Arboviruses represent major challenges to public health, particularly in tropical, and subtropical regions, and a substantial risk to other parts of the world as respective vectors extend their habitats. In recent years, two viruses transmitted by Aedes mosquitoes, Chikungunya and Zika virus, have gathered increased interest. After decades of regionally constrained outbreaks, both viruses have recently caused explosive outbreaks on an unprecedented scale, causing immense suffering and massive economic burdens in affected regions. Chikungunya virus causes an acute febrile illness that often transitions into a chronic manifestation characterized by debilitating arthralgia and/or arthritis in a substantial subset of infected individuals. Zika infection frequently presents as a mild influenza-like illness, often subclinical, but can cause severe complications such as congenital malformations in pregnancy and neurological disorders, including Guillain–Barré syndrome. With no specific treatments or vaccines available, vector control remains the most effective measure to manage spread of these diseases. Given that both viruses cause antibody responses that confer long-term, possibly lifelong protection and that such responses are cross-protective against the various circulating genetic lineages, the development of Zika and Chikungunya vaccines represents a promising route for disease control. In this review we provide a brief overview on Zika and Chikungunya viruses, the etiology and epidemiology of the illnesses they cause and the host immune response against them, before summarizing past and current efforts to develop vaccines to alleviate the burden caused by these emerging diseases. The development of the urgently needed vaccines is hampered by several factors including the unpredictable epidemiology, feasibility of rapid clinical trial implementation during outbreaks and regulatory pathways. We will give an overview of the current developments. Aedes species mosquitos, in most cases by Aedes aegypti and Aedes albopictus. Arboviruses are a major threat to human health. In addition to CHIKV and ZIKV, the family of these viruses comprise different human pathogens that can cause acute infections including Dengue Virus (DENV), Yellow Fever Virus (YFV), West Nile Virus (WNV), Japanese Encephalitis (JEV), Ross River Virus (RRV), and Eastern Equine Encephalitis virus (EEEV). Over 100 arboviruses are known to date that can cause infections in humans, and it is a widely accepted belief that many more such viruses remain to be discovered  is a flavivirus of African origin which was first identified in a rhesus monkey in the Zika Forest in Uganda 1947 . The virAedes . In addiAedes  that canAedes , 12. SucAedes . In natuAedes .Chikungunya virus (CHIKV) is an alphavirus transmitted by mosquitoes that causes a febrile disease referred to as Chikungunya fever. Like ZIKV, CHIKV was first isolated in Africa, in Tanzania in 1952. The disease is characterized by high, transient fever, polyarthralgia, and skin manifestations . While mZIKV and CHIKV have gathered increased interest in recent years due to several massive outbreaks. Climate change and increased travel activities have led to unprecedented spread of these viruses, particularly throughout tropical and subtropical regions, but also to temperate zones. Autochthonous transmission of CHIKV was reported in several European countries including Spain, France and Italy , 20. In Vaccination is the most effective defense against unpredictable outbreaks of emerging infectious diseases. Currently, there is no treatment or vaccine available to prevent CHIKV or ZIKV disease. Here, we give a brief overview on the molecular virology, epidemiology, pathogenesis and the immune response to ZIKV and CHIKV, followed by a summary of past and current efforts to develop vaccines against these diseases. Finally, we will discuss the current regulatory and policy framework that will facilitate and accelerate the development of a ZIKV and a CHIKV vaccine.The ZIKV genome consists of single stranded positive sense RNA of about 11 kB in length which harbors one single open reading frame flanked by 5′ and 3′ non-coding regions . TranslaZIKV exhibits a very broad tissue tropism in humans ranging from the brain, skin and immune cells to placenta, testis, kidney, and retina. The virus enters the cell via clathrin-dependent endocytosis following interaction of the receptor protein E with cell surface receptors. Multiple cell surface receptors facilitate viral entry, among these are tyrosine-protein kinase receptors AXL, Tyro3, DC-SIGN, and TIM-1 –33. UponFollowing the discovery of ZIKV in 1947, only sporadic cases of natural ZIKV infection of human beings were reported over a period of 60 years. In 2007, the first large ZIKV outbreak occurred on Yap Island (Micronesia) during which three quarters of the population were affected within a few months . ZIKV inZika virus can be grouped into two major lineages, African and Asian. Strains identified during the epidemic in South America all belong to the Asian lineage and have high degrees of similarity with strains from Polynesia . DistincTogaviridae family. The genome is about 12 kb, encoding four non-structural genes (nsP1-4) and five structural genes (C-E3-E2-6K-E1) expressed from a subgenomic RNA .CHIKV was first described after a 1952/1953 outbreak in Tanzania. Several outbreaks of varying size were reported in Africa and Asia in the following decades, with the first report in Asia occurring in the Philippines in 1954 . After aAedes albopictus (Chikungunya viruses have been divided into three genotypes based on phylogeny: West African (WAf), East Central South African (ECSA), and Asian genotype, with the Asian genotype likely derived from ECSA . During bopictus . This gebopictus –65.Originally, ZIKV infection in humans was described as mostly asymptomatic or mild with self-limiting flu-like symptoms in about 20–25% of infected people following an incubation period of 4–10 days , 66. TheThe recent, large outbreak in the Americas drastically showed the ZIKV infection is also associated with severe disease. The virus can cause thrombocytopenia, subcutaneous bleeding  and multZIKV infection in adults has been associated with the development of Guillain-Barré syndrome (GBS), an autoimmune disease causing peripheral nerve damage by the immune system that leads to muscle weakness and paralysis of the limbs , 75. TheChikungunya fever symptoms commence after an incubation period of 2–12 days following transmission from an infected mosquito. The majority of patients then enter the acute phase of Chikungunya fever, characterized by high fever, severe myalgia and arthralgia, and rashes as the most common manifestations. The rate of asymptomatic infection seems to vary from 4 to 28%, which depends on the CHIKV lineage and age of the patient, with higher rates of asymptomatic disease in children . The inf−/−) or wild-type C57BL/6 mice treated with an IFNAR1 receptor blocking monoclonal antibody– are frequently used to study Zika virus infections in adults. Dependent on the ZIKV strain and the age of the animals, infected mice demonstrate signs of tremor, ataxia, paralysis, conjunctivitis, damage of the central nervous system as well as inflammation of the male reproductive tract. In addition, neonate C57BL/6 mice are a useful model to study mechanisms of pathogenesis as well as neurodevelopmental processes. Pregnant IFNAR1 knockout mice and pregnant C57BL/6 mice treated with anti-IFNAR1 monoclonal antibody represent a good model to investigate ZIKV infection during pregnancy. In these animals, ZIKV induces pathological changes in the placenta, intrauterine growth restriction of the fetuses and fetal brain development defects. Recently, BALB/c mice treated with an anti-IFNAR1 monoclonal antibody as well as neonate BALB/c mice have also been used to model ZIKV infection in adults as well to study neuropathogenesis, respectively , type II (IFN-γ), and type III IFNs (IFN-λ1–4) followed by the induction of IFN stimulated genes such as small membrane-associated interferon-inducible transmembrane proteins (IFITMs) that interfere with specific steps of the viral life cycle , 88, 89.learance –94. Desp in mice . B-cell  in mice . These a in mice . In addiresponse . Moreoveresponse . All theresponse –102. It in vitro, no signs of ADE have been observed so far in vivo . Antibodies that are developed during the first DENV infection enhance disease burden in the context of a secondary infection with a heterologous DENV serotype . Althoug in vivo . Of note in vivo , 106.−/−, are similarly susceptible and are frequently used in lethal challenge models  and National Institute of Allergy and Infectious Diseases (NIAID) have developed two other DNA based vaccine candidates, VRC5283 and VRC5288. Both candidates encode a codon-optimized form of the prM-E sequence derived from the French Polynesian strain 2013 . The ZIKAnother appealing platform technology for the development of vaccines against infectious diseases is represented by mRNA vaccines. This technology has been improved over the last years by developing techniques to remove double stranded RNA product, by inserting modifications to increase RNA stability and by developing different formulations for delivery . In contDiverse ZIKV mRNA vaccine candidates have been developed and studied in animals. One of the first was described by Pardi et al. . The mRNWhole inactivated vaccines have been successfully developed for other flaviviruses including TBEV and JEV. This approach was taken up by several groups. Immediately after the 2015 outbreak, the first preclinical studies using a purified inactivated ZIKV vaccine were described by Larocca et al. . The aluBharat Biotech International (India) has started its ZIKV vaccine development after the French Polynesian outbreak in 2013. In preclinical mice studies using immunodeficient AG129 mice, the formalin-inactivated whole virus vaccine (BBV121) demonstrated its immunogenic potential by protecting against Asian and African challenge strains . BBV121 Vaccination experiences with other live attenuated flaviviral vaccines like YFV and JEV propose that a live attenuated ZIKV vaccine could be a promising approach for generating a robust immune response. In contrast to the traditional approach used for the 17D YF vaccine in which attenuation was achieved by several passages on different animal tissues, ZIKV attenuation was achieved by direct manipulation of the viral genome. This was only possible due to the fact that ZIKV infectious cDNA clones were available shortly after the first outbreaks . AttenuaAttenuated viruses carrying a 10 nucleotide deletion in the 3'UTR of a Cambodian Strain provided sterilizing immunity in A129 mice and rhesus macaques . FurtherXie et al. at the University of Texas Medical Branch have been involved in the generation of a chimeric virus containing the Zika prM-E in a Dengue Virus 2 (DENV-2) backbone. The chimeric vaccine protected A129 mice against ZIKV challenge . A seconAnother vaccine approach evolved over the last decades deals with the expression of ZIKV genes in the context of viral vectors, either replication competent or defective. Different viral vectors including adenovirus, vesicular stomatitis virus (VSV), vaccinia virus, or measles vaccine virus are used as a delivery platform for the production of heterologous antigens upon cell infection, making them a powerful plug and play technology for the rapid development against emerging diseases.A rhesus adenovirus based vaccine candidate (RhAd52) expressing the ZIKV prM-E proteins was assessed for its immunogenicity and efficacy in rhesus monkeys . This vaA live attenuated measles Schwarz vaccine vector expressing the ZIKV prM-E was developed by Themis Bioscience and was evaluated in a Phase 1 clinical trial in Austria (NCT02996890). Preclinical studies in an allogenic mouse pregnancy model have shown that vaccination with this candidate reduced the ZIKV load in distinct organs and prevented fetal infection . RecentlBesides adeno- and measles virus-based vectors, a further promising vaccine candidate based on a vaccinia virus vector directed against both emerging diseases ZIKV and CHIKV was successfully tested in preclinical studies. The vector has incorporated ZIKV prM-E as well as the structural proteins of CHIKV. A single immunization of this multivalent vaccine induced neutralizing antibodies toward both viruses in mice and protected against CHIKV viremia