” signal sent by apoptotic cells and the “eat me” signal on ApoBDs and phagocytize them  to complAlthough ApoBDs are easily engulfed by phagocytes, intact apoptotic cells can also be engulfed by phagocytes . MoreoveAn increasing amount of evidence has shown that the transfer, circulation, and even the reuse of the contents of ApoBDs widely affects the functions of phagocytes . Like exDuring apoptosis, the nuclear materials will be distributed into ApoBDs . The horAccumulated reports have revealed the critical role of ApoBDs in intercellular communications by transporting intracellular signaling molecules. The investigation and discovery of ApoBDs has offered new insights for pathological therapeutic targets and explorations for effective treatments .Staphylococcus aureus infections due to surgery, blood transfusions, radiotherapy and chemotherapy, as well as indwelling catheters and drainage tubes [Staphylococcus aureus (MRSA); however, it can have serious side effects during systemic treatment, including thrombophlebitis, renal injury, and epidermal necrolysis, which can result in more serious poisoning symptoms than the original infection [Staphylococcus aureus [Anti-cancer treatments often have a high risk of ge tubes . Vancomynfection . Loadings aureus .Staphylococcus aureus by using vancomycin loaded ReApoBDs. ReApoBDs were prepared from different cancer cells  and were used for vancomycin delivery. In vitro cell culture studies showed that in Staphylococcus aureus-infected macrophages, U87-MG and LN229 glioblastoma cell models, ReApoBDs effectively killed intracellular bacteria compared with free vancomycin treatment [Staphylococcus aureus-infected macrophages and cancer cells. ReApoBDs loaded with vancomycin have the potential to kill intracellular Staphylococcus aureus infections and that strategy can be used to eliminate treatment-related MRSA infection during anti-cancer treatment.Studies have shown that cell-derived vesicles (CDVs) extracted from invasive cancer cells can be used for the targeted delivery of miRNAs and nano-contrast agents for treatment and imaging ,86. Therreatment . TherefoThe development of atherosclerosis is mainly due to the accumulation of lipids and inflammatory debris in vascular walls; however, it is also related to the apoptosis of macrophages, smooth muscle cells (SMCs), and endothelial cells. Studies have shown that ApoBDs generated by apoptotic endothelial cells possess the ability to regulate atherosclerosis .Pro-inflammatory cytokine IL-1α was found in endothelial cell-derived ApoBDs, and moreover, the ApoBDs can induce monocytes to secrete chemokines, as well as to mediate neutrophil inflammation . AlthougDuring the bone remodeling process, the lifespan of osteoclasts is about 2 weeks after which they undergo apoptosis ,89, whic−/− mice revealed that a reduced generation of ApoBDs significantly impairs the self-renewal and osteogenic/adipogenic differentiation of bone marrow MSCs. The systemic infusion of exogenous ApoBDs rescued the impairment of MSCs and ameliorated the osteopenia phenotype. MSCs were able to engulf apoptotic bodies via integrin αvβ3 and reuse ApoBD-derived ubiquitin ligase RNF146 and miR-328-3p to inhibit Axin1. MSCs thereby activate the Wnt/β-catenin pathway. This suggests the potential use of ApoBDs in the treatment of osteoporosis [Hence, ApoBDs are necessary to maintain systemic bone density. Studies of Fas-deficient MRL/lpr and caspase 3oporosis .Hepatic stellate cells (HSCs) play a key role in the process of liver fibrosis. Under the induction of fibrogenic stimulation, HSCs were activated and differentiated into myofibroblasts. HSC survival is central to the progression of liver fibrosis and the induction of HSC apoptosis can rescue liver fibrosis. Phagocytosis of ApoBDs promotes HSC survival via the JAK1/STAT3 and PI3K/Akt/NF-κB pathways . TargeteChemotherapeutic nanomedicines can exploit the neighboring effect to increase tumor penetration; however, the neighboring effect is limited due to the consumption of chemotherapeutic drugs and the drug resistance of internal hypoxic tumor cells. ApoBDs were shown to carry the remaining drugs into neighboring tumor cells after apoptosis . It was In pathological conditions such as cancers and infections, ApoBDs contain antigens from cancer cells and pathogens, which can be recognized by the immune system. Since ApoBDs are easily engulfed by antigen-presenting phagocytes such as DCs, they can activate an adaptive T cell response through the cross antigen-presenting process ,91. MacrMeningeal epithelial cells (MECs) are the cellular components of the meninges, which work as a barrier for the central nervous system, establish an interface between neuronal tissues and cerebrospinal fluid, and are also a part of the immune system. MECs are highly activated phagocytes, which can engulf and digest apoptotic cells. MECs are immune suppressive via the inhibited secretion of pro-inflammatory, chemoattractant cytokines and chemokines following the uptake of apoptotic bodies to shut down immune responses in the brain .Type I diabetes is due to the autoimmune destruction of insulin-producing B cells in the islets. An ideal immunotherapy