Rationale: The endemic of peri-implantitis affects over 25% of dental implants. Current treatment depends on empirical patient and site-based stratifications and lacks a consistent risk grading system.Methods: We investigated a unique cohort of peri-implantitis patients undergoing regenerative therapy with comprehensive clinical, immune, and microbial profiling. We utilized a robust outlier-resistant machine learning algorithm for immune deconvolution.Results: Unsupervised clustering identified risk groups with distinct immune profiles, microbial colonization dynamics, and regenerative outcomes. Low-risk patients exhibited elevated M1/M2-like macrophage ratios and lower B-cell infiltration. The low-risk immune profile was characterized by enhanced complement signaling and higher levels of Th1 and Th17 cytokines. Fusobacterium nucleatum and Prevotella intermedia were significantly enriched in high-risk individuals. Although surgery reduced microbial burden at the peri-implant interface in all groups, only low-risk individuals exhibited suppression of keystone pathogen re-colonization.Conclusion: Peri-implant immune microenvironment shapes microbial composition and the course of regeneration. Immune signatures show untapped potential in improving the risk-grading for peri-implantitis. Dental implants are biocompatible medical devices that support the restoration of missing teeth. Dental implants-supported crowns offer esthetic, functional, and natural-feeling replacement options whose market expenditure is estimated to reach $6.8 billion USD by 2024. Despite how dental implants have transformed the reconstructive options, the emerging endemic of peri-implantitis has severely compromised the long-term success of implant dentistry with nearly 25% of all patients receiving implants experiencing some forms of peri-implantitis Fusobacterium nucleatum, Treponema denticola, and Porphyromonas gingivalisHowever, the use of clinical criteria alone is insufficient for the risk assessment and grading of peri-implantitis, which limits the design of an optimal preventive recall schedule and the risk stratification for treatment planning. Several systematic reviews with meta-analyses show that the outcomes of peri-implantitis therapies are highly variable and unpredictable with the current tools H1 populations are often seen in early or stable periodontal diseases; and TH2 populations are more often identified during disease progression As inflammatory responses wax and wane, an array of immune cells participates in shaping the peri-implant immune microenvironment A common challenge in profiling peri-implant infiltrating leukocytes (PILs) in clinical specimens is the rapid reproducible rendering of the peri-implant immune landscape. Immunohistochemical (IHC) staining of immune markers offers a convenient tool for immune subsets quantitation. However, there are two important caveats that limit its value. First, IHC can only stain a small panel of markers, which are insufficient to reconstruct the composition of multiple immune subsets simultaneously. Second, there is considerable inter-observer and inter-institutional variation in antibody clone selection, quantitation method, and cutoff calling. Thus, the introduction of immune deconvolution offers an unprecedented opportunity to interrogate the immune profiles based on signature gene matrices Fast And Robust DEconvolution of Expression Profiles (FARDEEP), to ensure the highest accuracy in immune subset calling in silico simulations in datasets with different levels of Gaussian or heavy-tailed noise, we showed that FARDEEP consistently outperforms other methods including a robust method CIBERSORT A main challenge for transcriptome-driven immune deconvolution is that whole tissue RNA-Seq frequently contains data outliers, which substantially reduce estimation accuracy. Therefore, we engineered a novel robust pipeline, + T-cells, CD8+ T-cells, regulatory T-cells (Tregs), natural killer cells (NKs), dendritic cells (DCs), monocytes, neutrophils, M1-like macrophages, M2-like macrophages, and B-cells. We treated the absolute quantities of each immune subset as features for each patient and then performed hierarchical clustering using the R package (cluster) to group individuals with a similar immune composition at the peri-implant site. The cluster package measured the immune cell abundance differences between patients and clustered patients with similar profiles. Then, we utilized the dendrogram to separate patients into three risk groups  cytokine and chemokine levels were measured by Luminex Multiplex assays. The peri-implant microbiome at each follow-up visit was sampled by paper points and 16S rRNA sequencing  to identify significant signaling pathways that associates with the favorable treatment outcome. Utilizing the annotation profiles published by the Broad Institute, we discovered four critical and novel pathways, including coagulation/complement pathways, inflammatory pathways, epithelial-mesenchymal transition pathway, and K-RAS signaling B. In agrOribacterium was preferentially identified in the low-risk group. The high- risk group show significantly elevated total abundance of the microbes belonging to the genera Eggerthia and Rikenellaceae. In addition, the high-risk group shows significantly higher levels of microbes in the Anaerovoracaceae and Erysipelatoclostridiaceae families  scores. Porphyromonas-like species were also preferentially identified in the high-risk group. Microbes belonging to the genera Desulfobulbus, Gemella, Pseudomonas, Chloroplast, and Filifactor were enriched in the intermediate-risk group , an intra-sulcular incision with full thickness flap reflection was performed to allow optimal access to the peri-implant bone defects. Granulation tissue was carefully procured using curettes  and placed immediately into RNAlater solution, completely submerged and stored at 4°C until the end of the procedure. The tissue was subsequently stored at -20°C until needed for further processing. The implant surface was mechanically debrided with metal curettes and rotary titanium brushes . Then, the supracrestal portion of the exposed implant surface was treated with implantoplasty. Er:YAG laser  was used to decontaminate implant surface in half of the cases. After decontamination, a composite bone allograft  was used to fill the infrabony defect and a dermal matrix membrane  was placed to cover the graft and a few millimeters of healthy bone. Finally, tissues were securely sutured with modified horizontal mattress using PTFE sutures (Cytoplast). The treated area was protected with a non-eugenol surgical dressing and the patients were dismissed with the prescriptions for antibiotic regimen (amoxicillin 500mg tid/week), pain medication (ibuprofen 600mg prn pain every 6 h) and oral rinsing with chlorhexidine gluconate (0.12%) for 2 weeks. Sutures were removed after 2 and 4 weeks.PICF samples and bacterial plaque were collected as previously described Escherichia coli was used as standard. The sample was run on quantitative PCR in triplicate. The PCR reaction was carried out with an initial holding stage of 50 °C for 2 min followed by 95 °C for 10 min. The cycling stage consisted of 40 cycles of 95 °C for 15 s, followed by 60 °C for 1 min.Patients' bacterial samples were collected by paper points at baseline, 2-week, 4-week, 3-month, and 6-month follow-up visits. DNA was extracted from the paper points with DNeasy PowerSoil Kit (Qiagen) and the qPCR reaction was performed using Taqman® Master Mix (Applied Biosystems) in StepOnePlus Real-Time PCR System (Applied Biosystems). Bac2F and Bac2R primers and corresponding Bac2 probe were used for the reaction. DNA from Regarding 16S rRNA paired-end sequencing, the sequence reads were analyzed using QIIME2 pipeline (core 2019.7 distribution), in which the denoising algorithm DADA2 was implemented to filter low quality sequences + T cells, CD8+ T cells, regulatory T cells, NK cells, dendritic cells, monocytes, neutrophils, M1-like macrophages, M2-like macrophages, and B-cells by employing the FARDEEP pipeline with quanTIseq signature matrix https://github.com/YuningHao/FARDEEP.git. PIL landscape estimates from FARDEEP were used for hierarchical clustering to separate patients into three risk groups. The hierarchical clustering algorithm was implemented in R using package stats based on the Euclidean distance of PILs between the 24 patients.Peri-implant granulation tissues of all 24 patients was collected and subjected to RNA-Seq for transcriptome profiling. Raw sequencing files are available at the NCBI Sequence Read Archive (SRA) with an accession #: PRJNA667664. RNA-seq data were then analyzed using R package Salmon to quantify gene expression in terms of Transcripts Per Million (TPM). The resulting gene expression data were used to estimate the abundance of ten PILs including CD4In order to characterize novel genetic features monotonically associated with different immune risk groups, Kruskal-Wallis test using R package dplyr were used to identify genes significantly associated with risk groups . Among those significant genes, Dunn post hoc test using R package dunn.test was used to select genes monotonically increasing or decreasing among the three groups. GSEA was performed on the list of genes significantly correlated with immune base risk groups. The genes ranked by the p-value from Dunn post hoc test were analyzed by GSEA (v4.0.3) with GseaPreranked analysis, and the significant gene sets were retrieved from the hallmark gene sets. Kruskal-Wallis rank sum test was performed to compare the probing depth, gene expressions, and immune subsets among different immune based risk groups. Patients were divided into three groups according to PICF cytokine and chemokine levels, which were used to test for the association with probing depth using Kruskal-Wallis rank sum test.The clinical protocol to obtain per-implant tissue specimens was approved by the University of Michigan Institutional Review Board (HUM00181139 and HUM00124386), with informed consent obtained from all patients.Supplementary figures and tables.Click here for additional data file.