and arthritis as well as ZIKV viremia and fetal/placental infection or testis infection .Another approach utilizes recombinant VSV vectors expressing ZIKV prM-E proteins. One candidate showed a good humoral and cellular T cell response in wild-type mice, another protected from lethal challenge , 159.In vitro purified virus-like particles (VLP) represent an alternative approach for the development of a ZIKV vaccine. As already mentioned above expression of ZIKV structural proteins give rise to the development of non-infectious virus-like particles which present antigens in their native confirmation leading to the development of high neutralizing antibody titers. VLP vaccines are efficiently produced by generation of stable cell lines. Some groups have explored their immunogenic potential in preclinical animal studies  together with WRAIR developed a formalin-inactivated CHIKV vaccine which showed encouraging immunogenicity and efficacy data in mice and NHP using four different CHIKV strains . In furtDevelopment of a live-attenuated vaccine initiated by USAMRIID yielded the strain CHIK 181/clone 25 . A laterTargeted attenuation of CHIKV by means of genetic engineering gave rise to several other promising candidates. Partial deletion of the gene encoding the non-structural protein nsP3 resulted in a live-attenuated vaccine showing immunogenicity and efficacy in the mouse footpad swelling model , 170 and−/− mice and Cynomolgus macaques  induced neutralizing antibodies to the homologous CHIKV S27 strain in immunocompromised AG129 mice. However, titers elicited by the MVA-6KE1 and MVA-E3E2 were significantly lower than titers induced by the full envelope cassette. Interestingly, both the MVA-E3E26KE1 and MVA-E3E2 fully protected the CHIKV susceptible AG129 mice against lethal challenge with CHIKV S27 strain . Similarng model . All MVAAnother candidate was created by replacing the glycoprotein G of Vesicular Stomatitis Virus (VSV) with the CHIKV structural cassette, resulting in a chimeric virus shown to be immunogenic and protective in the mouse footpad swelling model. In addition, the VSV-CHIK constructs induced a CHIKV E1- and E2-specific IFNγ-producing T-cell response after a single immunization .Two further pre-clinical candidates were created based on adenoviruses. One study showed that a vaccine based on the non-replicating Complex Adenovirus vaccine and expressing the CHIKV structural polyprotein induced antibodies and protected mice in the footpad swelling model . AnotherUsing an attenuated strain of Venezuelan equine encephalitis virus (VEEV) or Sindbis virus as backbone to generate a potent CHIKV vaccine has been used by the University of Texas Medical Branch. The structural proteins of VEEV or Sindbis were replaced by the structural proteins of CHIKV, rendering the virus highly attenuated. Immunization of mice resulted in protection from viremia post-challenge . One of −/− mice as well as in the mouse footpad swelling model and Cynomolgus macaques and found to be protective  represent an optimal antigenic structure without exposing the vaccine to replicating CHIKV. The epitopes are presented in the correct structure for immune recognition. A VLP vaccine program was developed at the vaccine research center (VRC) at NIH. The vaccine candidate VRC-CHKVLP059-00-VP is comprised of VLPs expressed from human embryonic kidney cells transfected with plasmid encoding all structural protein genes (C-E3-E2-6K-E1) from CHIKV strain 37997 (West African lineage). In cynomolgus monkeys, these VLPs were highly immunogenic and the antibodies produced were cross reactive to all CHIKV lineages . This cain vitro and in vivo neutralization activity and conferred protection in CHIKV infected neonatal mice  by usage of a cytomegalovirus promotor. In BALB/c mice, this iDNA vaccine induced neutralizing antibodies and protected from viremia upon challenge . Interesin vitro transcribed RNA. One mRNA based prophylactic vaccine candidate VLA-181388 was developed by Moderna Therapeutics. The company has publicly announced that a single dose induced protection against challenge in mice and neutralizing antibodies in NHP. So far, no pre-clinical data have been published. Currently, a Phase 1 clinical trial is evaluating the safety and immunogenicity of this candidate (NCT03325075).mRNA vaccines represent one of the newest strategies in the development of vaccine candidates against infectious diseases which are comprised of Subunit vaccines generally have a good safety profile compared to other vaccine candidates as no viral DNA/RNA or infectious particles are present. In addition, the production of single proteins is scalable and can be easily adapted to large-scale manufacturing purposes. A disadvantage of this vaccine candidate may be the limited immunogenic potential due to the fact that quaternary epitopes are not present. In addition, subunit vaccine necessitates the use of vaccine adjuvants that potentially increase production costs. Different groups have followed this strategy and have generated E1 and E2 subunit vaccines by the baculovirus system or by bacterial expression system , 203. BaFour vaccine candidates for the prevention of Chikungunya disease have progressed past phase 1 clinical studies, all of which look promising in terms of safety and immunogenicity. While the candidates are based on very different platform technologies, they all induce neutralizing antibodies, a reasonably likely correlate of protection. Thus, all these candidates are probably suitable for the prevention of Chikungunya disease. However, the unpredictable epidemiology of CHIKV remains a challenge in bringing any of these candidates to licensure, as discussed in greater detail below.The development of safe and effective vaccines suitable for the use in all age groups is of utmost importance to prevent the spread of CHIKV and ZIKV and for successful outbreak intervention. Regulatory bodies including European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) agree on the urgent medical need for CHIKV and ZIKV vaccines , 206. ThThe development of vaccines from early development to the availability on the market is a timely process that can take more than 10 years with cost >$100 Million US Dollars . The lichttps://www.fda.gov/advisory-committees/advisory-committee-calendar/vaccines-and-related-biological-products-advisory-committee-november-8-2019-meeting-announcement). In addition, the WHO has assembled experts in for a R&D blueprint meeting on the same topic. The experts gave guidance on current epidemiological features of the disease and on potential clinical trial designs  to discuss topics on the licensure of CHIKV vaccines. The purpose of the meeting was to publicly discuss the feasibility of vaccine efficacy trials and use of a non-human primate animal model to assess vaccine efficacy. The recently published meeting minutes state that the epidemiology of CHIKV does not allow for the conduct of randomized controlled clinical efficacy trials in the immediate future, and the combination of seroepidemiological knowledge can be combined with animal models to identify an immune marker reasonably likely to predict vaccine effectiveness are supported ( designs .In 2016, the WHO declared a public health emergency during the ZIKV outbreak in the Americas. To facilitate continued ZIKV vaccine development the WHO and NIH/NIAD co-hosted an expert and regulatory meeting in 2018. The experts concluded that vaccine efficacy trials are not feasible, and a discussion was held on the use of immunological markers that predict protection . Taken tSS mainly contributed on the Zika virus and Zika vaccine section by literature review, manuscript writing, and final editing. RT mainly contributed to the Chikungunya virus and Chikungunya vaccine sections by literature review, manuscript writing, and final editing. ET reviewed the content and contributed to Zika and Chikungunya vaccine development status. KR contributed to the regulatory review and introduction, and final review of the manuscript content.All authors are employees of Themis Bioscience GmbH, a company developing vaccines to prevent Chikungunya and Zika.


Responsive nerve stimulation (RNS) represents a safe and effective treatment option for patients with medically refractory temporal lobe epilepsy. In cases of long intraparenchymal course and posterior-anterior electrode direction through occipital burr holes, disciplined stereotaxy is essential for stimulation of the appropriate target.A 13-year-old female with a history of multifocal, independent, bitemporal-onset seizures since 12 months of age showing evidence of left-sided mesial temporal sclerosis on MRI, underwent placement of bilateral mesial temporal RNS leads. An O-arm spin was performed after the placement and the images obtained were fused to the preoperative CT images. It demonstrated curvature of the leads, with some deviation from the planned trajectory, but no deviation from the target, that was worse on the left side, compared to the right; the left lead was placed first, followed by the right lead. Following discussion with our epilepsy neurology colleagues in the operating room, electrophysiological measurements from the implanted leads showed cleared epileptic activity and therefore no repositioning was pursued. Our hypothesis at that time was that cerebrospinal fluid leakage distorted the underlying ventricular anatomy causing some curvature in the lead during transventricular course and prolonged consideration during surgery.In conclusion, transventricular trajectories during RNS lead placement may lead to cerebrospinal fluid loss and associated lead deformation. The distal aspect of the lead may nonetheless reside in the desired surgical target. Neuromonitoring for epileptic signature can provide reassurance with regard to accurate lead placement, obviating the need for lead repositioning. Surgeons should also recognize that fused imaging may confuse inferred anatomic position from preoperative MRI with actual anatomy post brain shift. It has been estimated that up to one-third of patients with epilepsy have medically refractory seizures . SeveralThe RNS system is composed of an implanted cranial programmable neurostimulator connected to depth or subdural cortical strip leads . In bilaThe patient is a 13-year-old right-handed female who presented with drug-resistant seizures since the age of 12 months. Her initial seizure was a prolonged, right-sided hemiclonic event, which was quickly followed by focal, non-motor, impaired awareness seizures characterized by staring, behavioral arrest, manual automatisms, chewing and right-or-left gaze deviation. The seizures occasionally evolved to hemiclonic or bilateral tonic-clonic activity. Her past medical history is significant for moderate intellectual disability  and attention deficit and hyperactivity disorder. The preoperative MRI demonstrated abnormal T2 hyperintensity within the left hippocampus and mesial left temporal lobe as well as mild atrophy of the temporal portion of the left lateral ventricle, which are findings suggestive of mesial temporal sclerosis. There was also evidence of a small focal nonspecific area of T2 hyperintensity seen within the right frontal white matter and congenital ventriculomegaly of occipital horns  3D pictures were obtained to verify placement and were fused to the preoperative imaging. These demonstrated local curvature and concern for deviation from the planned trajectory, left being worse than right , which showed the leads in the hippocampi and amygdalae Figure . Hardware complications in functional neurosurgery are not uncommon; lead-related problems comprise the second most common complication following surgical site infections, with an estimated incidence of 3.8% . They moThe majority of evidence regarding the inaccuracy of anatomical targeting during neurostimulation is derived from DBS literature. In the RNS publication of long-term outcomes, there was no mention of complications related to transventricular trajectory; “device lead damage” occurred in 3.5% . FurtherZrinzo et al. later reported a 42% rate of transventricular electrodes in a cohort of 109 DBS patients . InvolviLead placement during deep brain or responsive nerve stimulation procedures can be distorted during transventricular trajectories to the surgical target. Neurophysiological recordings can provide reassurance with regard to accurate lead placement and prevent unnecessary and possibly counterproductive lead repositioning.