should inhibit the autoimmune destruction, avoid systemic side effects, and promote islet regeneration. Apoptotic cells, as the source of autoantigens, are cleared rapidly by macrophages and DCs through an immunologically silent process that contributes to maintaining tolerance. DCs that produce peripheral immune tolerance may open new therapeutic approaches to prevent or alleviate autoimmunity.In one study, immature DCs were obtained from the bone marrow of non-obese diabetes (NOD) mice and were pulsed with antigen-specific ApoBDs from the beta cell line NIT-1 . The DCsTo identify new treatments for large-area skin wounds, an increasing number of studies has shown that bone marrow MSCs can accelerate the healing of skin wounds and restore an intact and orderly skin structure . ApoBDs Engineered and recombinant EVs carrying effective therapeutic molecules are ideal candidates for the treatment of diseases  residual contents irrelevant to the purpose of treatment cannot be completely removed, which may pose a potential threat to the body ; (b) EVsExtensive research studies on EVs have proven that ApoBDs not only phagocytize residual materials of apoptotic cells, but, like exosomes and microvesicles, ApoBDs also play an important role in intercellular communications by transporting signaling molecules. The natural combination of ApoBDs and phagocytes provides a unique opportunity for future treatment schemes. They are bound to provide new ideas for immunotherapy, vaccine development, tissue regeneration, drug delivery, and disease diagnosis, which can maximize the therapeutic effects and specificity.Although the applications of ApoBDs have great prospects, there are still some key points that need to be further investigated, including their heterogeneity, storage conditions, quality control, standardized isolation, and purification. It is necessary to further understand the biological mechanism of ApoBDs. Various molecules have been proposed as signals of phagocytosis; however, the consensus remains unclear. Further investigation of the targeting, classification, and engulfing mechanism of ApoBDs will magnify their therapeutic effects and overcome those limitations. ApoBDs play regulatory roles in multiple physiological and pathological processes, and the manipulation and reconstruction of ApoBDs hold great promise for curing systemic diseases.


Psoriatic arthritis is a heterogenic, immune-mediated musculoskeletal disease that presents with inflammation of joints, enthesis, and axial skeleton, and is associated with cutaneous psoriasis.Anti-TNF therapy has been known to trigger autoantibody response. Several cases of Autoimmune Hepatitis (AIH) have been reported. Anti-TNFα therapy in AIH is mainly attributed to the disruption of the regulatory role of TNFα signalling on the immune system. Close monitoring is essential to identify any evolving autoimmune events while on TNF antagonists. Treatment requires discontinuing the agent in mild cases and in those with systemic involvement, treatment with steroids and specialist referral is warranted.This 39-year-old South African origin gentleman was initially referred to the Rheumatology department in 2013 with throbbing pain in his hands and stiffness in heels associated with scalp and nail changes secondary to psoriasis. His past medical history included psoriasis and sports trauma related left anterior cruciate ligament (ACL) tear. He also had a strong family history of psoriasis.His initial autoimmune results were negative including a negative ANA. The Magnetic Resonance Imaging (MRI) of his right ankle at the time, revealed enthesopathy with surrounding oedema, consistent with psoriatic arthropathy.The patient was managed initially with methotrexate. Over following months, he experienced significant fatigue and hypersomnia, and stopped the medication. He re-presented one and half years later with a flare on his hands and feet. The clinical examination showed onycholysis, guttate pattern psoriasis and synovitis in his PIPJs. He was started on Leflunomide, and his symptoms improved, however he suffered loose stools. The drug frequency was tapered, but the efficacy was poor leading to recurrent flares.Subsequently, he was switched to Adalimumab in 2016 and his symptoms improved significantly. However, after a few months his monitoring bloods showed that abnormal Alanine Transaminase (ALT) levels soaring to 263 U/L, with normal albumin, bilirubin and alkaline phosphatase. A repeat autoimmune study showed strongly positive Anti-Nuclear Antibody (ANA) Hep2 cells . His Adalimumab was stopped and within 2 months his ANA became negative and ALT normalized. He was then switched to Ustekinumab.In 2018, he started to notice finger swellings especially on the left hand and ultrasound showed paratenonitis of the left third and fourth proximal interphalangeal joints and synovitis of the left 1st distal interphalangeal joint consistent with active psoriasis. He was then switched to Secukinumab which he has remained on with sustained remission and no adverse effects.We present a rare case of adalimumab induced autoimmune hepatitis in a patient with psoriasis, who developed strongly positive ANA during the