Understanding SARS-CoV-2 transmission dynamics is critical for controlling and preventing outbreaks. The genomic epidemiology of SARS-CoV-2 on college campuses has not been comprehensively studied, and the extent to which campus-associated outbreaks lead to transmission in nearby communities is unclear. We used high-density genomic surveillance to track SARS-CoV-2 transmission across the University of Michigan-Ann Arbor campus and Washtenaw County during the Fall 2020 semester.th – November 25th, 2020 (n = 2245). We extracted viral RNA, amplified the SARS-CoV-2 genome by multiplex RT-PCR, and sequenced these amplicons on an Illumina MiSeq. We applied maximum likelihood phylogenetic analysis to whole genome sequences to define and characterize transmission lineages.We retrieved all available residual diagnostic specimens from the Michigan Medicine Clinical Microbiology Laboratory and University Health Service that were positive for SARS-CoV-2 from August 16We assembled complete viral genomes from 1659 individual infections, representing roughly 25% of confirmed cases in Washtenaw County across the fall semester. Of these cases, 468 were University of Michigan students. Phylogenetic analysis revealed 203 genetically distinct introductions of SARS-CoV-2 into the student population, most of which were singletons (n = 171) or small clusters of 2 – 8 students. We identified two large SARS-CoV-2 transmission lineages , including individuals from multiple on-campus residences. Viral descendants of these student outbreaks were rare, constituting less than 4% of cases in the community.We identified many SARS-CoV-2 transmission introductions into the University of Michigan campus in Fall 2020. While there was widespread transmission among students, there is little evidence that these outbreaks significantly contributed to the rise in COVID-19 cases that Washtenaw County experienced in November 2020.Adam Lauring, MD, PhD, Roche (Advisor or Review Panel member) Sanofi (Consultant)


An 84-year-old Japanese woman presented to our hospital with a month-long dry cough during the coronavirus disease 2019 (COVID-19) pandemic. She also had skin lesions on her fingers from 3 months prior. A chest computed tomography (CT) scan showed bilateral ground- glass opacities with a subpleural distribution, similar to the findings of COVID-19. The results of COVID-19 tests were negative. The titer of the anti-melanoma differentiation-associated gene 5 (MDA5) antibody was elevated. Consequently, we confirmed the diagnosis of clinically amyopathic dermatomyositis (CADM) and then administered oral prednisolone combined with tacrolimus. After the treatment, her symptoms, skin lesions and CT findings were gradually resolved. An 84-year-old Japanese woman presented to our hospital with a month-long dry cough during the coronavirus disease 2019 (COVID-19) pandemic. She also had skin lesions on her fingers from 3 months prior; she attributed the rash to frequent hand washing and disinfection with alcohol for the prevention of COVID-19.Physical examination revealed fine bilateral crackles on her chest, palmar papules on her fingers  and then administered oral prednisolone combined with tacrolimus. After the treatment, her symptoms, skin lesions and CT findings were gradually resolved.There are many diseases that affect both the lung and the skin including infection, collagen vascular disease and sarcoidosis . It is cTherefore, it is more critical than ever to diagnose CADM comprehensively using various approaches, including tissue biopsy.


Neuroimaging has transformed the way brain tumors are diagnosed and treated. Although different non-invasive modalities provide very helpful information, in some situations, they present a limited value. By merging the specificity of antibodies with the resolution, sensitivity, and quantitative capabilities of positron emission tomography (PET), “Immuno-PET” allows us to conduct the non-invasive diagnosis and monitoring of patients over time using antibody-based probes as an in vivo, integrated, quantifiable, 3D, full-body “immunohistochemistry”, like a “virtual biopsy”. This review provides and focuses on immuno-PET applications and future perspectives of this promising imaging approach for glioblastoma.Neuroimaging has transformed neuro-oncology and the way that glioblastoma is diagnosed and treated. Magnetic Resonance Imaging (MRI) is the most widely used non-invasive technique in the primary diagnosis of glioblastoma. Although MRI provides very powerful anatomical information, it has proven to be of limited value for diagnosing glioblastomas in some situations. The final diagnosis requires a brain biopsy that may not depict the high intratumoral heterogeneity present in this tumor type. The revolution in “cancer-omics” is transforming the molecular classification of gliomas. However, many of the clinically relevant alterations revealed by these studies have not yet been integrated into the clinical management of patients, in part due to the lack of non-invasive biomarker-based imaging tools. An innovative option for biomarker identification in vivo is termed “immunotargeted imaging”. By merging the high target specificity of antibodies with the high spatial resolution, sensitivity, and quantitative capabilities of positron emission tomography (PET), “Immuno-PET” allows us to conduct the non-invasive diagnosis and monitoring of patients over time using antibody-based probes as an in vivo, integrated, quantifiable, 3D, full-body “immunohistochemistry” in patients. This review provides the state of the art of immuno-PET applications and future perspectives on this imaging approach for glioblastoma. Glioblastoma is the most common and aggressive tumor of the central nervous system in adults . With anThe current standard-of-care for treatment of newly diagnosed glioblastoma has remained relatively unchanged since 2005 and consists of maximal safe resection followed by concomitant chemoradiation with the alkylating agent temozolomide (TMZ), and subsequent adjuvant TMZ .6-methylguanine-DNA methyltransferase (MGMT) impairs the killing of tumor cells by alkylating agents chemotherapy fluoro-2-deoxy-D-glucose ([18F]FDG), a fluorine-18 glucose analog [11C]methionine ([11C]MET), [18F]fluoroethyl-tyrosine ([18F]FET), [18F]L-fluoro-dihydroxyphenylalanine ([18F]FDOPA), or [18F]fluoro-thymidine ([18F]FLT), that show high diagnostic performance [11C]choline or [18F]choline, as tumor cells increase choline uptake since they experience high phospholipid turnover in their membranes [11C]choline can be used for tumor monitoring, as it presents high diagnostic accuracy for the differentiation of glioma relapses from radiation-induced necrosis [18F]choline could be potentially used as a surgical imaging biomarker, as it was proven helpful for the discrimination of the highly proliferative peripheral area of the tumor and intraparenchymal hemorrhage FDG is ineffective for diagnosing gliomas due to the high glucose metabolism in the normal brain, which results in suboptimal tumor detection and delineation, especially upon treatment Fluoromisoinodazole ([18F]FMISO), which can be used to visualize hypoxia [18F]FMISO in glioma imaging is limited for its low sensitivity differentiating normoxic and hypoxic tissue and its low BBB permeability Zr-DFO-fresolimumab, an immuno-PET tracer based on a monoclonal antibody that can neutralize all mammalian isoforms of TGF-β, was assayed in humans and penetrated recurrent high-grade gliomas  and the blood–cerebrospinal fluid (CSF) barrier (BCSFB) . These dCamelidae  In-DTPA-pa2H [111In]In-DTPA-pa2H-Fc Ga-NOTA-anti-HER2 VHH1) has demonstrated its efficient diagnosis of primary breast carcinoma patients by PET/CT in a phase I study [99mTc labeled anti-PD-L1 nanobody ([99mTc]Tc-NM-01) in non-small cell lung cancer patients by SPECT/CT imaging Zr-DFO-fresolimumab, an immuno-PET tracer based on a monoclonal antibody that can neutralize all mammalian isoforms of TGF-β was assayed in humans, penetrated recurrent high-grade gliomas but did not result in clinical benefit [To date, several immuno-PET imaging tracers have been designed to target glioblastoma and have already proven successful in detecting gliomas in multiple preclinical models. These tracers target membrane proteins whose expression is altered in glioblastoma  ,106,107: benefit . Other i benefit ,111,120  benefit ,114,125.—- or α-emitter) [Targeted-radionuclide therapy is a strategy for the treatment of glioblastoma. This nuclear medicine approach enables the visualization of molecular biomarkers and pathways on a subcellular level using a biochemical vector coupled to a radionuclide that could work either for diagnosis (positron- or gamma-emitter) or for therapy  ; when themitter) .The administration of targeted-radionuclide therapy requires to confirm the presence of the glioblastoma target to determine on treatment options. To this end, immuno-PET emerges an opportunity as a possible quantitative imaging procedure to investigate the different biological properties and pharmacokinetics of tumor-targeted radiolabeled macromolecules including antibody fragments or engineered antibodies.In addition to antibodies, multiple molecules  have been designed to target specific biomarkers in the gliomas to develop probes of interest that can now be non-invasively imaged with multimodality molecular imaging techniques including MRI, CT, PET, single-photon emission computed tomography (SPECT), bioluminescence imaging, and near-infrared fluorescence to guide targeted therapies with a potential survival benefit and monitor patients’ response .BBB permeability to the antibody-based probes remains a hurdle for immuno-PET applications. Glioblastomas are highly infiltrative and frequently alter the integrity of the BBB, resulting in leakiness, even though all glioblastomas may have clinically significant regions with an intact BBB . These iThe development of immuno-PET probes based on antibody subunits targeting glioblastoma biomarkers can overcome BBB selectivity emerging as promising probes for the non-invasive diagnosis of gliomas . Among tVarious molecular mechanisms for the transportation of nanobodies through the Blood–Brain Barrier (BBB) have been extensively described and include adsorptive and receptor-mediated transcytosis, somatic gene transfer, and the use of carriers or shuttles such as cell-penetrating peptides, extracellular vesicles, liposomes, and nanoparticles as well as device-based and physicochemical disruption of the BBB ,145,146.The development of nanobody-based radiotracers for the non-invasive diagnosis of glioblastoma by immuno-PET may also involve some potential challenges. First, suboptimal imaging of glioblastoma may be achieved due to the low penetration of nanobodies through the BBB , althougFurthermore, the production of nanobodies in the current good-manufacturing-practice (cGMP) grade is essential for their application in clinics. cGMP includes meeting preclinical quality standards, validating the nanobody format without tags utilized for production  and the 68Ga or 44Sc, which can be produced in a generator [Recently, a bivalent nanobody for the treatment of patients suffering from thrombotic thrombocytopenic purpura  ,169 receenerator ,80,81, mIn contrast to a reduction in tumor size as observed in MRI, which usually represents late treatment effects, biomarker changes can occur earlier. Immuno-PET allows the quantification of biomarkers in a non-invasive manner in the whole body and holds the potential of detecting functional glioblastoma biomarker changes helping to an earlier diagnosis of glioblastoma, surveillance of patients, and monitoring of treatment response.The current diagnosis of glioblastoma by MRI in some situations does not allow proper discrimination of the tumoral tissue from concurrent processes and can be confounding with other lesions and post-therapeutic effects .Immuno-PET represents an attractive and innovative option for diagnosing gliomas, allowing the analysis of biomarkers in a non-invasive manner. By merging the target selectivity and specificity of antibodies and derivatives toward a biomarker with the high sensitivity, spatial resolution, and quantitative capabilities of PET , allowinTo date, several immuno-PET imaging tracers have been designed to target glioblastoma and have already proven successful in detecting gliomas in multiple preclinical models, and they are advancing through clinical trials. The development of immuno-PET probes based on antibodies and nanobodies can overcome BBB selectivity emerging as promising probes for the non-invasive diagnosis, surveillance of patients, and monitoring of treatment response of gliomas .