Ingestion of Western-diets enriched in long chain saturated fatty acids (LCSFA) are associated with increased risk of blood-brain barrier (BBB) dysfunction and neurovascular inflammation. Potential mechanisms include vascular insult as a consequence of metabolic aberrations, or changes in capillary permeability resulting in brain parenchymal extravasation of pro-inflammatory molecules. Bovine dairy milk (BDM) is potentially a significant source of dietary LCSFA, however, BDM contains an array of bioactive molecules purported to have vascular anti-inflammatory properties. This study investigated the effects of full cream  and delipidated (skim) BDM on BBB integrity and neuroinflammation in wild-type mice. Mice consuming substantial amounts of full cream or skim BDM with LCSFA-enriched chow were dyslipidemic compared to control mice provided with standard chow and water. However, there was no evidence of BBB dysfunction or neuroinflammation indicated by parenchymal abundance of immunoglobulin G and microglial recruitment, respectively. Positive control mice maintained on an LCSFA-enriched diet derived from cocoa-butter and water, had marked BBB dysfunction, however, co-provision of both full cream and skim milk solutions effectively attenuated LCSFA-induced BBB dysfunction. In mice provided with low-fat chow and full cream BDM drinking solutions, there were substantial favorable changes in the concentration of plasma anti-inflammatory cytokines. This study suggests that consumption of BDM may confer potent vascular benefits through the neuroprotective properties exuded by the milk-fat globule membrane moiety of BDM. Risk factors for vascular dysfunction such as dyslipidaemia and obesity are associated with early onset and progression of neurodegenerative disorders such as Alzheimer's disease and vascular dementia . ChronicDietary LCSFA's are commonly found in animal products such as meat and dairy, staple food commodities in Western styled diets. Standard bovine dairy milk (BDM) preparations contain ~4% w/w fat, of which 70% is principally palmitic and stearic fatty acids , 10. HowBovine dairy milk is rich in phospholipids which have been reported to suppress inflammatory reactions. Bovine dairy milk phospholipids reduce endoplasmic reticulum stress in neuronal cells, implicated in development of neurodegenerative disorders . MoreoveCalcium enriched foods such as BDM may confer vascular benefits indirectly, by positively modulating metabolism, obesity and lipid homeostasis. Bruckbauer et al.  suggesteDairy milk contains compounds and macromolecules which potentially promote, or inhibit vascular inflammation, however the synergistic net effect of BDM on cerebral capillary integrity has not been previously reported. This study explored putative effects of alternate BDM formulations on indices of the BBB and neurovascular inflammation in wild-type mice. Moreover, potential synergistic effects with a pro-atherogenic LCSFA enriched chow diet was also considered.n = 10). Low-fat control group was fed standard low-fat maintenance chow containing 4% (w/w) fat as monounsaturates . The high LCSFA positive control group was maintained on a semi-synthetic diet containing 40% of digestible energy derived from cocoa butter (23% (w/w), SF07, Specialty Feeds, WA, Australia). Two other groups were allocated a 20% full cream (FC) milk solution diluted with water, with one group receiving high LCSFA diet (LCSFA + FC) and the other receiving low-fat control chow (LF + FC). The final group was 20% skim milk with a high LCSFA diet (LCSFA + Skim). Each group was sacrificed at 13 weeks from the start of the dietary intervention. The mice were held in Curtin University Animal Facility with controlled air temperature (22°C), air pressure and a 12-h light/dark cycle. All mice had ad libitum access to food and liquid. Milk solutions were replaced daily to prevent rancidity. Liquid consumption was monitored and recorded daily and food consumption was measured weekly for each group . Mice were randomly allocated into 5 experimental groups  extravasation, as established previously , 4, 8. BThe expression of ionized calcium-binding adaptor protein-1 (Iba-1) was determined as a measure of neuroinflammation by indicating microglial recruitment, as described previously . Twenty An average of 15 × 3-D images were captured to represent the cerebral cortex. The voxel intensity of Iba-1 staining was determined by using Volocity 5.4.2 image analysis software .TM multimode plate reader  at a wavelength of 546 nm. Assay standards were used to develop a linear regression line in which sample lipid concentrations were interpolated from.Serum triglycerides and total cholesterol concentrations were determined by using colorimetric assays according to the manufacturer's instructions . Briefly, 2 μL of serum was added to 200 μL of reagent and incubated for 10 min at room temperature (RT). Optical density was measured using an EnSightTM multimode plate reader  at a wavelength of 450 nm. Assay standards were used to develop a linear regression line in which sample concentrations were interpolated from.Serum levels of insulin were measured using an Ultrasensitive Mouse Insulin ELISA kit , as per the manufacturer's instructions. Briefly, 25 μL of serum was added to 100 μL of enzyme conjugate and incubated for 2 h on a plate shaker. Samples were washed and then combined with TMB substrate for 15 min at RT. Fifty Microliter of stop solution was added to each well and samples were then read on an EnSightDetection of cytokines was carried out using the commercially available BD cytometric bead array (CBA) Mouse Th1/TH2/TH17 cytokine kit  as established previously with some minor adaptions . All data that were not normally distributed were analyzed by Krusal-Wallis test with Dunn's post hoc test . Statistical significance was detected at p < 0.05 (n = 10).All data were calculated and expressed as mean ± SEM. In order to assess the effects of LCSFA diet and dairy milk consumption on cerebrocapillary BBB, neuroinflammation and peripheral cytokine profile, all normally distributed data were analyzed with one-way analysis of variance (ANOVA) followed by Fisher's least significant difference (LSD) This study investigated the putative effects of chronic ingestion of full cream or skim BDM on neurovascular integrity and neuroinflammation in genetically unmanipulated wild-type mice. The milk formulations were diluted to either 20% of commodity strength and provided with standard low-fat chow, or to mice maintained on a pro-atherogenic LCSFA- enriched diet. The milk drinking solutions were well-tolerated and no adverse events were reported for the 12-week treatment period. The ingestion of milk solutions compared to treatment groups provided with water is indicated in ad libitum provision of LF + FC did not increase net energy intake. Conversely, LCSFA-fed mice provided with skim milk had ~20% greater energy intake compared to the low-fat controls for the duration of the study. Mice provided with diets enriched in LCSFA had greater total energy intake, principally as a consequence of the higher caloric content of the chow formulation.Average chow consumption and the total energy intake for treatment groups  and was sufficient to induce modest changes in their cholesterol and triglyceride response. Despite comparable levels of hypercholesterolemia in BDM-fed groups maintained on LCSFA chow and LCSFA-only mice, and pronounced triglyceride concentrations in full-cream BDM groups, the changes in lipid indices were not associated with cerebral capillary dysfunction or neurovascular inflammation. The attenuation of capillary dysfunction and marked attenuation of neuroinflammation occurred concomitant with increased levels of anti-inflammatory cytokines in mice maintained on the LF+FC formulation. However, low-fat and skim milk interventions were found to reduce inflammation without significant effects on anti-inflammatory cytokine abundance. The latter suggests that BDM may also regulate neurovascular function and inflammation through other mechanisms, including perhaps via direct modulation of the capillary endothelium. Milk formulations were provided in 20% dilutions and would be physiologically relevant if intake aligned with that of the control mice. However, intake of milk formulations were significantly greater. Liquid consumption was the highest among the LF + FC group, followed by LCSFA + FC and LCSFA + Skim. Mice provided milk solutions concomitant with LCSFA-enriched chow showed modestly higher ingestion of drinking solutions compared to the LCSFA and water, but markedly less than the LF + FC group. Treatment effects on satiety signaling may underlie differences observed in chow and supplementary liquid (milk formulations/water) intake. Individual consumption of liquid, chow and energy intake could not be deliberated due to grouped housing/diet conditions. The consumption rates of both chow and liquid depict the cumulative energy consumption, in that the LCSFA-chow receiving groups had the greatest overall energy intake.Groups maintained on full cream BDM formulations  developed an increased dyslipidemic response that was dose dependent as indicated by serum triglyceride levels. The liquid consumption data  suggestsDespite the notable effect of BDM on an abnormal lipid and insulin profile, immunomicroscopic analysis revealed there was no evidence of BBB leakage, characterized by extravasation of IgG from blood to brain parenchyme, within these groups. More intriguingly, both LCSFA-fed full cream and skim BDM groups revealed modest reductions in BBB indices and indeed indicative that BDM may attenuate dietary LCSFA-induced BBB dysfunction. However, IgG has a molecular weight of 166 kDa, thus the possibility of more subtle changes in BBB permeability cannot be excluded. Although extravasation of smaller biomarker molecules may not be detected, subtle changes in BBB permeability can be indirectly detected through Iba-1 expression, a biomarker of microglial recruitment, of which extravasation is normally associated with. Interestingly, Iba-1 expression is consistently lower for all BDM-maintained groups compared to the LCSFA positive control. Iba-1 is expressed in microglia during their activation by infection or damage to cells, indicating the correlation of chronic consumption of LCSFA with an inflamed state, as previously published .Although LCSFA is known to induce BBB dysfunction, this study suggests that if consumed in conjunction with full cream BDM, both BBB dysfunction and neuroinflammation are modestly attenuated. This provides evidence that BDM may exude neuroprotective effects on BBB integrity. Furthermore, the skim BDM group also showed a decrease in both IgG extravasation and neuroinflammation, suggesting that neuroprotective components could potentially be within the non-lipid moiety of milk. The possibility of certain lipids having neuroprotective effects on the BBB cannot be excluded, however, the skim milk phase reportedly contains a small percentage of phospholipids .As discussed earlier, an increase in pro-inflammatory cytokines can ultimately cause a breakdown of endothelial cell structure. A diet rich in LCSFA can lead to endothelial dysfunction through dylipidemia and obesity effects and is likely to be contributing to the BBB dysfunction observed in the LCSFA control group , 18, 22.An increase in anti-inflammatory cytokines and protective effect of BDM on the BBB may be the product of phospholipids that are contained within the MFGM. A study by Nagai  identifiBDM contains a significant amount protein as part of its macronutrient profile, with ~20% of the total contributed by whey protein. BDM is also a rich source of calcium and riboflavin, notably in the whey protein fraction, which has proven antioxidant and inflammatory effects, principally through its effects on glutathione activity. Recent studies have implicated the association between cerebral glutathione concentration and consumption of BDM-enriched foods. Dairy products purportedly act as exogenous substrates for glutathione synthesis in the brain and whilst glutathione status was not assessed in this study, it is reasonable to infer that increased consumption of BDM may have contributed to vasculo-protection and attenuation of glial activation, mediated by cerebral antioxidant pathways .Streptococcus pneumoniae, the ability to phagocytose and clear the bacteria was suppressed compared to wild-type mice, indicating a protective role of mucin 1 (Furthermore, whey protein is known to have anti-inflammatory effects, with studies indicating significant reductions in C-reactive protein as a response to whey supplementation . It is u mucin 1 . Lactadh mucin 1 , 12. ColPreviously, a diet high in LCSFA has been described in inducing BBB dysfunction, though the direct mechanism is unknown. The present study provides evidence that BDM may attenuate dietary LCSFA induced BBB dysfunction and neuroinflammation through the bioactive protein moieties of BDM. Furthermore, evidence of BDM induced dyslipidemia had no effect on BBB dysfunction or neuroinflammation, most likely due to the protective effects of anti-inflammatory cytokines, or by an increase in other BDM bioactive compounds such as phospholipids, calcium and lactadherin. Collectively, the findings in this study further supports the neuroprotective value of BDM-derived compounds and may provide insight into future preventative treatments to help reduce the risk of vascular-related neurodegenerative diseases.All datasets generated for this study are included in the article.The animal study was reviewed and approved by Curtin University Animal Research Ethics Committee.ZD'A, VL, MN, and LG were involved in data collection, data analysis, and manuscript preparation. ZD'A, VL, RT, and JM were involved in study design, results interpretation, data analysis, preparation, and writing of 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.


Here we report a case of a middle-aged man who complained of blurred vision in his left eye 1 h post cardiac catheterization and proved to have central retinal artery occlusion, a dangerous but potentially treatable sight-threatening complication of cardiac-catheterization. The patient was successfully treated through an Ophthalmological surgical intervention.A 49- year-old male patient admitted to the coronary care unit as a case of non-ST-elevation-myocardial infarction. The patient underwent cardiac catheterization and stenting of the right coronary artery. One hour later, he complained of blurred vision in his left eye.Ophthalmological examination showed an inferior visual field defect in the left eye. Fundus fluorescein angiography revealed that the patient had a hemi-central retinal artery occlusion, a rare complication of cardiac catheterization. A pars plana vitrectomy eye surgery was performed with an excellent result.This case highlights the importance of early recognition and treatment of central retinal artery occlusion post cardiac catheterization •Rarely, central retinal occlusion can complicate cardiac catheterization.•Meaningful spontaneous recovery in vision in cases of central retinal artery occlusion occurs in less than 10%.•What is peculiar about our case is that prompt surgical intervention resulted in an unexpectedly excellent result.•The case highlights the need for prompt diagnosis and referral of patients with visual symptoms post cardiac catheterization. Rarel2A 49-year-old male patient hypertensive, smoker with positive family history of premature coronary artery disease who presented with heavy retro-Sternal chest pain.At admission, the patient had no chest pain. Physical examination revealed a blood pressure of 130/85 mmHg and heart rate of 85/minute. Cardiac and respiratory examination were unremarkable and there was no carotid bruit. Electrocardiogram showed sinus rhythm with Q waves and T-wave inversion in lead II, III, and aVF . EchocarThe patient underwent cardiac catheterization and was found to have one vessel disease with 90% stenosis of the right coronary artery. A drug-eluting stent was successfully implanted in the right coronary artery .Fig. 2CoOne hour after intervention, the patient reported acute painless blurred vision in the left eye associated with a lower visual field defect. History from the patient revealed no prior visual complaints or amaurosis fugax. Ophthalmological examination of the left eye showed visual acuity of 6/60 with normal anterior segment and intraocular pressure. Visual field testing showed complete inferior visual field defect in left eye. Fundoscopic exam showed moderate macular edema and a small yellow arterial embolus at the level of the optic nerve head .Fig. 3FuFundus fluorescein angiography  revealedA pars plana vitrectomy eye surgery under local anesthesia was performed. Massaging of the superior retinal artery wall over the embolus using a silicone-tipped aspiration cannula was performed in conjunction with induced ocular hypotony, which resulted in dislodgment of the embolus and migration into the peripheral circulation. The patient noted an immediate marked improvement in his vision. Four weeks later, the patient had a visual acuity of 20/25 and normal visual fields. There was complete resolution of the embolus and the artery occlusion and restoration of retinal blood flow .Fig. 5FuThe study was approved by the Institute Research Board at King Abdullah University Hospital and Jordan University of Science and Technology. The work has been reported in line with the SCARE 2020 criteria . Written34).Retinal artery occlusion is an uncommon sight-threatening condition, with an incidence of about 1/100,000 in the general population and usually presents with a sudden painless severe loss of vision or a visual field defect in the affected eye. The central retinal artery is most commonly occluded by a cholesterol embolus (Hollen-horst plaque) which usually originates from atherosclerotic plaques in the carotid arteries, aortic arch, or ophthalmic artery with an average age of 62 years at presentation. Embolic occlusions that affect younger people, however, are commonly cardiac in origin and emboli may arise from calcific heart valves, atheromas, or myxomas in cardiac chambers  arteritis, polyarteritis nodosa, Behcet disease, and syphilis, or hypercoagulable states such as polycythemia vera, sickle cell anemia, multiple myeloma, factor V Leiden, and antiphospholipid syndrome. Rapid differentiation between thrombo-embolic versus arteritic central retinal artery occlusion is important as optimal therapy differs and the rapid administration of steroids for vasculitic causes is associated with improved outcomes . The ratDuring cardiac catheterization the central retinal artery is most commonly occluded by a cholesterol embolus which are thought to be caused by the disruption of a vascular plaque usually in the aortic arch (mainly by catheter manipulation) with the release of subendothelial cholesterol crystals into the blood stream ,8.Cerebral blood flow as well as the diameters of the cerebral and the central retinal blood vessel are thought to play major role in determining the destination of the embolus. Whereas large atheromas are big enough to lodge in the small retinal artery, very small one cannot occlude it .Meaningful spontaneous recovery in vision in cases of central retinal artery occlusion occurs in less than 10%.Treatment for embolic central retinal artery occlusion is typically conservative and includes ocular massage, ocular anterior-chamber paracentesis, sublingual isosorbide dinitrate, intravenous acetazolamide, intravenous mannitol or oral glycerol, hyperbaric oxygen, and isovolumic hemodilution. None of these treatments have proven effective and their use is largely based on anecdotal reports and small case series .In addition, some of these interventions such as intra-arterial thrombolysis and intraocular surgery may carry high rates of complications and are usually not adopted by most ophthalmologists .The utilization of vitrectomy has been reported with variable success rates and visual benefit and a potential for complications. Surgical techniques included stylet cannulation of the central retinal artery, intra-operative controlled hypotony, and retinal wall massage .4What is peculiar about our case is that prompt surgical intervention by the ophthalmology service resulted in an unexpectedly excellent result with complete restoration of blood flow in the retinal arteries and almost complete visual recovery.This case highlights the need for prompt recognition and urgent referral of any patient with visual symptoms post cardiac catheterization.Not commissioned, externally peer-reviewed.All of the authors declare that they have no conflict of interests.