course of treatment. Following cessation of adalimumab, his liver profile returned to normal limits and ANA became negative.Autoimmune hepatitis (AIH) is a chronic inflammation of the liver caused by hepatocyte-specific autoantigens. Not all pathophysiologic mechanisms of AIH are fully understood however, there is emerging evidence that genetic predisposition, molecular mimicry, and an imbalance between effector and regulatory immunity are important factors for disease development and progession. Liver biopsy results for autoimmune hepatitis classically reveal interface hepatitis, focal necrosis, portal inflammation, and plasma cells. Hepatotoxicity is an expected adverse reaction of tumour necrosis factor-α (anti-TNF-α) blocking agents including, infliximab, etanercept and adalimumab. The majority of cases appeared between one month and one year after initiation of the biological agent and complete resolution was observed in nearly 75% of cases after cessation of therapy. Evidence suggests that patients with psoriasis have a higher risk of AIH.Drug induced AIH and drug induced liver injury (DILI) can be quite challenging to diagnose due to the similar presentations and a recent study also suggest that infliximab was associated with a higher risk of DILI than other agents like adalimumab and etanercept.Our literature search has shown, that TNF-alpha antagonists, especially adalimumab and infliximab are known to trigger many autoimmune conditions like Psoriasis, Inflammatory Bowel disease (IBD), Systemic Lupus Erythematosus (SLE), and vasculitis, with the majority of cases having good outcomes following cessation of drug. Drug induced demyelinating disease and interstitial lung disease is noted to have poor outcomes. Interestingly, biologic agent induced IBD is more common than AIH.The number and complexity of drug-induced autoimmune syndromes with biologics has increased in the recent years. Anti-TNF-induced AIH has occurred most commonly with Infliximab and much less commonly with adalimumab or etanercept.Physicians dealing with patients treated with biological therapies should be aware of the possible development of several autoimmune phenomena, and close follow-up and monitoring is essential.Before commencing anti-TNF therapy in patients with any autoimmune conditions, it is imperative to have a baseline liver profile and autoimmune profile so as to differentiate between AIH and other causes of liver derangement including genetic predisposition. It is also crucial to rule out other potential causes of hepatitis including alcohol use, viral infection, and hepatotoxic medications.Clinical response is excellent in almost all cases following discontinuation of anti TNF therapy and in severe cases low or moderate steroid therapy and hepatologist input is needed. Close monitoring and regular follow-up with blood tests would help in minimising adverse drug reactions. Referral to hepatologist is recommended if ALT> 3 times the upper limit of normal or when there is an increase in bilirubin or onset of jaundice.


Nanophotonics has been widely utilized in enhanced molecularspectroscopy or mediated chemical reaction, which has major applications in the field of enhancing sensing and enables opportunities in developing healthcare monitoring. This review presents an updated overview of the recent exciting advances of plasmonic biosensors in the healthcare area. Manufacturing, enhancements and applications of plasmonic biosensors are discussed, with particular focus on nanolisted main preparation methods of various nanostructures, such as chemical synthesis, lithography, nanosphere lithography, nanoimprint lithography, etc., and describing their respective advances and challenges from practical applications of plasmon biosensors. Based on these sensing structures, different types of plasmonic biosensors are summarized regarding detecting cancer biomarkers, body fluid, temperature, gas and COVID-19. Last, the existing challenges and prospects of plasmonic biosensors combined with machine learning, mega data analysis and prediction are surveyed. With continuous development of physics, materials, medicine, communication, computer and other disciplines, we have higher requirements and expectations for daily healthcare and disease diagnosis . At presIn this context, plasmonic biosensors have been particularly favored in the fields of healthcare monitoring and disease diagnosis owing to their preponderance of low detection limit, low cost and convenience ,5,6 The In this review, we will briefly summarize the common manufacturing methods for fabrication of a plasmonic biosensing structure, such as chemical synthesis, direct writing lithography, nanosphere lithography and so on. Thereafter, we will present advanced applications of plasmonic biosensors in the fields of detection of cancer markers, gas, temperature, body fluid, COVID-19, etc., containing the principles, fabrications and prospects. To conclude, we will probe the future prospects of plasmonic biosensors combined with machine learning.Sensing material with nanopatterned arrays is the most vital part of plasmonic sensors, which accomplishes the task of enhancing biosensor through its unique