Anti-CD20 antibodies such as rituximab proved powerful yet remained as an off-labeltreatment in multiple sclerosis (MS) until the approval of ocrelizumab in 2018. Thedrug-free intervals between two courses of B-cell depletion are long, as treatment efficacyis determined by prolonged immunosuppression, which can eventually be assessed by peripheralB-cell reconstitution. This provides the opportunity to individually delay therapy, known asextended interval dosing (EID), which is likely to be beneficial in view of safety concerns.Specifically, aside from frequent infusion reactions, continuous B-cell depletion might beassociated with long-term immunological complications, such as an increased risk ofmalignancy or hypogammaglobulinemia. The latter, in turn, is likely to result in higherinfection rates and reduced vaccine efficacy. Finally, EID may also provide sufficient timefor a drug-free pregnancy, while under ongoing protection from disease activity.+ repopulation took longer than one year. Although the potential of anti-CD20 antibodies to deplete B-cell subsets has yet tobe fully elucidated, both agents further deplete memory B-cells, which can last for years after treatment. Marked depletion of memory B-cells alsoappears to be a common feature contributing to the efficacy of so-called “immunereconstitution therapies” (IRT), which show long-term efficacy after short treatmentcycles.In light of notable recent studies, a number of mechanistic features argue in favor ofdurable efficacy beyond the standard 6-monthly dosing; only ~20% of patients treated withrituximab and even fewer (~5%) with ocrelizumab began to repopulate by 6 months andCD19 There were no differences between both groups in terms of relapses, confirmedprogression of disease or NEDA-3 status, suggesting that EID does not affect efficacy, atleast after short-term evaluation. Similar findings were observed by Baker et al. for longer follow-up periods. Specifically, using data from the open-label, phase IIocrelizumab extension trial, they demonstrated that 12 to 18 months after the last infusionfollowing three cycles of ocrelizumab, the levels of disease activity appear to be similarto that seen in the phase III extension studies following six cycles of ocrelizumab. Likewise, smaller real-world studies supported longer treatment-free intervals ofocrelizumab in MS without lack of efficacy. In line with this, the phase I extension study of rituximab in MS reported amaintained benefit 12 months after the last infusion. Moreover, off-label studies with rituximab, where treatment was halted, demonstrateda long-acting benefit and an absence of rebound disease activity after stoppingtherapy.7Several studies showed that EID between two infusions of rituximab or ocrelizumab in MS isassociated with a low risk of disease activity. A recently published large multicenter studycompared ocrelizumab-treated MS patients on EID with a control group on standard intervaldosing, 3 months after the last treatment cycle. In addition, it has recently been indicated that delaying anti-CD20 infusions by 3 to6 months increases the likelihood of developing an adequate humoral response to COVID-19 vaccination.Regarding safety concerns, one study showed that there were fewer overall adverse eventsand infections in the EID cohorts.Taken together, these findings make a convincing argument that B-cell-depleting agentsinduce durable inhibition of relapsing disease. Consequently, it is likely that efficacy canbe maintained by reducing the frequency of dosing, while limiting infections and other risksassociated with continued immunosuppression and allowing for more effective vaccination.+ B-cells may be a sufficient guide for individualized decision-makingregarding reinfusion intervals. Indeed, studies investigating rituximab in other autoimmuneconditions have indicated that low levels of CD19+ B-cells serve as a surrogatemarker to justify delaying B-cell-depleting infusions. However, evidence of personalized,B-cell-tailored EID in MS patients remains controversial: Although EID of anti-CD20antibodies represented a predictor for repopulation of CD19+ B-cells in severalstudies,9 only one study showed that the therapeuticeffect was closely associated with the absence of CD19+ B-cells. In contrast, no association between the absolute CD19+ B-cell number andre-emerging disease activity was evident in other studies,7 suggesting that the effects of rituximaband ocrelizumab in MS are maintained after CD19+ B-cell repopulation.It has been repeatedly hypothesized that continuous monitoring of peripheralCD19+CD27+ memory B-cells was shown to be more reliable in the prediction ofclinical activity. Indeed, a preliminary study with rituximab suggests that dosing accordingto memory B-cell population kinetics reduces dosing frequency while maintaining efficacy in MS. Monitoring of peripheral B memory cells further demonstrated biomarker activity inother autoimmune diseases, including neuromyelitis optica and myasthenia gravis.Of note, a more detailed assessment of B-cells in the periphery including CD19Overall, these findings indicate that EID can be performed without a detrimental impact oneffectiveness, and while improving patient safety. Although the complex mechanismsunderlying the observed effects remain unclear, the findings suggest that anti-CD20antibodies likely possess features of an IRT.A major challenge for MS research now is to elucidate the exact impact of anti-CD20antibodies on the immune system to determine surrogate markers for individualtime-to-infusion decision-making. In this context, the memory B-cell repopulation rateappears to be a promising candidate to appraise individually adapted therapy intervals.Thus, we should further concentrate our research efforts on head-to-head studies ofanti-CD20 therapy with extended doses compared to the current standard, includingimmunophenotyping analysis. Moreover, future prospective studies should investigate thelong-term impact of continuous EID in terms of clinical outcomes and patient safety.If the promising durable effect of CD20 depletion is confirmed, ocrelizumab would have agreater utility in the treatment of MS due to relatively low side effects and limited needfor monitoring compared to other highly effective therapies. However, even if ocrelizumabrequires repeated treatments, a lower dosing frequency is beneficial as it lessens thelikelihood of infusion-related events and the development of severe infections.


OBJECTIVES/GOALS: Normal fear learning produces avoidance behavior that promotes survival, but excessive and persistent fear after trauma can lead to development of phobias and post-traumatic stress disorder (PTSD). Our goal is to understand the mechanism and identify novel genetic targets underlying fear responses. METHODS/STUDY POPULATION: Involvement of the amygdala in fear acquisition is well established and requires activation of N-methyl-D-aspartic acid receptors (NMDARs). At a cellular level, NMDAR activation leads to production of nitric oxide (NO) by a process mediated by interaction between postsynaptic density protein 95 (PSD95) and neuronal nitric oxide synthase (nNOS). To elucidate mechanisms underlying the role of the PSD95-nNOS-NO pathway in conditioned fear, here we use rodent behavioral paradigms, pharmacological treatment with a small molecular PSD95-nNOS inhibitor, co-immunoprecipitation, Western blotting, and RNA-sequencing. RESULTS/ANTICIPATED RESULTS: We show that fear conditioning enhances the PSD95-nNOS interaction and that the small-molecule ZL006 inhibits this interaction. Treatment with ZL006 also attenuates rodent cued-fear consolidation and prevents fear-mediated shifts in glutamatergic receptor and current densities in the basolateral amygdala (BLA). With RNA-sequencing, expression of 516 genes was altered in the BLA following fear expression; of these genes, 83 were restored by systemic ZL006 treatment. Network data and gene ontology enrichment analysis with Ingenuity Pathway Analysis and DAVID software found that cell-cell interaction, cognition-related pathways, and insulin-like growth factor binding were significantly altered. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results reveal novel genetic targets that underlie plasticity of fear-memory circuitry via their contribution of NMDAR-mediated fear consolidation and can inform future strategies for targeting fear related disorders like PTSD. CONFLICT OF INTEREST DESCRIPTION: Anantha Shekhar and Yvonne Lai are co-founders of Anagin, Inc., which is developing some of the related molecules for the treatment of PTSD.