Methods: This was a longitudinal cohort study comparing the nutritional outcomes of HPV-positive and negative OPSCC patients undergoing (chemo) radiotherapy. The primary outcome was nutritional status as measured using the Patient Generated-Subjective Global Assessment (PG-SGA). Secondary outcomes included loss of weight, depression, QOL and adverse events. Results: Although HPV-positive were less likely to be malnourished according to PG-SGA at the beginning of treatment, we found that the difference between malnutrition rates in response to treatment was not significantly different over the course of radiotherapy and 3 months post treatment. HPV-positive participants had significantly higher odds of experiencing >10% weight loss at three months post-treatment than HPV-negative participants  p ≤ 0.01). Conclusions: The nutritional status of HPV positive and negative patients were both negatively affected by treatment and require similarly intense nutritional intervention. In acute recovery, HPV positive patients may require more intense intervention. At 3- months post treatment, both groups still showed nutritional symptoms that require nutritional intervention so ongoing nutritional support is essential.Background: Patients undergoing (chemo) radiotherapy for oropharyngeal squamous cell carcinoma (OPSCC) are at high risk of malnutrition during and after treatment. Malnutrition can lead to poor tolerance to treatment, treatment interruptions, poor quality of life (QOL) and potentially reduced survival rate. Human papillomavirus (HPV) is now known as the major cause of OPSCC. However, research regarding its effect on nutritional outcomes is limited. The aim of this study was to examine the relationship between HPV status and nutritional outcomes, including malnutrition and weight loss during and after patients’ (chemo) radiotherapy treatment for OPSCC.  The prevalence of malnutrition in people with head and neck cancer (HNC) is one of the highest of all cancers ,2. PeoplThe prevalence of OPSCC is increasing in higher socioeconomic countries despite the relatively stable incidence of other HNC . This isPatients with HPV-positive OPSCC are known to have better nutritional status at diagnosis than people with HPV-negative OPSCC ,14,15. SWhile research is emerging regarding the impact of HPV status on malnutrition, there are several gaps including a lack of focus on patients specifically with OPSCC and inconsistencies in regard to measurement tools and data collection time points. Early studies often compared HPV-positive and negative patients in a heterogeneous HNC population but we now know that the causal link between HPV and survival is unique to OPSCC . Recent Despite the better prognosis associated with HPV-positive OPSCC, treatment remains the same and the risk of poor nutritional outcomes is high. A better understanding of nutritional outcomes in relation to HPV status could help to inform targeted nutritional interventions and therefore contribute to better nutritional and treatment outcomes for people who are undergoing treatment for HPV-positive OPSCC. The aim of this study was to examine the relationship between HPV status and nutritional outcomes, including malnutrition and weight loss during and after patients’ (chemo) radiotherapy treatment for OPSCC. Based on previous research, we hypothesized that patients with HPV-positive status would experience more malnutrition (primary outcome), more weight loss, more depression, lower QOL and more adverse events (secondary outcomes) during and after treatment compared to patients with HPV-negative status.This longitudinal, cohort study was a sub-study of a stepped wedge, cluster, randomized controlled trial titled, ‘Eating As Treatment (EAT)’. Detailed information on the original study protocol is published elsewhere . Data coEligible participants were selected from the two participating sites. Informed, written consent was provided in the original trial for data to be used for related studies. EAT was approved by Hunter New England Health (HREC/12/HNE/108).All outcome assessments were conducted by an independent researcher at four time points: first week of radiotherapy (RT), last week of RT, one-month post-RT and three-months post-RT. All baseline characteristics and covariates such as tumor stage and treatment type were collected by a research assistant via chart review with the exception of p16 status which was retrospectively collected from confirmed histopathology reports.Eligible patients were aged 18 years or older who had pathologically confirmed diagnosis of OPSCC. Immunohistochemistry (IHC) staining using Roche CINtec histology kit, clone E6H4was used to confirm p16 status. They were planned for definitive or postoperative radiotherapy with curative intent (chemoradiation permitted including neoadjuvant and adjuvant chemotherapy), the prescribed radiation dose being at least 60 gray as regional nodal irradiation. They were available for follow-up for at least 6 months post study initiation and provided written, informed consent. Patients were excluded if they were unable to communicate in English; had organic brain disease (impairing ability to complete questionnaires), had T1 or T2 glottic carcinoma undergoing small fields radiation therapy or T1 and T2 tonsil cancer undergoing unilateral treatment.The primary outcome of nutritional status was measured using the Patient Generated -Subjective Global Assessment (PG-SGA) . The PG-Secondary outcome measures included loss of weight (LOW), depression, QOL and adverse events including treatment interruptions, unplanned hospital admissions and length of stay (LOS) of these admissions, reactive nasogastric tube (NGT) insertions for feeding, dietary adequacy, nutrition impact symptoms (NIS) and mortality at two years post-treatment.The weight (kg) of participants was recorded using electronic standing scales, at all-time points. Percentage LOW from baseline was calculated at each time point. LOW was also categorized into >5% LOW and >10% LOW as these are cut off points used in other literature for important weight loss and used clinically to indicate significant weight loss. Depression was assessed using the validated Patient Health Questionnaire 9 (PHQ-9) . This isQOL was assessed using the European Organization for Research and Treatment of Cancer, Core Quality of Life Questionnaire (EORTC QLQ-C30). This is a 30-item self-report questionnaire designed and validated to measure QOL in patients with cancer . It inclDietary adequacy was obtained from a section (Box 2) of the PG-SGA. Scores for Box 2 are measured on a scale of 0–5 . NIS were obtained from Box 3 of the PG-SGA. Scores for Box 3 are measured on a scale of 0–24 (0 indicating the patient has no symptoms that are affecting intake and 24 indicating that intake is severely impacted by symptoms). Reactive NGT insertions were measured as the proportion of patients with NGT inserted after the start of treatment in relation to poor oral intake.Treatment interruptions were measured as total number of participants with unplanned interruptions to their radiotherapy schedule. Unplanned hospital admissions were measured as total number of admissions from start of radiotherapy to 3 months post treatment and LOS was calculated as a total of all participants’ hospital stays for each HPV status group. Mortality was measured as proportion of participants who had died by two-years post treatment.This exploratory study used an opportunistic sample of patients with OPSCC who participated in a large multi-center trial, examining the entirety of OPSCC patients from two of the sites. All analyses were conducted using STATA 13 . The priSecondary outcomes were assessed using the same model to analyze the differences across all time points. Percentage LOW, depression (PHQ-9) and QOL (QLQ C30) were analyzed using LMM while the categorical variables such as the PG-SGA, >5 and >10% weight loss were analyzed using logistic mixed effects models. Treatment interruption was analyzed using a logistic regression. Unplanned hospital admissions were analyzed using a negative binomial regression. Both models included HPV status, intervention group, baseline nutritional status, hospital and calendar time.n = 2). Both of these were lost to follow-up post-treatment, the reasons stated were; living in rural areas and not able to return to hospital for follow up assessments. None of the participants died during the course of the study. Patient characteristics at baseline can be seen in Data was available from 83 participants (70 HPV-positive and 13 HPV-negative) from the two sites between July 2013 and January 2016. There was one dropout from each of the p16 positive and negative groups , found no significant difference in PG-SGA category and scores between HPV-positive and negative patients over the course of treatment and recovery .p ≤ 0.01)   ≤ 0.01) . The aveAcross all time points, there were no statistically significant differences in depression scores between HPV-positive and HPV-negative patients .p = 0.03) (p < 0.01).HPV-positive patients had statistically significant higher odds of unplanned admissions than HPV-negative patients ,  = 0.03) . MortaliThere was no statistically significant difference in reactive NGT insertions, dietary adequacy and NIS between groups .p < 0.01).There was no statistically significant difference in global QOL scores, symptom and functional scale scores between groups across all time points . The excThis is the first longitudinal study, to our knowledge, in which the impact of HPV status on malnutrition and related factors has been investigated at various time points during and after RT treatment using a diagnostic tool validated in the oncology population. The detrimental effects of treatment in the heterogenous HNC population has been widely studied and, as a result, nutritional guidelines have been established . These gIn contrast to previous studies ,14, we dThe PG-SGA is the recommended tool for malnutrition assessment in radiotherapy patients in Australia . It has Differences in treatment modality may be an additional explanation for the difference between the results of our study and previous studies. Concurrent chemotherapy administration and intensity of RT are known to worsen nutritional outcomes ,32. WhilWe did find evidence of more severe weight loss at 3 months post treatment in HPV-positive OPSCC patients suggesting a slower recovery for these patients. This more severe weight loss was unlikely due to worse toxicities as patient-reported symptoms were similar for HPV-positive and negative patients overall and unplanned admissions, an indirect measure of treatment toxicity, were predominately seen during treatment. HPV-positive patients did have significantly higher BMI at the beginning and end of treatment which may have lessened both patients’ and clinicians’ concern regarding weight loss and reduced likelihood of adherence to nutritional guidelines. Future research is needed to confirm this potential slower recovery post treatment in HPV-positive patients and the reasons for this so that the appropriateness of current recommendations for nutritional and supportive care post-treatment for HPV-positive patients can be investigated. This is especially important given that the frequency of follow up recommended for HNC patients is based on patients undergoing radiotherapy alone and current best practice treatment for this patient group has the added detrimental impact on nutrition associated with chemotherapy treatment . Given tPrevious studies ,15 suggeThe significant difference in unplanned admissions is interesting. It may be an indirect marker for clinician-reported treatment toxicity given patients are usually admitted for management of side effects. This is supported by previous evidence that clinician-reported toxicities are worse for HPV-positive patients though this same study reported no difference in unplanned admissions . Given wn = 70 vs. n = 13). The study had 90% power to find a large effect (Cohen’s d = 0.8), 53% power to find a medium effect (d = 0.5) and only 12% power to find a small effect (d = 0.2). As such, non-significant findings should be viewed in the context of this sample not being powered to find smaller effect sizes and the subsequent higher risk of Type 2 error. Further research should address this limitation.The higher proportion of HPV-positive patients than HPV-negative patients in the OPSCC population is a limitation of this and earlier studies. The difference in sample size between groups could explain the lack of statistical significance found. This study was a post-hoc sub-study of a previous work which meant the sample is limited and a prospective sample size calculation was not conducted. The sample, while not small, was not balanced (The current study was conducted in tertiary treatment centers with well-established multidisciplinary teams, so findings may not be generalizable to smaller treatment centers. There may have been individual differences in nutritional intervention as nutritional interventions were undertaken by different clinicians. As mentioned in the introduction, bimodal detection of HPV status is now recommended and this was not available in this study which could potentially overestimate positive HPV cases.In this first study of longitudinal change of nutritional status in relation to HPV status in OPSCC using validated measures, two main outcomes are seen. Firstly, we found the impact of treatment on HPV-positive patients’ nutritional status, whose baseline nutrition was superior, was not significantly different by the final week of RT treatment to that of HPV-negative patients and this continued into the post-treatment period. Secondly, HPV-positive patients had significantly greater patients with >10% LOW at 3 months post treatment and significantly more unplanned admissions. We found similar rates of malnutrition were associated with similar % weight loss, depression scores, QOL and reactive NGT rates. Our results differ to other studies in that HPV-positive patients did not have worse nutritional outcomes by the end of treatment. This is likely due to difference in research methodology and potentially due to better adherence to guidelines for HPV-positive patients in our study. Nevertheless, the nutritional status of HPV positive and negative patients were both negatively affected by treatment and require similarly intense nutritional intervention during treatment. In acute recovery, HPV positive patients may require more intense intervention. At 3-months post treatment, both groups still showed nutritional symptoms that require nutritional intervention so ongoing nutritional support is essential. Further prospective research with a larger sample size is needed to confirm the results found in this study and could include sociological and psychological exploration with a focus on interventions to improve adherence to nutritional guidelines and support to help manage patient expectations. The superior survival outcomes for HPV-positive patients mean these patients are living longer so a study of long-term effects of treatment on nutritional status and contributing factors along with appropriate nutritional intervention is warranted.