electrical or optical properties . Thanks 4 for Au and AgNO3 for Ag) and appropriate reductant , it is possible that noble metal NPs are reduced directly from metal–salt solution [Noble metal nanoparticles (NPs) are widely used in plasmonic structure because of its relatively simple process. Usually, by using metal–salt solution  ,29, focuThe desired pattern can be written by EBL, FIB or DLW on the substrate or photoresist directly. EBL and DLW write patterns on photoresist and then transfer the patterns to the substrate by plasma-reactive ion etching (RIE). Due to the large ion mass, FIB can directly write patterns on the sample surface without photoresist. However, since direct writing lithography is a kind of processing method based on point scanning, although it has high flexibility and resolution for graphic design, it also leads to limitations, such as slow producing speed and small producing area. Among three methods, EBL has the highest resolution, FIB has the least processing steps and DLW has the highest throughput. Additionally, the high cost of direct writing lithography is an impediment for industrial production. To address these problems, several groups have employed holographic lithography, nanosphere lithography or nanoimprint lithography to construct plasmonic structures over large areas.Recently, Esposito et al. prepared a three-dimensional chiral plasmonic spiral structure  by focusThe desired pattern is formed by interference of multiple laser beams in holographic lithography, so holographic lithography is also called interference lithography . Adjusti−1 in plasmonic refractive sensing applications at 570–610 nm wavelength and achieves the function of biosensors by monitoring the LSPR peak shift of the polymer interaction between bovine serum albumin (BSA) and anti-BSA protein [A gold nano-checkerboard structure based on holographic lithography for biomolecular sensing is developed by Cai’s group. The specific preparation method is shown in  protein .NSL is a widely used manufacturing method to produce large-scale biosensor array structures with a relatively simple process . OrderedCompared to other photolithography methods, NSL can manufacture a large area of plasmonic sensing structures, which has lower production cost and is more practical ,48. In pRecently, Yang and co-workers demonstrated a football-like antireflective surface microstructure inspired by leafhoppers. This unique structure is obtained by electrochemical deposition on the template of double-layer colloidal ball. The specific preparation process is shown in −1) is weakened.In order to realize integration of plasmonic nanostructure and optical fiber, Manago’s group fabricated three highly ordered plasmon arrays: close-packed array (CPA), sparse array (SA) and ball removal (SR) by nanospheres lithography technology and researched their effects on three representative biological probes: ultra-low-molecular-weight molecule biphenyl-4 mercaptan (BPT), Bovine serum albumin (BSA) and red blood cells . In addiNIL makes templates by EBL or other lithography methods and then transfers the structures on the templates to the photoresist on the substrate by mechanical pressing ,54. AfteZhao and his colleagues proposed a method to prepare multilaminar nanopores using layer by layer nanoimprinting, which simply achieved controlling the gap of nanopores on a large area . They usIn addition, a flexible plasmonic sensor composed of Au nanocones based on nanoimprinting technology is demonstrated by Suresh’s group. The geometry of the Au nanocones is adjusted by the mold of NIL, and the gaps between nanocones are controlled by the thickness of the deposited gold . The plaDALI method is to assemble different patterns and structures by DNA origami and then transfer the structures to the substrate by combining deposition, RIE, wet etching and other steps . DNA ori2O3)/silicon nitride (Si3N4) substrate by PECVD. (2) The DNA origami nanostructures are drop-casted on the substrate treated by oxygen plasma. (3) The silicon dioxide (SiO2) layer is selectively grown on the bare silicon that is out of DNA structure by CVD. (4) Silicon is etched by RIE with mask that SiO2 is regarded as. (5) Depositing a layer of metal to obtain metal DNA structures. (6) Removing SiO2 and residual Si. Authors showed the practicability of this method by manufacturing metal nanostructures with a chiral plasmonic response and bowtie-shaped nanoantennas [Combining the high resolution and multifarious structure of DNA origami with conventional lithography techniques, Shen’s group reported a DALI method that can create elaborate structures on the scale of 10 nm. The specific preparation process is as follows : (1) an antennas .2. Yet, it should be noted that there is a characteristic of anisotropy in the alkaline solution wet etching of monocrystalline silicon. Thus, wet etching can be used to fabricate three-dimensional structures, such as grooves and pits, on the silicon substrate.After pattern templates are prepared by various photolithography methods, the patterns on the templates need to be transferred to the substrate, which requires RIE, deposition, wet etching and other methods for auxiliary