However, remdesivir’s clinical effects are controversial and new antiviral drugs are still urgently needed. We will discuss the current status of the drug discovery efforts against COVID-19 and potential future directions. With the ever-increasing movability of human population and globalization of world economy, emerging and reemerging viral infectious diseases seriously threaten public health. Particularly the past and ongoing outbreaks of coronaviruses cause respiratory, enteric, hepatic and neurological diseases in infected animals and human (pro), 3-chymotrypsin-like protease (3CLpro), helicase, and RNA-dependent RNA polymerase (RdRp) are key enzymes involved in viral transcription and replication. The spike and the four key enzymes were considered attractive targets to develop antiviral agents (SARS-CoV-2 belongs to the family of enveloped, single-strand RNA viruses known as Betacoronavirus in Coronaviridae, first reported late 2019 in China. It has since been circulating world-wide, causing the COVID-19 epidemic with high infectivity and fatality rates. As of the beginning of April 2021, pandemic SARS-CoV-2 has infected more than 130 million people and led to more than 2.84 million deaths. Given the severity of the epidemic, scientists from academia and industry are rushing to identify antiviral strategies to combat the disease. There are several strategies in antiviral drugs for coronaviruses including empirical testing of known antiviral drugs, large-scale phenotypic screening of compound libraries and target-based drug discovery. To date, an increasing number of drugs have been shown to have anti-coronavirus activities nd human . The humnd human , severe nd human , the SARnd human  highlighnd human . This dil agents . The catl agents . Besidesl agents . Therefol agents , these i Currently, there is no highly efficacious and specific treatment for SARS-CoV-2. Therefore, it is urgent need to identify effective antiviral agents from existing drugs to combat the infection. These drugs can be rapidly repurposed to clinic application for treating COVID-19 patients given their proven safety. These compounds include direct acting antivirals  and hostThe functional domains of the spike protein of SARS-CoV-2 are highly conserved with those of SARS-CoV . These din vitro via impeding spike trimerization , which belongs to adenine or guanine derivatives, block viral RNA synthesis by targeting viral RdRp and in a broad spectrum of RNA viruses, including human coronaviruses, human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV). Up to now, NAs favipiravir, ribavirin, remdesivir, galidesivir, sofosbuvir, tenofovir, NHC  and EIDD-2801 have potential to treat SARS-CoV-2 , since tin vitro . Patientin vitro  or in coin vitro . SeveralRibavirin, a guanine derivative approved in combiantion with other anti-medication for treating HCV and respiratory syncytial virus (RSV), has been evaluated in patients with SARS and MERS, but some patients may manifest side effects such as severe anemia at high doses  and we din vitro and in vivo   . Darunav4252274) . It indi4252274) .pro) is also reported as an attractive target for inhibiting viral replication and transcription. Currently known Mpro inhibitor-based structures include N3, 11a, 11b, Camostat Mesylate, Carmofur. The crystal structure of inhibitors-Mpro complex has been reported , which is highly expressed in lung, small intestine, brain, testis, kidney , is the in vitro , includiin vitro  and widein vitro , as wellin vitro  likely bin vitro . An openin vitro  and WHO in vitro .Two mucosa-specific transmembrane serine protease 2 (TMPRSS2) and 4 (TMPRSS4) catalyze the cleavage of SARS-CoV-2 spike and trigger virus entry into host cells . Particuin vitro  targeting viral RNA structure units and FDA-approved drugs Deguelin, Nilotinib, and Sorafenib inhibiting the predicted RNA binding protein expression and showed these compounds decreased SARS-CoV-2 infection in human cells  algorithms are increasingly being applied for rapid and cost-effective drug discovery . The scain vivo . From anin vivo . Among oin vivo , SARS-Coin vivo , and SARin vivo  by blockin vivo . Beck etd values . Gysi etin vitro. In addition it also suppressed IL-6 production. Molecular docking simulation showed that meguparib can bind to the nucleoprotein of SARS-CoV-2, which might mediate the inhibition of viral replication 1/JAK2 inhibitor for treating rheumatoid arthritis, to be effective at inhibiting SARS-CoV-2 infection as well as reducing virus induced inflammations . Althouglication .In silico molecular modeling suggests that several FAD-approved anticancer drugs  might be able to inhibit COVID-19 by docking on Mpro and spike of SARS-CoV-2 , 2,800 mg dose of SARS-CoV-2 neutralizing antibody LY-CoV555 from Lilly declined the viral load in patients , but LY-SARS-CoV-2 infection causes severe acute pneumonic processes with pathological damages, including inflammatory cytokine storm characterized by heightened levels of C-reactive protein, ferritin, interleukin (IL)-1, IL-6, IL-8, and TNF-α . SeveralDampening IL6 is a potential therapeutic avenue for immuno-modulation because IL6 is elevated in patients suffering from ARDS and its level correlated positively with viral loads . TocilizIn order to cope with emerging and re-emerging infectious diseases, challenges and opportunities abound for developing antiviral therapeutics. A large number of anti-viral agents are currently being explored for treating SARS-CoV-2 infection. Repurposing of existing drugs have demonstrated power by bringing several drugs to approval for treating COVID-19 patients, such as remdesivir and dexamethasone. However, these drugs still suffer from suboptimal therapeutic effect or known strong side-effect. Direct acting antivirals (DAAs) are rapidly being developed and there is great hope for producing highly potent antivirals from these efforts. Structure-based studies have significantly boosted the efficiency and precision of these developments. However, drug resistant strains are going to arise sooner or later. The recent rises of several high transmissible strains sounded alarms for currently used vaccines and neutralizing antibodies. Combinations of DAAs or neutralizing antibodies will likely be required for effective control of the viruses, much like the development of HIV cocktail therapies. On the other hand, development of broad-spectrum antiviral drugs not only fight against COVID-19 but also provide arsenals for protection from future viral outbreaks. The wealth of knowledge that has been and continues to be gleaned on the interaction between viruses and hosts will guide and prod the development of host-targeting antivirals, which have the potential advantages of broad-spectrum therapeutic effect and insensitivity to viral evasion. Antiviral drug development in general will benefit from the heightened level of social awareness of the COVID-19 epidemic and hopefully provide a safeguard against future viral epidemics for the humankind.MM and XT wrote the manuscript. Both authors contributed to the article and approved the submitted version.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.