Metabolomic analyses of human gut microbiome samples can unveil the metabolic potential of host tissues and the numerous microorganisms they support, concurrently. As such, metabolomic information bears immense potential to improve disease diagnosis and therapeutic drug discovery. Unfortunately, as cohort sizes increase, comprehensive metabolomic profiling becomes costly and logistically difficult to perform at a large scale. To address these difficulties, we tested the feasibility of predicting the metabolites of a microbial community based solely on microbiome sequencing data. Paired microbiome sequencing  and metabolome (mass spectrometry and nuclear magnetic resonance spectroscopy) datasets were collected from six independent studies spanning multiple diseases. We used these datasets to evaluate two reference-based gene-to-metabolite prediction pipelines and a machine-learning (ML) based metabolic profile prediction approach. With the pre-trained model on over 900 microbiome-metabolome paired samples, the ML approach yielded the most accurate predictions  of metabolite occurrences in the human gut and outperformed reference-based pipelines in predicting differential metabolites between case and control subjects. Our findings demonstrate the possibility of predicting metabolites from microbiome sequencing data, while highlighting certain limitations in detecting differential metabolites, and provide a framework to evaluate metabolite prediction pipelines, which will ultimately facilitate future investigations on microbial metabolites and human health. The scientific community has only recently begun to realize and fully appreciate the significant role of the microbiome in human health . Increasin silico means of predicting metabolic capacity from nucleic acid sequences would embolden drug discovery by generating testable hypotheses sans cost-prohibitive upstream metabolome profiling analyses.Untargeted metabolomic analyses greatly facilitate the detection and characterization of a wide range of metabolites, affording researchers a comprehensive understanding of the metabolic pathways invoked within a microbial community. Such techniques have bolstered and accelerated mechanistic studies and biomarker identification strategies across a variety of diseases . With maRecent years have seen advances in linking microbiome sequencing data to metabolome data. One such strategy relies on the network of connections linking a given gene to reactions and compounds in a database. These linkages are used to infer molecular compound identities from the genetic information housed within a microbial community. A method called predicted relative metabolomic turnover (PRMT) was used to predict metabolites from a coastal marine metagenomics dataset, and the predicted metabolites correlated strongly with environmental factors . MIMOSA Via MelonnPan, a model trained from paired microbiome and metabolome datasets can be used to predict metabolites from a novel microbiome dataset san a priori knowledge regarding relationships between genes and metabolites. This approach circumvents the limitations of the reference-based methods discussed above and has been used to generate promising results between two inflammatory bowel disease (IBD) cohorts  consisted of samples other than stool or intestinal tissue, (4) had microbiome data other than NGS sequencing, (5) had targeted metabolomics data, and (6) had fewer than 30 microbiome-metabolome paired samples. Notably, as different metabolome generation and processing methods have significant impact on results, we only selected studies which applied untargeted metabolome profiling with multiple liquid chromatography-mass spectrometry (LC-MS) methods or nuclear magnetic resonance (NMR) for a comprehensive view of the metabolite pool. Eighteen studies were retained and six were used in this evaluation due to data accessibility  using USEARCH (de novo OTU clustering) and determined a representative consensus sequence per de novo OTU. Piphillin (Raw sequencing data were either downloaded from NCBI Sequence Read Archive (SRA) for public studies or generated in-house using the Illumina platform . We anal at 0.97 . For 16S USEARCH . We assiiphillin  was thenFor metagenomic and metatranscriptomics reads, adapter sequences and low-quality ends were trimmed with Trimmomatic <Q20; . We then1, and the Chemical Translation Service2 were used to identify and convert metabolites.Detailed LC-MS/NMR conditions, software and library databases for metabolite data generation per each study are listed in A relationship table linking KEGG orthologs (KO) and BioCyc reactions (RXNs) to the compounds housed in KEGG (release date 2019 May) and BioCyc (version 23.0) databases was compiled. Specifically, if KOs have corresponding KEGG reactions which produce or consume KEGG compounds, we directly linked those KOs to KEGG compounds. An example is that K24443 corresponds to two reactions R02428, R02526, which produce or consume compounds C02753, C00001, C00502, C01114, and C00545. Thus, K24443 is linked to those compounds. There are cases when KOs do not have corresponding reactions but KEGG Enzyme numbers, and we would link those enzymes to reactions and further to compounds. An example is that K00046 has no corresponding reactions but the enzyme number EC:1.1.1.69, through which we linked to reactions R01738, R01740 and the reactions produce or consume compounds C00257, C00003, C01062, C00004, C00080, C00006, C00005, thus those compounds are linked to K00046. Manual effort was also made to link transporter KOs (BRITE class: ko02000) to their associated compounds. For the BioCyc database, RXNs and their associated compounds were inferred from “core_description_of_RXNs” directly, which included reactions from both MetaCyc and all PGDBs in BioCyc. This collection of reactions is referred to as “BioCyc reactions” in the remainder of the text. Reaction directionality was not taken into consideration when constructing the relationship table because many biochemical reactions are reversible depending on the reactant and product concentrations.via DESeq2 analysis  between case and control samples 3, we updated the community metabolic network template to the May 2019 version of the KEGG database. Microbiome functional abundance data were normalized by means of MUSiCC using default settings . As the abundance matrix generated with MIMOSA includes both positive and negative values, we elected to apply Wilcoxon rank sum tests with Benjamini-Hochberg FDR correction . Wilcoxon signed rank tests were used to comparatively evaluate precision, recall, and F1 scores across pipelines and databases between paired datasets.Prevalence filtering was applied to measured metabolome data, as well as MIMOSA and MelonnPan predicted metabolome data, and metabolites detected or predicted in fewer than 5% of the samples were omitted from consideration. Data resulting from empirically measured- and MelonnPan predicted-profiles were log transformed after imputing zeros with the minimum non-zero value per metabolite. We then conducted Student’s Random sampling analysis was also performed and compared to the pipeline prediction. For coverage evaluation, the same number as the predicted metabolites were randomly selected from the in-house chemical dictionary and compared with the pipeline predicted results. For differential metabolite identification, gene labels were randomly shuffled prior to DESeq2 and used for prediction for Mangosteen. For MIMOSA and MelonnPan, predicted metabolite labels were shuffled in the predicted matrices, followed by the same procedure for differential metabolite identification. The results were further evaluated against the measured metabolites and performance was represented in precision, recall and F1 score. The procedure was repeated for 99 times.n = 648 from five studies), while a smaller subset (n = 83) arose from intestinal tissue samples of the CRC study. All samples were subjected to metabolomic analysis via either mass spectrometry  or NMR spectroscopy . For microbiome profiling, half of the datasets originate from 16S rRNA gene amplicon sequencing (four datasets), followed by shotgun metagenomic analysis (two datasets), and metatranscriptomic sequencing (two datasets).Among the 6 studies that fit our selection criteria as described in n = 1,273) and NMR the least  identified the greatest number of metabolites . While Mangosteen-B predicted significantly fewer unique microbial functions than Mangosteen-K , it linkMetabolites bearing KEGG identifications were then assigned to their corresponding BRITE classifications . Melonnpp = 0.01) and overall F1 scoring  compared to its BioCyc-referenced counterpart. With respect to ML-based MelonnPan, these two reference databases used for model building performed equally well and yielded no significant differences in precision, recall, or F1 score. We also compared the prediction results to random sampling, which randomly predicted the same number of metabolites, and all pipelines showed significantly better performance regarding precision, recall and F1 score (p < 0.01).Predicted metabolite profiles were compared to empirically measured metabolite profiles. MelonnPan exhibited the greatest precision  in predicting the presence/absence of metabolites in the human gut, followed by MIMOSA (0.08) and Mangosteen , with squared m12 values  compared to random prediction while MIMOSA and MelonnPan did not .We evaluated these pipelines on their ability to identify differentially abundant metabolites between case and control groups as shown in a priori microbiome sequencing data affords researchers the ability to generate hypotheses in a rapid, cost-effective, and relatively reliable fashion. We systematically compared the ability of metabolite predictions by two published tools alongside a newly developed reference-based pipeline using more than 900 paired microbiome-metabolome stool/intestinal tissue samples from six different studies on various human diseases. Resulting metabolite profile predictions were compared to one another and contrasted alongside an empirically measured metabolome dataset, focusing primarily on occurrence  and identification of differentially abundant metabolites.Gaining insight into the metabolite pool of a microbial community is of paramount importance to understand its ecological role(s), not to mention its potential as a source of therapeutics and other invaluable molecules . As an avia these reference-based pipelines likely contributed to the elevated recall values. In addition, any given KO may participate in any number of distinct reactions, bearing the potential to dramatically increase the number of associated metabolites. With regard to reference-based methods, MIMOSA surpassed Mangosteen-K in both precision and F1 score. This speaks to the benefit of considering the directionality as well as the network of connections between reactions within a community. Although Mangosteen showed worse performance in metabolite prediction compared to MIMOSA, it was able to link more metabolites and could be useful for mining metabolites outside of known metabolic network. While the type of microbiome data examined