manufacturing. Most photolithography methods require their assistance to prepare metal plasmonic structures. Due to most lithography technologies only being able to produce two-dimensional patterns on photoresist, patterns must be transferred to the substrate and manufactured to the third direction, so RIE is required to transfer patterns and fabricate patterns in the depth direction during the manufacturing process. RIE is isotropic etching, so it can retain the pattern better and transfer it to the substrate. In addition, for plasmonic structures, the position of the gap as “hot spot” is vital; therefore, RIE is very critical as a step controlling the size of gap manufacturing in NSL. Generally, metal is deposited on the substrate by magnetron sputtering, thermal evaporation or electrochemistry. In most structures, the metal is deposited perpendicular to the substrate surface to obtain the same or complementary shape as template. However, changing the direction of substrate placement can make the metal grow obliquely, which may result in some unique plasmon structures. Wet etching is usually used to remove the sacrificial layer of Si or SiOWe expect a cheap and fast manufacturing method that can control the size, shape and distribution of the structures in a large area, yet advantages and limitations exist at the same time in each fabricating method. Hence, it is critical to select the appropriate manufacturing techniques for every specific application. Moreover, a combination of multiple manufacturing methods may be more effective than a single technique for sophisticated structures.In clinical health monitoring and disease diagnosis, it is of importance to detect patients’ physiological signals and biomarkers . More anCancer has a high mortality rate, high metastasis rate and high recurrence rate and remains among the most common causes of death worldwide . The traSurface-enhanced Raman spectroscopy (SERS) can provide unique molecular “fingerprint” information by identifying the shift and intensity change in spectral signal . The preSome researchers have shown that, since the exosomes from tumor cells can carry the information of tumor cells, exosomes show potential for early detection and diagnosis of tumors. Recently, Lee and his colleagues developed a Au nanopillar SERS biosensor with ultra-high sensitivity and high specificity for quantitative determination of exosomal miRNAs from breast cancer cells. Highly uniform plasmonic Au nanopillars generate dense hot spots; thus, coupling of local plasma fields is enhanced and targeting miRNAs hybridized is specifically locked with nucleic acid (LNA) probes. The sensor can distinguish single base mismatch in miRNA molecules and shows an exceedingly low detection limit as little as 1 am, 100 times lower than other SERS methods to detect miRNA, a wide dynamic range (1 am to 100 nm), multiplex sensing capability and sound miRNA recovery in serum. The detection level of exosomal miRNAs from breast cancer cells by this sensor is consistent with the detection level of traditional QRT-PCR. More importantly, this SERS sensor can classify the molecular subtypes of breast cancer cell lines in light of the expression mode of exosomal miRNAs, which indicates that it provides a new method for classification of molecular subtypes of breast cancer .Further, Hyungsoon and co-workers described a label-free high-throughput approach based on surface plasmon resonance (SPR) for quantitative analysis of exosomes. Detection of exosomes (nPLEX) by this plasmonic biosensor is based on light transmission of periodic nanopore arrays instead of total internal reflection commonly used in industry since the detection depth (<200 nm) of periodic nanopores arrays and the size of exosome (nPLEX) match each other. In order to biosensor can be more convenient to operate, they combined the sensing device with the integrated circuit to design a sensor chip, which can display the spectral shift or intensity change in direct proportion to the target labeled protein level by targeting specific exosome binding. Compared with traditional methods, it can continuously and real-time monitor molecular binding with highly sensitivity and without label .Toxic and dangerous gases in the environment and human health go hand in hand owing to not only their irritation of the upper respiratory tract but also the possibility of causing accidents. Therefore, detection of toxic gases in the living environment is very necessary for prevention of diseases and maintenance of human health . Plasmon2 nanofibers, indium tin oxide (ITO) transparent electrodes and glass substrate for detecting toxic NO2 gas at room temperature under visible light illumination. The plasmonic NO2 sensor has exceedingly low coverage of sensing materials (≈0.3%), resulting in high transparency (≈93%) of the entire biosensor. In particular, thanks to the surface plasmon effect, the Au nanofiber arrays in the gas sensor significantly enhance the response signal without external heaters or light sources, and its detection limit is as low as 6 ppb [Kyeorei and his colleague reported transparent nanopatterned chemiresistors composed of aligned 1D Au–SnOas 6 ppb .In order