Berberis amurensis. Berbamine has been shown to modulate different oncogenic cell-signaling pathways in different cancers. In this review, we comprehensively analyze how berbamine modulates deregulated pathways  in various cancers. We systematically analyze how berbamine induces activation of the TGF/SMAD pathway for the effective inhibition of cancer progression. We also summarize different nanotechnological strategies currently being used for proficient delivery of berbamine to the target sites. Berbamine has also been reported to demonstrate potent anti-cancer and anti-metastatic effects in tumor-bearing mice. The regulation of non-coding RNAs by berbamine is insufficiently studied, and future studies must converge on the identification of target non-coding RNAs. A better understanding of the regulatory role of berbamine in the modulation of non-coding RNAs and cell-signaling pathways will be advantageous in the effective translation of laboratory findings to clinically effective therapeutics.Natural product research is a cornerstone of the architectural framework of clinical medicine. Berbamine is a natural, potent, pharmacologically active biomolecule isolated from  The deregulation of cell-signaling pathways is a central area of research in molecular oncology, and scientists have developed a nearly complete resolution of the multiple signaling cascades in different cancers. JAK/STAT and CAMKII/c-Myc are central drivers of carcinogenesis and metastasis. Moreover, TGF/SMAD has been shown to play a significant role in the regulation of carcinogenesis. Additionally, autophagy and apoptosis have fundamental roles in molecular oncology.Nature has evolved over billions of years to generate an extra-ordinarily diverse array of natural products of wider structural variations and incredible complexities. Such widely heralded landmark discoveries as etoposide, teniposide, vinblastine and vincristine serve as monuments to the contributions of medicinal chemistry research over the decades.Berberis amurensis. Berbamine has a molecular weight of 608.7. There are some good reviews that have generally explored the pharmacological properties of berbamine . Th. Th37]. Preliminary studies on berbamine indicate its role in cancer treatment. It is efficient because other natural compounds could be limited by reduced water solubility, low absorption rate in the bowels and rapid metabolism. The role of berbamine in autophagy is still unclear. Further studies are needed to elucidate the effects of this natural compound on autophagy regulation. Berbamine efficiently suppressed the migratory and invasive capacity of highly metastatic breast MDA-MB-231 cancer cells via the inhibition of pro-MMP2/MMP9 activation. It also reduced the phosphorylated levels of AKT and c-Met in MDA-MB-231 cancer cells . Aspirin dose-dependently induced the phosphorylation of CREB and ATF1 . ImportaBerbamine concentration-dependently inhibited the migratory and invasive potential of SMMC-7721 cells and increased expression of Cx32 in SMMC-7721 cells. However, after silencing Cx32, berbamine failed to inhibit cell invasion and metastasis .The novel berbamine derivative time- and dose-dependently induced apoptosis in cancer stem-like cells (CSCs) of human glioblastoma. The berbamine derivative induced an upregulation of microRNA-4284 of more than fourfold in GBM stem-like cells [However, the role of miR-4284 needs to be tested more effectively in various cancers. Certain hints that point towards the oncogenic role of miR-4284 in gastric cancer have emerged. miR-4284 directly targeted TET1 (ten-eleven translocation 1) and promoted the tumor growth, migration and invasion of gastric cancer cells . Therefoβ-cyclodextrin/chitosan nanoparticles are developed to enhance oral bioavailability and improve small intestinal absorption [Nanotechnology is widely applied in delivery systems to effectively improve the therapeutic index and reduce side effects in a lower drug load context than the native compound. Various materials can be made to nanoscale and used as drug carriers in many applications. Polymeric nanoparticles are one such delivery system due to their biocompatibility, biodegradability, tunable pharmacokinetics and circulation. Sulfobutylether-sorption . Sulfobusorption . FurtherIn another study, poly(caprolactone) (PCL) was conjugated with methoxy poly (ethylene glycol) (mPE) as copolymers to utilize the hydrophobic portion of PCL for drug encapsulation and the hydrophilic part of mPE for drug delivery providing suitable aqueous solubility. The amphilic mPE-PCL block copolymers were self-assembled into nanospheres with a hydrophilic outer shell made of mPE and a hydrophobic inner core. The mPE-PCL copolymer is reported to construct polymeric nanoparticles co-delivering paclitaxel (PTX) and berbamine. PTX and berbamine were able to enter the cells via the nanoparticles, as confirmed by fluorescence caused by coumarin-6. The berbamine-PTX-loaded mPE-PCL nanoparticles demonstrated effective anti-cancer properties in human gastric cancer BGC823 cells in vitro, as well as superior tumor suppression, compared with free drugs in vivo. The paclitaxel/berbamine-loaded nanoparticles showed the best tumor suppression compared with free PTX, berbamine or paclitaxel/berbamine compound . HoweverLipid nanoparticles are alternative, rapidly developing carriers within the nanotechnology field in drug delivery due to their unique size-dependent properties and biodegradability. Parhi et al. reported that berbamine-loaded lipid nanoparticles with a diameter of 75 m showed effective anti-metastatic and anti-tumorigenic properties both in vitro and in vivo. The berbamine nanoparticles were made from a two-step process, in which berbamine was first incorporated into the fluid phase of glyceryl mono-oleate (GMO) and further emulsified with α-tocopherol poly (ethylene glycol) 1000 succinate (TPGS). The 6-coumarin-loaded nanoparticle showed that GMO-TPGS lipid nanoparticles were able to access A549 and MDA-MB-231 cells. The berbamine-GMO-TPGS nanoparticles showed superior cellular toxicity, as well as an inhibition of migration and invasion in metastatic breast cancer MDA-MB-231, lung cancer A549 and melanoma B16F10 cell lines when compared to native berbamine. An in vivo mouse study with a melanoma B16F10 subcutaneous tumor model demonstrated that berbamine-GMO-TPGS nanoparticles effectively suppressed primary tumor growth when compared with free berbamine. Berbamine-GMO-TPGS nanoparticles inhibited lung metastasis after 45 days of cell injection through the tail vein in the C57BL/6 mouse model . Berbamine effectively inhibited cell survival pathways. The PI3K/AKT-signaling axis was reported to be reduced . The inhBerbamine increased the intracellular ROS levels via the downregulation of antioxidative genes such as NRF2, SOD2, GPX-1 and HO-1. ROS accumulation led to the activation of the intrinsic apoptotic pathway by an increase in the ratio of Bax/Bcl-2 . A rapid+/K+-ATPase-dependent manner, consequently resulting in the activation of p38-MAPK and EGFR-ERK transduction cascades [+/K+-ATPase ligand ouabain also induced EGFR, IGF1R, Src, ERK1/2 and p38-MAPK phosphorylation in HCC cells. Intriguingly, the treatment of HCC cells with EGFR or Src inhibitors blocked berbamine-mediated ERK1/2 phosphorylation. Furthermore, sorafenib inhibited the activation of EGFR, Src, p38-MAPK and ERK1/2 by berbamine and ouabain. Berbamine and sorafenib combinatorially inhibited the growth of tumors in mice xenografted with HepG2 cells [Berbamine induced the phosphorylation of the non-receptor tyrosine kinase Src in Nacascades . ImportaG2 cells .−CD8− T cells was found to be considerably enhanced in the combinatorically treated group, signifying an antitumor role played by CD4−CD8− T cells in the combinatorically treated group. Furthermore, the ratio of Treg (regulatory T) cells was reported to be notably suppressed in mice combinatorically treated with ΔA146Ply and berbamine. Pulmonary metastatic lesions were also noted to be reduced in mice combinatorically treated with ΔA146Ply and berbamine [Detoxified pneumolysin derivative ΔA146Ply, a pneumolysin mutant, has been shown to be effective against cancer . ΔA146Plerbamine .The loss of apoptosis is a critical step that fuels carcinogenesis and metastasis. Researchers are focusing on different strategies to restore apoptosis in resistant cancer cells ,54,55.2-Methylbenzoyl berbamine (BBD24) time- and dose-dependently inhibited the growth of the osteosarcoma HOS and MG63 cells. BBD24 induced an increase in the levels of BAX in HOS cells . FurtherBerbamine led to a reduction in the levels of survivin in prostate cancer cells. The berbamine-mediated activation of caspase-9 and caspase-3 was blocked by methazolamide (inhibitor of cytochrome c). Berbamine inhibited tumor growth rates in athymic mice injected subcutaneously with PC-3 cells .Berbamine downregulated the levels of anti-apoptotic proteins Bcl-XL and Bcl-2 in imatinib-resistant BCR-ABL-positive K562 cells . BerbamiBerbamine induced apoptosis in HepG2 cells. It upregulated the expression of Fas in HepG2 cells. Berbamine induced tumor regression in HepG2 xenograft models . BerbamiRapidly emerging, cutting-edge research works have provided new horizons for preclinical-to-clinical translatability of molecularly targeted cancer therapies. Based on the sizzling insights gleaned from decades of cutting-edge research, researchers are developing a better understanding of the pharmacological properties of myriad natural products. Overwhelmingly increasing, high-quality, scientifically verified information has enabled interdisciplinary researchers to unravel remarkable medicinal properties of natural products for the treatment of different diseases. Although researchers have reported the promising potential of berbamine as a cancer-chemopreventive agent, berbamine has been shown to pharmacologically target different cellular pathways shown in .There are some major knowledge gaps in our understanding of the regulation of transduction cascades by berbamine. The regulation of non-coding RNAs by berbamine is still inadequate. Identification of the most plausible target miRNAs, lncRNAs and circRNAs will be effective, and rationally designed preclinical studies using oncogenic non-coding RNA inhibitors and tumor suppressor non-coding RNA mimics will be beneficial. Likewise, the use of nanotechnologically delivered berbamine will be effective in targeted therapy in tumor-bearing mice. Although intriguing evidence indicates Berbamine as a good candidate for the adjuvant of canonical cancer therapies, further studies and clinical trials are needed to better understand the effects of this natural compound in humans.For future studies, it is imperative to have more pre-clinical and clinical evaluations on the efficacy and safety of berbamine by integrating novel technologies in precision medicine and pharmacology.