also affects prediction performance, we lacked an appropriate number of independent datasets to evaluate this aspect in our study (one study for metagenomic to metatranscriptomic and another for 16S rRNA amplicon to metatranscriptomic sequencing comparisons). A pipeline’s ability to accurately distinguish differential metabolites between case and control groups is typically a sound foundation from which hypotheses are structured and biomarker and therapeutics discovery initiatives expound. Such evaluations are predicated upon accurate predictions of metabolite abundance, and as such, are much more stringent than occurrence prediction. While we did not observe promising results in any of the pipelines, MelonnPan predictions yielded the highest mean F1 scores (around 0.1 in both MelonnPan-K and MelonnPan-B).When used to predict the presence or absence of given metabolites within a community, the ML-based MelonnPan approach significantly outperformed its reference-based competitors, with respect to overall precision and F1 score. However, the reference-based Mangosteen and MIMOSA pipelines did exhibit high recall despite their lower precision and F1 scores. As recall is calculated based on true-positive and false-negative tallies, the high number of metabolites predicted Machine-learning-based MelonnPan outperformed the reference-based methods in predicting both occurrence and differential metabolites. In contrast to the reference-based pipelines, this powerful technique considers the impact of host metabolism and hitherto undiscovered interactions that do not exist in reference databases. Like all ML strategies, MelonnPan relies heavily on the accuracy of training data. As such, while untargeted metabolome profiling strategies generate the most comprehensive overview of the metabolites present within a sample, coverage is unavoidably limited due to the inherent technical biases associated with either MS or NMR . Hence, While conducting the differential metabolite evaluation, we observed the highest F1 score in the MelonnPan-predicted metagenomic dataset of via conventional untargeted metabolome profiling as well as ML-based prediction , either differential ones between case and control or any features the researchers are interested in. Ultimately, caution and common sense go a long way in interpreting the predicted metabolite profiles resulting from reference-based pipelines.As opposed to ML-based and data-driven approaches, reference-based methods can potentially identify/predict metabolites whose concentration or specific chemical properties precluded them from detection ediction . Referenediction  and BioCediction  are two ediction , but thiediction . There aThere are several limitations in the current evaluation that need to be taken into consideration when researchers try to choose the optimal prediction tool for their research. While we treated the empirically measured, untargeted metabolome as a “gold standard” and directly compared all pipeline prediction results to this profile, biases in technology render even this portrayal imperfect in its attempt to unveil the true and complete metabolite pool . In realPredicting metabolic capacity and metabolite diversity is of paramount consequence to better understanding and manipulating the function(s) of microbial communities, all of which bears immense significance to improving human health and empowering environmental microbiome research . UltimatPRJNA668188.Metadata, 16S rRNA gene amplicon and metatranscriptomic sequencing data generated in this study (SG_IBD) have been deposited in the NCBI database under BioProject ID The studies involving human participants were reviewed and approved by University of Calgary, Conjoint Health Research Ethics Board (ID:18142 and 14-2429). The patients/participants provided their written informed consent to participate in this study.SI, TD, and KD conceived and supervised the project. XY, TA, TD, and SI designed the study. XY, ER, KW, YW, JC, PB, and GK performed data collection and processing. XY conducted the analysis and wrote the first draft of the manuscript. All authors edited and approved the manuscript.XY, ER, KW, YW, JC, KD, TD, and SI are employees of Second Genome Inc., which provides salaries and stock options. Second Genome Inc. is an independent therapeutics company with products in development to treat intestinal disorders and other human diseases. TA is an employee of Altman Analytics LLC. The remaining 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.


Glioblastoma is the most common and lethal primary brain malignancy. Despite major investments in research into glioblastoma biology and drug development, treatment remains limited and survival has not substantially improved beyond 1–2 years. Cancer stem cells (CSC) or glioma stem cells (GSC) refer to a population of tumor originating cells capable of self-renewal and differentiation. While controversial and challenging to study, evidence suggests that GCSs may result in glioblastoma tumor recurrence and resistance to treatment. Multiple treatment strategies have been suggested at targeting GCSs, including immunotherapy, posttranscriptional regulation, modulation of the tumor microenvironment, and epigenetic modulation. In this review, we discuss recent advances in glioblastoma treatment specifically focused on targeting of GCSs as well as their potential integration into current clinical pathways and trials. Glioblastoma (GBM), a World Health Organization grade IV astrocytoma, is the most common primary brain malignancy with an incidence of 3.22:100,000 annually in the U.S , a small subpopulation of cells that are implicated in tumor invasiveness, recurrence, and chemo(radio)resistance. The GSC population remains challenging both to define empirically and treat. GSCs are described by their ability to self-renew and differentiate to reform the heterogeneity of GBM . MultiplGBM development originally was defined by two divergent but interconnected models, namely, the stochastic model and CSC model. The stochastic or clonal evolution model suggests that all cells have the equal capacity for undergoing transformation based on accumulated mutations and/or epigenetic changes that confer a survival benefit . The CSCin vitro has depended on multiple molecular mechanisms, genetics, epigenetics, cellular states, intrinsic cell stimuli, microenvironmental influences, and other factors  have not reproducibly supported the isolation of fully competent CSCs. Three functional tests that are considered the gold standard for validating CSCs are: 1) self-renewal, 2) tumor initiation upon transplantation, and 3) differentiation into distinct progeny that can recapitulate the initial tumor’s heterogeneity upon serial transplantation  utilized single cell sequencing technology to investigate 430 cells in each of 5 GBM tumors, and elucidated a stem-like population of cells which existed within a stemness gradient . Further6-methylguanine-DNA methyltransferase (MGMT) at levels 32–56 times those of CD133(−) cells  cells . The hig−) cells . By indu−) cells —TMZ may −) cells . Resista−) cells , and CD1otherapy . Hypoxicotherapy . GSC radotherapy  as well otherapy .Scientists have attempted to target GSCs through a multitude of avenues Figure 2via the immune system. However, GBMs are known to exert immunosuppressive effects systemically and in the tumor microenvironment through a combination of decreased immunogenicity and active suppression of T cells that exceeds the immunosuppressive capacity of non-stem glioma cells . ICD induces the translocation of calreticulin to the surface of the tumor cells surface, causing APCs to phagocytose tumor cells. ICD also promotes the release of HMGB1, encouraging dendritic cell maturation and tumor antigen presentation . RadiatiIn this section, we will discuss immunotherapy clinical trials which target GSC-specific and GSC-overexpressed targets with a focus on ongoing clinical trials Table 1.Passive immunotherapy utilizes antibodies to bind to oncomodulatory signaling molecules or target proteins on cancer cells and disrupt cellular function without producing a memory immune response in the patient. The most significant improvement in standard of care treatment in the United States recently has been the addition of the monoclonal antibody bevacizumab, or Avastin, which was granted accelerated approval by the FDA in 2009 . BevacizEpidermal growth factor receptor (EGFR) is molecular target overexpressed in GSCs which confers chemo- and radioresistance in GBM tumors, resulting in poorer outcomes among GBM patients . The EGFEGFR variant III (EGFRvIII) is a constitutively active mutated form of EGFR that is highly expressed in many GBM tumors . Though via cellular membrane damage. Jing et al. demonstrated that CD133-targeted NIR-PIT induced rapid cell death of CD133(+) GSCs in vitro and in orthotopic GSC tumor mouse models  and novel technologies have shown promise in bolstering the anti-GSC effects of passive immunotherapies. A bispecific antibody against CD133 and EGFRvIII was demonstrated to be highly cytotoxic against GSCs (but not NSCs) and significantly more effective in prolonging OS in mice as compared to CD133 or EGFRvIII mAbs alone . Though e models . ImportaWhile research into GSC-specific passive immunotherapy is sorely lacking, additional research is warranted given their demonstrated superiority to bulk tumor-targeting mAbs in preclinical applications. New antibodies which can eliminate chemo(radio)resistant GSC populations, such as one against anti-apoptotic protein CD47, are being developed constantly and warrant optimism . Given tvia two primary mechanisms: 1) promotion of a broad immunity against GSCs by GSC lysate-pulsed DCs and 2) activation of immunity against specific GSC antigens by synthetic peptide/RNA/mRNA-pulsed DCs.Both dendritic cell (DC) vaccines and antibody-mediated T cell immunotherapies rely on the activation of host immunity in order to target specific cancer cells. These approaches have demonstrated safety and efficacy for treatment of GBM in both preclinical and clinical trials . Given tPromoting immunity against GSC lysate trains the immune system against any antigens associated with GSCs. Murine models which utilize this technique have highlighted the potential for anti-GSC DC vaccines and have served as the basis for multiple clinical trials. Dendritic cells pulsed with GSC tumor lysate have shown to be highly effective at preventing viability of murine GBM tumors grown as both neurospheres (which preferences GSC growth) , and havOther groups are utilizing patients’ surgical specimens to culture tumor-initiating GSCs and train autologously derived dendritic cells (NCT03400917). Along with GSC lysate, it is also possible to extract mRNA from patient derived GSCs and produce personalized vaccines. One phase I trial, NCT00846456, demonstrated the safety of this approach as well as a nearly three-times longer PFS compared to matched controls .Much effort has been taken over the past three decades to define GSC specific peptides in order to decrease the risk for off-target effects, and numerous clinical studies are now assessing the effectiveness of DC vaccines which