to achieve ultra-high sensitivity and high selectivity detection of multiple gases, combining with microfluidic chips and surface-enhanced Raman spectroscopy (SERS), Yang and his co-workers designed a plasmonic gas biosensor containing three different detection units to identify the gas through physical adsorption and chemical adsorption at the same time. The preparation step of plasmonic structure is to fabricate mold by NSL method first and then deposit bimetallic (Au@Ag@Au) nanocubes by self-assembly method . A gas bTemperature sensing is vital in daily healthcare monitoring, disease diagnosis and complex medical solutions due to abnormality of human body temperature possibly being clinically confirmed as a major symptom of major public health events, such as SARS and COVID-19. Plasmonic temperature sensors can provide stable and sensitive temperature sensing signals, which has great development potential ,82,83.−4/°C in the temperature range of 35–185 °C [A type of Tamm-temperature sensor was reported based on surface plasmon resonance , as desc5–185 °C .A highly sensitive fiber temperature sensor based on photon crystal fibers (PCFs) with periodic microscopic pore structure was reported by Abu Bakar Siddik et al. Compared with conventional fiber sensors, the temperature sensor can adjust structural parameters, such as the thickness of gold, to control sensitivity and performance, which shows high sensitivity at temperatures ranging from 0 °C to 60 °C. The temperature sensor has sufficient temperature monitoring potential in healthcare monitoring and medical treatment . Han et Body fluids, including urine, sweat, saliva, tears, etc., carry abundant proteins and metabolites, which contain the basic information of personal health and the markers of some diseases . CurrentIt will increase the refractive index of the plasmonic material around the captured analyte that the plasmonic material decorated with specific molecular recognition agents that can capture the corresponding analyte. Thus, it causes a red shift in the LSPR wavelength and achieves sensing of the analyte. One example is the AuNS@PNM composite material for detecting chronic dry eye in tears proposed by Culver’s group , which sFor sweat detection, a wearable sensor device, which can make real-time and portable sensing come true, is one of the hottest research directions at present . Mogera As a result of the continuous spread of COVID-19 that has brought heavy losses to our physical, mental health and economic development, COVID-19 detection has gradually become normalized in countries around the world ,95. It rLeong and co-workers designed handheld COVID-19 biosensors based on specific spectral variations in surface-enhanced Raman scattering (SERS) due to interactions between respiratory metabolites and multiple molecular receptors on the substrate of silver nanocubes . The COVRecently, Qiu et al. reported a two-functional plasmonic biosensor with gold nanoislands (AuNIs) for rapid screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on a large scale, combining the plasmonic photothermal (PPT) effect and localized surface plasmon resonance (LSPR) sensing transduction. Using two different incident angles, the plasma resonance of PPT and LSPR can be excited at two different wavelengths. AuNI with complementary DNA receptors based on the self-assembly process of thermal dewetted Au nanofilm was used for detecting specific sequences in SARS CoV-2 through nucleic acid hybridization. The thermoplastic heat generated from PPT can improve the temperature of in situ hybridization, which further assists to promote accurate identification of two similar gene sequences. The plasmonic coronavirus sensor is still sensitive when the virus concentration is as low as 0.22 PM and can accurately capture the desired analyte in the polygenic mixture .In summary, plasmonic biosensors have various manufacturing methods, such as chemical synthesis of nanoparticles, direct writing lithography, nanosphere lithography, etc., which can be selected and combined according to the advantages and limitations of each method to manufacture more sophisticated sensing units that can be applied to cancer marker detection, gas sensing, temperature sensing, body fluid sensing, COVID-19 detection and other scenarios. Furthermore, plasmonic biosensors can also be extended to more challenging and potential fields such as gene sequencing, which will contribute to risk assessment of complex genotypic diseases and development of personalized medicine. With continuous innovation and development of various micro/nano fabricating methods, as well as wide application of plasmonic biosensors, we are closer and closer to an ideal healthcare approach that can prevent diseases through real-time monitoring of individual body physiological index as well as diagnosing and treating diseases early through convenient screening of disease markers. Nevertheless, materialization of this wonderful prospect requires joint efforts and interdisciplinary support. For instance, the data collected by plasmonic biosensors need to be transmitted quickly to intelligent portable devices to enable real-time analysis of these data . The lar