While CRISPR has been actively explored as a strategy for treating inherited retinal diseases, with the first human trial recently initiated, its applications for complex, multifactorial conditions such as ocular angiogenesis has been relatively limited. Currently, neovascular retinal diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and neovascular age-related macular degeneration, which together constitute the majority of blindness in developed countries, are managed with frequent and costly injections of anti-vascular endothelial growth factor (anti-VEGF) agents that are short-lived and burdensome for patients. By contrast, CRISPR technology has the potential to suppress angiogenesis permanently, with the added benefit of targeting intracellular signals or regulatory elements, cell-specific delivery, and multiplexing to disrupt different pro-angiogenic factors simultaneously. However, the prospect of permanently suppressing physiologic pathways, the unpredictability of gene editing efficacy, and concerns for off-target effects have limited enthusiasm for these approaches. Here, we review the evolution of gene therapy and advances in adapting CRISPR platforms to suppress retinal angiogenesis. We discuss different Cas9 orthologs, delivery strategies, and different genomic targets including VEGF, VEGF receptor, and HIF-1α, as well as the advantages and disadvantages of genome editing vs. conventional gene therapies for multifactorial disease processes as compared to inherited monogenic retinal disorders. Lastly, we describe barriers that must be overcome to enable effective adoption of CRISPR-based strategies for the management of ocular angiogenesis.Among genome engineering tools, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based approaches have been widely adopted for translational studies due to their robustness, precision, and ease of use. When delivered to diseased tissues with a viral vector such as adeno-associated virus, direct genome editing can be efficiently achieved  Ocular angiogenesis, which is characterized by the formation of new blood vessels from pre-existing vasculature in the eye, underlies the leading causes of blindness across different age groups, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and neovascular age-related macular degeneration (nAMD) , fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), transforming growth factor- β (TGFβ) superfamily, endothelins, galectins, and integrins , antibody fragments , and recombinant decoy receptors (aflibercept)  Todoric. IntraviAnother mode of therapy for ocular angiogenesis is photodynamic therapy (PDT), which combines intravenous delivery of a porphyrin-based photosensitizer (verteporfin) with focal low-intensity light exposure to trigger singlet oxygen release within the CNV, causing vascular occlusion and ablation of the lesion. In clinical trials, PDT with verteporfin reduces vision loss and CNV in eyes with nAMD [Treatment of Age-Related Macular Degeneration With Photodynamic Therapy (TAP) Study Group, The pursuit of gene therapies for ocular angiogenesis was born out of a need for more sustained treatments to overcome the burden of repeated injections. Ocular gene therapy has gained renewed interest since the approval of the first retinal gene therapy using an AAV2 vector to express the RPE65 gene encoding a retinal isomerase for patients with type 2 Leber Congenital Amaurosis . Another strategy employing subretinal AAV8 to express a monoclonal anti-VEGF antibody fragment (RGX-314) has been found to be comparable to anti-VEGF Fab expression  permanent suppression of angiogenic signals, (2) potential disruption of both extracellular and intracellular targets, and (3) possible cell-specific delivery aimed at more pathologically relevant sources.Derived from prokaryotic adaptive immune systems, CRISPR-associated Cas9 endonucleases can induce a site-specific cleavage in the target DNA with programmable guide RNAs (gRNAs), creating double-strand breaks (DSBs) that can be repaired by error-prone non-homologous end-joining (NHEJ) or homology directed repair (HDR) when paired with donor DNA template Yiu, . The fasStreptococcus pyogenes (SpCas9) has been the most well-characterized ortholog, its larger size (4.2 kb) limits its ability to be packaged with gRNAs into a single AAV vector for in vivo use. Strategies to circumvent this limitation include packaging in lentiviral vectors, employing dual AAV vectors to express full-length SpCas9 separately from the gRNAs, or using “split-Cas9” by dividing the expression of SpCas9 at its disordered linker (V713–D718) and reconstituting the full-length protein by split-intein protein trans-splicing  and Campylobacter jejuni (CjCas9) can be packaged along with gRNAs in an “all-in-one” AAV vector  to the eye, which can induced DNA cleavage almost immediately and degrade rapidly in cells, helping to minimize off-target effects and cellular toxicity  transcription factor, or the hypoxia response element (HRE) in the VEGF promoter to which Hif-1α binds. CRISPR-Cas9 may also target VEGF receptors or downstream signals. Finally, CRISPR-based strategies enable simultaneous and multiplexed targeting of several factors using an array of gRNAs  which creates staggered DNA cuts   and VEGFR-2 (KDR). Although VEGFR-1 has higher binding affinity, VEGFR-2 has greater kinase activity and mediates most of the pro-angiogenic signal Shibuya, . Early gHif-1α is a major regulator of the cellular hypoxic response  reduce off-target activity without compromising on-target specificity by minimizing their binding affinity to DNA (Pattanayak et al., + cytotoxic T cell activity which eliminated the edited cell population despite still showing efficient genome editing (Li et al., S. pyogenes and S. aureus from which the most commonly-used SpCas9 and SaCas9 endonucleases are derived (Attenello, The foreign nature of bacterial-derived CRISPR-Cas9 proteins has the potential to elicit unwanted host immune responses that may limit therapeutic efficacy. In mice, AAV-mediated Cas9 delivery triggered both humoral and cellular response against Cas9 (Chew et al., The application of genome editing for retinal diseases has received significant attention since the recent initiation of the first human trial using CRISPR technology. Genome editing pose unique advantages as well as challenges when compared to anti-VEGF pharmacotherapies or current gene therapy approaches. Genomic ablation can permanently suppress pro-angiogenic pathways, providing a true cure for ocular angiogenic conditions. Viral-mediated delivery of CRISPR components also enables efficient targeting of not only secreted factors such as VEGF, but also intracellular targets including upstream transcription factors and regulatory elements, as well as downstream signal mediators. Yet, the needs to overcome a high therapeutic threshold and maximize cellular specificity, while minimizing off-target activity and host immune responses, must be adequately addressed to facilitate more rapid translation of research outcomes into real world therapies.SC and GY conceived the project. SC, T-NS, and TN collected literature. SC, T-NS, and GY wrote the manuscript. GY oversaw the project. All authors contributed to the article and approved the submitted version.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.


Immune checkpoint inhibitors (ICI) have reshaped treatment paradigms of multiple solid organ malignancies. In genitourinary malignancies (GU), ICI provide significant clinical benefit and are approved for use in localized and metastatic renal cell carcinoma and urothelial carcinoma. Investigational approaches to maximize clinical benefit and expand use of ICI across GU malignancies are actively being pursued. In addition, biomarkers predictive of clinical benefit to ICI have been identified; however, further validation and incorporation into guideline-based management remain active areas of investigation.Cancers of the genitourinary (GU) tract are common malignancies in both men and women and are a major source of morbidity and mortality. Immune checkpoint inhibitors (ICI) targeting CTLA-4, PD-1 or PD-L1 have provided clinical benefit, particularly in renal cell and urothelial carcinoma, and have been incorporated into standard of care treatment in both localized and metastatic settings. However, a large fraction of patients do not derive benefit. Identification of patient and tumor-derived factors which associate with response have led to insights into mechanisms of response and resistance to ICI. Herein, we review current approvals and clinical development of ICI in GU malignancies and discuss exploratory biomarkers which aid in personalized treatment selection. Cancers of the genitourinary (GU) tract are a major source of cancer morbidity and mortality. GU malignancies include prostate adenocarcinoma, the most common malignancy in men, urothelial and renal cell carcinoma (RCC), the sixth and eighth most common cancer among men and women combined, and testicular cancer, the most common solid tumor malignancy among men younger than 35 years old . There hTherapies which harness anti-cancer immune responses, referred to broadly as immunotherapy, have changed the paradigm of treatment for many cancers in recent years. Immune checkpoint inhibitors (ICI) are a class of immunotherapy which block immune-inhibitory receptors, or immune checkpoints, and re-invigorate anti-cancer immunity and facilitate tumor elimination . Within Unfortunately, practice-changing efficacy of ICI has been limited to a subset of tumor types, such as melanoma, kidney, bladder and non-small cell lung cancer (NSCLC), and many tumors fail to derive benefit, such as tumors of the gastrointestinal tract . FeatureHerein, we review current FDA approved indications and active areas of clinical development of ICI in GU malignancies . In addiRenal cell carcinomas (RCC) are the eighth most common cancer diagnosis with an estimated 74,000 new cases diagnosed in 2019 . RCC is ICI is approved for the treatment of untreated and refractory metastatic clear cell RCC. ICI use in the adjuvant and neoadjuvant setting are in advanced stages of clinical development. In metastatic disease, treatment guidelines and FDA indications take into account International Metastatic RCC Database Consortium (IMDC) Risk groups, which assign a risk score based on clinical variables such as time from diagnosis to systemic therapy greater than or less than 12 months, performance status, hemoglobin, neutrophil and platelet counts and corrected calcium level. Patients without any positive variables are considered favorable-risk, while those with one or two