target peptides that are highly expressed in GSCs. One phase III clinical trial, NCT02546102, which was suspended in 2017 for inadequate funding showed significant promise in early phases. DC vaccines were pulsed with 6 synthetic peptides overexpressed in GSCs: HER2, TRP-2, gp100, MAGE-1, IL13R⍺2, and AIM-2 and this therapy was given in conjunction with standard of care chemo(radio)therapy. Results from phase I of the trial suggested a powerful therapeutic effect: median PFS in newly diagnosed patients treated with the vaccine was 16.9 months and OS was 38.4 months, noticeably exceeding historical standards . Five pain vitro causes an upregulation of CD133, Notch1, Sox2, Oct4, and Nestin, and promotes the growth of GBM neurospheres, suggesting that pp65 may play a role in stemness , is an interesting DC vaccine target. A large fraction of clinically isolated CD133(+) cells are found to be positive for pp65, and infection of GBM cells with HCMV stemness . The prestemness . RegardlResearchers at Duke University have a number of ongoing clinical trials investigating the effectiveness of a pp65 RNA-pulsed DC vaccine in combination with various other treatments. One addition being tested is that of anti-IL-2R⍺ antibodies which researchers hope will decrease Treg function and improve the penetration of DC vaccinations. Early research demonstrated that TMZ-induced lymphopenia enhances vaccine responses but dramatically upregulates T-reg function. IL-2R⍺ therefore diminishes the T-reg response allowing for a more robust anti-tumor effect . InteresIn a phase I trial (NCT00626483), the group combined a pp65 DC vaccine with basiliximab, another anti-IL-2R⍺ antibody . InitialAnother promising ongoing clinical trial is that of a trivalent GSC-targeting DC vaccine which is now in stage II/III (NCT03548571). Researchers at Oslo University Hospital are targeting GSCs by administering DC vaccines transfected with GSC mRNA along with the anti-apoptotic peptide survivin and human telomerase reverse transcriptase (hTERT). Both survivin and hTERT have been found to increase stemness in GBM and are expressed in high levels in GSCs , 87. In Monoclonal and bispecific antibodies are also being investigated for their ability to activate T cell immune responses against GSCs, and once again survivin is a promising target. SurVaxM, a survivin vaccine, is being tested in combination with TMZ and granulocyte-macrophage colony-stimulating factor in a phase II clinical trial (NCT02455557). Initial results warrant optimism: of 55 patients with newly diagnosed GBM treated with the SurVaxM vaccine concomitant with standard of care therapy, 96% were progression free at 6 months and 93% were alive at 12 months, a substantial improvement over historical controls of 43 and 41%, respectively . SurviviThe recombinant bispecific antibody AC133CD3 targets T cells and the CD133 epitope AC133, redirecting human polyclonal T cells to patient derived AC133(+) GSCs, inducing GSC lysis, and preventing the growth of subcutaneous GBM xenografts . In tandThe successes of endogenous and virus-associated GSC antigen-targeted therapies indicate that GSC antigens may represent promising targets for various therapy modalities. Caution must be taken, however, when considering early clinical victories, particularly with antibody-mediated T cell therapies. Some trials which demonstrated promise in phase I and II have failed in phase III as they could not demonstrate survival benefit. In the context of their established success in recurrent GBM treatment, DC vaccines are a promising GBM immunotherapy approach and preclinical and clinical results of GSC antigen-specific and GSC lysate DC vaccine approaches should motivate further investigation.ex vivo before autologous reintroduction. CTL-mediated GSC targeting has demonstrated promise in preclinical applications and small human cohorts, but has yet to be put to test in a large clinical trial. Chimeric antigen receptor (CAR) T cells, in contrast, are genetically engineered to exert their cytotoxic effects against specific antigens and have been extensively studied in the context of targeting tumor-associated antigens in various types of cancer. Though CAR T cell therapies have been successful treating blood cancers—two different therapies were approved by the FDA in 2017 for treatment of acute lymphoblastic leukemia  cell therapies are of growing interest for various cancers and are being utilized to target GSCs. Cytotoxic T lymphocytes (CTL) can be used to target tumor-associated antigens by being drawn from the patient, selected for their existing antitumor specificity, and expanded leukemia  and diffleukemia —they havin vitro and in mouse models and improved OS in treated mice.CAR T cells have been developed to target several GSC-specific antigens. In a preclinical application of CAR T cell therapies targeting GSCs, therapies have been developed against the CD133 epitope AC133 . These ANon-GSC-specific peptides, which are also upregulated in GSCs, have demonstrated some efficacy in killing GSC populations. IL13Rα2 , 96, EGFAdoptive immunotherapies cannot always be easily subcategorized. Some groups of researchers are investigating technologies which utilize both genetic modification  as well as selection of T cells based on reactivity to particular antigens . A group at Baylor has an ongoing clinical trial (NCT01109095) aiming to improve the efficacy of a human epidermal growth factor receptor 2 (HER2) CAR T cell therapy by selecting for cytotoxic T cells which recognize the human cytomegalovirus (HCMV) protein pp65. The group has previously demonstrated the efficacy of the HER2-targeted CAR T cells in eliminating GBM cells irrespective of CD133 expression . Their iin vitro and in a mouse model  which utilized pp65-trained CTLs in patients with GBM were stopped due to poor subject recruitment, though other groups are pursuing this approach with promising preliminary results. The Duke group pursuing multiple pp65 DC vaccination trials has also demonstrated that training T cells with pp65-pulsed DCs increases the polyfunctionality of CTLs in a cohort of 11 patients (NCT00693095), increasing OS (Some groups also are investigating the HCMV protein pp65 as a target for CTL-mediated oncolysis. In one study, HCMV pp65-specific CTLs were comparably cytotoxic against both GSCs and differentiated cells both se model . Here, aasing OS . Anotherasing OS . Unfortuasing OS .Another peptide which is being utilized for CTL-mediated cytolysis is that of SOX6, an immunogenic peptide is involved in inhibition of neuronal cell differentiation and neuronal stem cell maintenance , 107. ItMost adoptive GBM immunotherapy research has been devoted to CAR T cell and CTL therapies as early research suggested NK cells were ineffective against GBM –110. Howin vitro (Finally, CAR-NK cells have also been explored for their ability to selectively target and eradicate GSCs. In 2015, a group from The Ohio State University demonstrated that CAR-NK cells targeting both EGFR and EGFRvIII effectively killed patient-derived GSCs in vitro . They alPreclinical insights for NK and CTL therapies warrant some optimism, and clinical evidence that utilizing DCs to train CTLs increases the treatment modality’s effectiveness supports further research into this strategy. However, in the absence of clinical evidence which demonstrates an improvement in OS or PFS, hope for approaching advancements in adoptive GSC therapies lies primarily with CAR T cells. Even these advancements, though, will take patience. None of the three ongoing CAR T cell clinical trials which target GSCs have yet to reach phase III, and therefore clinical adoption is unlikely for a number of years.A broad range of viruses have been explored for treating high grade glioma , and theThe growing body of knowledge regarding GSCs’ role in oncogenesis and tumor recurrence necessitates reevaluation of conventional cancer assessment and treatment methods, as lasting remission will likely remain elusive for many GBM patients without the means of reliably detecting and extinguishing GSC populations. Methods of targeting GSCs vary widely, both in terms of the vector used to exert cytolytic effects on this cell population as well as the antigen or cellular pathway targeted by such vectors. Over the past several years, immunotherapy has emerged as a promising method of culling GSC populations in GBM tumors and has increased survival in GBM patients. Nevertheless, immunotherapies targeting malignancies of the immune-privileged central nervous system present a host of unique challenges.in vitro may stumble in in vivo and clinical application. Direct introduction of immunotherapeutic agents into the tumor resection cavity effectively bypasses the blood-brain barrier but runs the risk of causing inflammation in the brain due to a productive immune response. The paradoxical need for a robust immune response with limited inflammatory changes to kill GBM while preserving the patient highlights just one of the complexities of immunotherapy in the context of intracranial neoplasia. Dexamethasone, the current standard of care agent to treat GBM-associated edema, exerts global immunosuppression and may thereby limit the efficacy of some immunotherapies (Due to the relative inability of many immunotherapies to penetrate the blood-brain barrier and subsequently localize to GBM, modalities which show promise herapies . This unAnother hurdle for future immunotherapies to clear is the necessity that therapies be highly specific for glioma stem cells, avoiding antigens shared by healthy neural stem cells or other normal stem cell populations throughout the body, in order to mitigate the risk of adversely affecting somatic stem cell function. Even with highly targeted therapies, however, GBM tumors’ innate immunosuppressive effects can dampen the benefit of immunotherapies. Although aberrant cell growth results in immune recruitment through production of chemotactic factors, GBMs antagonize this process by secreting other chemokines which recruit T regulatory cells and suppress immune effector cells . In GBM,Though many immunotherapeutic approaches are currently being investigated, some have shown greater promise in clinical application than others. The use of static monotherapies such as single-target antibodies have been repeatedly demonstrated to be ineffective in the long term due to cellular adaptation by GSCs and, in some cases, even differentiated tumor cells. However, the success of bevacizumab in slowing GBM progression and improving quality of life indicates that passive immunotherapies might be viable adjuncts to more aggressive chemo- or immunotherapeutic approaches.Further, research into the interaction between GSCs and the tumor microenvironment has shown us that stem cell phenotypes vary significantly throughout the tumor and that subpopulations of GSCs may be differentially susceptible to immunotherapeutic approaches . These sKP, LD, MK, and CC all contributed to the writing and editing of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Ben & Catherine Ivy Center for Advanced Brain Tumor Treatment General Fund.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.