positive variables are considered intermediate-risk, and those with three or more factors considered poor-risk ,29.In localized disease, neoadjuvant and adjuvant ICI is being investigated and treatment considers risk groups based on histology, grade and TNM stage. Subsequent sections referring to clinical trials and indications for RCC discuss clear cell histology and non-clear cell histology separately.First LineIn patients with previously untreated metastatic clear cell RCC, treatment indications are based on IMDC risk group. Combination anti-PD-1/PD-L1 plus VEGF TKI is approved in all risk groups while combination nivolumab plus ipilimumab is indicated in patients with intermediate- and poor-risk disease.Nivolumab plus ipilimumab was approved for first-line therapy based on the phase 3 CheckMate-214 trial versus sunitinib monotherapy . In thisMultiple anti-PD-1/PD-L1 plus VEGF TKI combinations have been approved for first-line treatment of advanced RCC regardless of risk group. These combinations include axitinib plus pembrolizumab evaluated in the KEYNOTE-426 clinical trial, lenvatinib plus pembrolizumab evaluated in the CLEAR clinical trial, axitinib plus avelumab evaluated in the JAVELIN Renal 101 clinical trial and cabozantinib plus nivolumab evaluated in the CheckMate-9ER clinical trial ,32. AcroDespite clear activity of anti-PD-1/PD-L1 plus VEGF TKI combinations, it is unclear if the activity is synergistic or additive. VEGF inhibitors have been shown to normalize tumor vasculature and increase immune cell tumor infiltration, decrease immunosuppressive cell populations and promote T cell priming and activation via dendritic cell maturation ,36,37. IIn patients with intermediate- and poor-risk disease, evidence-based selection of first-line treatment between nivolumab plus ipilimumab or anti-PD-1/PD-L1 plus VEGF TKI combinations remains undefined. Longer follow-up is required to determine if combination anti-PD-1/PD-L1 plus VEGF-TKI will produce as durable of complete responses as nivolumab plus ipilimumab since median OS has not been reached in many pivotal studies of anti-PD-1/PD-L1 plus VEGF TKI. These outcomes will provide basis for comparison and facilitate discussion with patients when selecting treatment; however, cross-study comparison has limitations. In the long-term, a head-to-head phase 3 clinical trial comparing these two strategies is warranted.ICI-specific outcome measures such as treatment free survival (TFS), defined as time from ICI cessation to subsequent systemic therapy initiation or death, should be considered in future clinical trials . In favoSecond LineIn current practice, most patients receive upfront anti-PD-1 in combination with either anti-CTLA-4 agent or VEGF TKI, and there is limited data to guide subsequent treatment selection. Investigators have begun to conduct clinical trials which specifically enroll patients who have progressed on anti-PD-1/anti-PD-L1 based combination as most recent therapy. In patients with metastatic RCC initially treated with anti-PD-1 therapy plus VEGF TKI, salvage nivolumab plus ipilimumab has demonstrated activity and is approved regardless of IMDC risk group ,41,42. AIn patients treated first-line with VEGF TKI monotherapy, nivolumab monotherapy is approved based on results from the phase 3 CheckMate 025 trial which demonstrated improved OS compared with everolimus ,45.Non-clear cell RCCAnalysis of the CheckMate 374 study of nivolumab monotherapy in previously treated RCC revealed meaningful responses in non-clear cell RCC . The KEYPerioperative ICISeveral large phase 3 clinical trials are being performed to evaluate perioperative ICI in patients with intermediate or high-risk localized RCC. In the phase 3 KEYNOTE-564 trial, adjuvant pembrolizumab versus placebo demonstrated significant improvement with a hazard ratio for disease recurrence or death of 0.68, making it the first trial to show benefit for adjuvant immunotherapy in localized RCC . The phaAlternative Therapy SchedulesAn ongoing phase 2 trial is evaluating intermittent nivolumab dosing in which patients who achieve >10% reduction in tumor burden enter a treatment-free observation period and are re-imaged every 3 months. In this study, out of five patients who met criteria for treatment-free observation, only one patient re-started therapy and the other four sustained responses for median of 34 weeks after therapy discontinuation .Triple TherapyTriple combination cabozantinib with nivolumab and ipilimumab in clear cell RCC demonstrated tolerability and encouraging outcomes and is being evaluated in a multi-center, randomized phase 3 clinical trial (NCT03937219) .There are no widely accepted models predictive of response and clinical benefit to ICI treatment in patients with RCC; however, prospective and retrospective analyses have identified demographic, clinical, histologic and molecular biomarkers which associate with treatment outcomes. Assocation between PD-L1 expression and outcomes to ICI treatment have been mixed in RCC. In the CheckMate 025 trial, patients found to have PD-L1 expression >1% had worse survival outcomes than those with low PD-L1 expression, even when treated with single agent anti-PD-1. This was not observed in the CheckMate 214 trial of combination anti-CTLA-4/PD-1 . Intereseff signature, TeffHigh) had improved ORR to atezolizumab plus bevacizumab treatment versus TeffLow. Similar benefit was observed in the JAVELIN Renal 101 trial, where higher-than-median Immuno Score resulted in more favorable outcomes in patients treated with avelumab plus axitinib . Multiple phase 1 and phase 2 trials are evaluating ICI in combination with treatments such as abiraterone and cabozantinib [NCT04477512], radiation therapy  and an experimental IL-8 directed monoclonal antibody [NCT03689699]. In addition, perioperative ipilimumab in combination with castration prior to radical prostatectomy has demonstrated feasibility with longer follow-up ongoing .Biomarker-directed treatment selection has become standard of care in prostate cancer. Two to five percent of prostate cancers have been found to have microsatellite instability-high status for which pembrolizumab has recently been approved in the refractory setting ,129,130.A single arm phase 2 study of ipilimumab in mCRPC found the presence of tumor-infiltrating lymphocytes by IHC and IFN-γ gene signatures associated with favorable outcomes . Loss-of-function alterations of tumor suppressor protein CDK12, found in approximately 5–7% of prostate cancers, results in genomic instability, increased neoantigen burden and T cell infiltration ,137. RetTesticular germ cell tumors (TGCTs) are highly sensitive to cisplatin-based chemotherapy and many achieve cure with either upfront or salvage chemotherapy upon relapse. In chemotherapy-refractory cases, ICI has been studied in multiple case series as well as early phase clinical trials ,141,142.While durable responses to ICI treatment have been observed in multiple cancer types, only approximately 15–30% patients achieve response in aggregate ,148. CorWith rapidly evolving treatment options and clinical trial landscape within GU malignancies, development and incorporation of prognostic and predictive biomarkers is critical to personalize treatment selection and optimize clinical benefit for patients. Large clinical trials of ICI in GU malignancies and other solid tumors have prospectively evaluated the predictive and prognostic value of histopathologic and molecular assessment of individual tumor characteristics and the TME. Some of these factors have been incorporated into guideline-based approvals of ICI. Herein, we review established and investigational biomarkers being utilized in the current landscape of ICI with a focus on GU malignancies.Tumor-promoting inflammation and avoidance of immune destruction are considered hallmarks of cancer . CharactThe presence of infiltrating T cells and supporting pro-inflammatory milieu, defined as the T cell-inflamed tumor microenvironment (TME), has associated with improved clinical outcomes in patients treated with ICI in a variety of solid tumors ,13. In cFactors associated with the development of T cell-inflamed TME remain areas of active research. To date, the field has recognized the importance of the presence of tumor-associated neoantigens sufficient to stimulate tumor-reactive T cells, the presence of antigen-presenting dendritic cells and a TME hospitable for accumulation of anti-tumor immune cells ,156,157.Advances in multiplex immunofluorescence (IF) and immunohistochemical (IHC) techniques have allowed for spatial interrogation of a broad scope of immune cell populations within the TME of solid tumor malignancies. These techniques represent important modalities to appropriately characterize complex immune TME and their relation to prognosis and treatment outcomes. During initial evaluation of anti-PD-1 agents in melanoma, IHC characterization of CD8 positivity and PD-L1 expression were significantly associated with response to ICI . CD8+ T Quantification of RNA expression of gene subsets in tumor samples can characterize aspects of underlying tumor and TME immune biology and serve as predictive biomarkers for ICI treatment. Gene expression signature analysis of IFN-γ associated genes, cytotoxic T cell effector genes, dendritic cell and antigen presentation associated genes and T cell chemo-attractants have been shown to correlate with T cell infiltration and clinical benefit to ICI and cancer vaccines ,162,163.High tumors. Alternatively, gene expression signatures which detect the presence of alternative immune checkpoints, such as LAG3 or TIM3, may inform selection of combination treatments.Certain gene expression signatures designed to assess activity of a specific biologic or signaling pathway, such as the angiogenesis signature or TGF-β signature have also been evaluated in pre-treatment tumor samples in large clinical trials of ICI in RCC and mUC, respectively ,164. TheNon-T cell-inflamed tumors identified by gene expression analysis may be tumors for which innate immune-stimulating agents, such as STING and TLR agonists, could stimulate T cell-inflammation and induce response to ICI . Serial Use of gene expression signatures as predictive biomarkers has thus far been limited to prospective correlative analysis. As gene expression signatures continue to mature, incorporation into both FDA-approved indications may assist in optimal personalized treatment choices. In addition, incorporation of gene expression signatures into clinical trial inclusion criteria will enable optimal identification of patients for appropriate clinical trials and accelerate the development of new, personalized therapies . No consensus has been reached on the predictive value of immunohistochemical assessment of PD-L1 expression and response to ICI in GU malignancies. In large trials in mUC, PD-L1 expression by immune cells, not tumor cells, correlated with response. In RCC, PD-L1 expression did not correlate with improved outcomes in ICI plus VEGF TKI combination treatment ,77,169. Tertiary lymphoid structures, organized lymphoid aggregates made up primarily of B cells, have been identified within tumors of a broad range of malignancies. TLS quantification by H&E and immunohistochemical detection of B cells (CD20), T cells (CD3), follicular dendritic cells (CD21) and high endothelial venules (MECA79) revealed increased TLS associated with response to ICI in melanoma and RCC ,171. In MicroRNA are small single-stranded non-coding RNA nucleotides which negatively regulate gene expression by post-transcriptional RNA silencing. Analysis of human cancers has identified miRNA signatures which associate with tumor behavior and clinical outcomes to treatment . ExpressTumor-intrinsic factors such as high somatic tumor mutation burden (TMB), defective mismatch repair pathways (dMMR) and dysregulated oncogenic signaling pathways have been shown to impact response to ICI.Tumors with defective genes associated with mismatch repair mechanisms (dMMR), such as MSH2, MSH6 and MLH1, or other DNA proofreading mechanisms have been shown to be sensitive to ICI ,176. MecTumors with high somatic mutation burden (TMB) have been found to have improved clinical outcomes to ICI across cancer types in part due to the association of TMB with neoantigen load and immune cell infiltration . PembrolIncreased tumor burden has correlated with worse outcomes to ICI treatment in solid tumors . In addiThe association between occurrence of immune-related adverse events (irAEs) and outcomes has been mixed. In patients with metastatic melanoma treated with ipilimumab, studies found conflicting associations between development and an irAE with duration of response ,187. In Patient demographic and clinical factors such as germline polymorphisms, gender, BMI and circulating factors have been shown to associate with response to ICI. Mechanistic exploration of each of these factors remains an active area of investigation.ERAP2 associated with enhanced immunogenicity and improved survival in ICI-treated patients with luminal subtype of bladder cancer. In RCC, although somatic PRBM1 mutations correlated with T cell infiltration, the infiltrated tumors were enriched for deleterious PRBM1 mutations as compared to non-infiltrated tumors, which negatively impacted survival [Gene expression analysis has demonstrated how genes related to immune response are under strong germline genetic control suggesting an individual’s anti-tumor immune response is, in part, determined by one’s genome . In addisurvival .Peripheral blood analysis represents a less-invasive method of personalized prediction and prognostication. In RCC and bladder cancer, a higher neutrophil-to-lymphocyte ratio associates with worse outcomes to ICI treatment ,192. CirT cell receptor repertoire has been found to predict response to ICI. In one study, increased peripheral T cell receptor diversity associated with improved outcomes to ICI treatment in melanoma . In contPatients with elevated BMI were found to have improved outcomes to ICI in retrospective analysis of large clinical trials in RCC and metastatic melanoma, but other studies have reported mixed results ,22,197. The commensal microbiome, which includes the gut and urinary microbiome, is known to have significant influence on tumorigenesis and the immune system, and there is a growing body of evidence demonstrating the impact of commensal microbiome composition on response to ICI ,207,208.Understanding the impact of diet and nutrient availability within the TME on immunometabolism and function of tumor and immune cells is also an area of active investigation. Studies of the ketogenic diet (KD), a high-fat, low carbohydrate diet has been shown to cause metabolic alterations in tumor cells and restrict tumor growth and sensitize tumors to chemotherapy and radiotherapy ,216,217.Immune checkpoint inhibitors have become standard of care for the treatment of GU malignancies in both localized and metastatic disease settings. In patients with metastatic disease, continued emphasis must be placed on the development of novel ICI and ICI-based combinatorial strategies to increase response rates and enhance depth of response. In localized disease, further clarification of the impact of adjuvant ICI on surgical outcomes or as an alternative to surgery is awaited. Lastly, continued validation of biomarker-based approaches will be critical to personalize treatment strategies and optimize treatment efficacy and clinical benefit for patients.


COVID-19 has impacted human life globally and threatens to overwhelm health-care resources. Infection rates are rapidly rising almost everywhere, and new approaches are required to both prevent transmission, but to also monitor and rescue infected and at-risk patients from severe complications. Point-of-care lung ultrasound has received intense attention as a cost-effective technology that can aid early diagnosis, triage, and longitudinal follow-up of lung health. Detecting pleural abnormalities in previously healthy lungs reveal the beginning of lung inflammation eventually requiring mechanical ventilation with sensitivities superior to chest radiographs or oxygen saturation monitoring. Using a paradigm first developed for space-medicine known as Remotely Telementored Self-Performed Ultrasound (RTSPUS), motivated patients with portable smartphone support ultrasound probes can be guided completely remotely by a remote lung imaging expert to longitudinally follow the health of their own lungs. Ultrasound probes can be couriered or even delivered by drone and can be easily sterilized or dedicated to one or a commonly exposed cohort of individuals. Using medical outreach supported by remote vital signs monitoring and lung ultrasound health surveillance would allow clinicians to follow and virtually lay hands upon many at-risk paucisymptomatic patients. Our initial experiences with such patients are presented, and we believe present a paradigm for an evolution in rich home-monitoring of the many patients expected to become infected and who threaten to overwhelm resources if they must all be assessed in person by at-risk care providers.The online version contains supplementary material available at 10.1186/s13089-021-00231-9. COVID-19 has fundamentally impacted all aspects of human life in 2020. As of December 6 2020, the World Health Organization reported that there had been over 66 million confirmed cases of COVID-19, including 1,523,583 deaths .The diagnosis of COVID-19 typically relies upon PCR-testing, which unfortunately may have significant false-negative test characteristics. Thus early chest computed tomography (CT) has been recommended as a screening tool for suspected COVID-19 patients with better sensitivity than PCR , but thiRemotely Telementored Self-Performed Ultrasound (RTSPUS) is an innovative technique that was first described as a solution to providing medical imaging onboard the International Space Station, wherein astronauts were guided from earth to image their own bodies under the direction of terrestrial ultrasound experts . We beliSterilized ultra-portable economical ultrasound probes interfacing with the patients own smartphones or computing devices can be delivered to patients by any means including commercial couriers and even unmanned aerial vehicles. Thereafter, the informatics can be easily provided using off-the-shelf technologies widely available on a global basis. For example, in our experience, the ultra-portable Philips L12-4 broadband linear array transducer , attached to a Samsung Galaxy Tablet A , communicating with a remote expert using Zoom Teleconferencing  through a standard smartphone  and with remote mentor control of the ultrasound knobology using the Teamviewer real-time remote access and support software , provided a simple off-the-shelf communications package to readily facilitate RTSPUS.Any conscious motivated patient who is not in severe respiratory distress and able and willing to follow instructions from a remote mentor is a candidate to participate in a remotely guided self-performed home-surveillance lung program. Musculoskeletal limitations of the arms may be a relative contraindication to performing a complete thoracic evaluation of the back, although recent studies have emphasized examinations limited to the anterior chest, with the most relevant areas of surveillance being the most accessible anterior lung segments in a study performed on hospitalized patients .We are currently conducting a prospective study describing the accuracy and logistics of augmenting RTSPUS to the routine surveillance of paucisymptomatic and at-risk COVID-19 patients. However, the practicality of this approach became dramatically apparent during the recent re-evaluation of our initial self-isolating and healthy volunteers after one of these subjects became infected with COVID-19. Neither subject had ever held an ultrasound prior to agreeing to be remotely mentored. Nevertheless, multiple and complete longitudinal RTSPUS examinations were easily accomplished using widely available cellular phone services, with seamless demonstrations of the pleural anatomy of both the infected patient, and the at-risk partner, during pre- and post-COVID infection examinations Figs. , 4 and 52 saturation or CXR in predicting the future occurrence of adverse outcomes including a requirement for mechanical ventilation and death [It should be understood that at all times this is an additive adjunct to home surveillance which otherwise consists of symptom and standard vital signs monitoring . At all nd death . The onlnd death –19. OnlyRTSPUS may potentially revolutionize home-self monitoring for complications of COVID-19. With this paradigm, remote subject matter experts in both infectious diseases and remote tele-ultrasound can easily conference , 20, to Additional file 1: Video of baseline self-performed lung ultrasound study of 32-year-old patient 6 months prior to COVID-19 Diagnosis.Additional file 2:  Video of first follow-up self-performed lung ultrasound study of 32-year-old patient 2 weeks after COVID-19 Diagnosis.Additional file 3: Video of second follow-up self-performed lung ultrasound study of 32-year-old patient 2 weeks 2 days after COVID-19 Diagnosis.Additional file 4: Video of baseline self-performed lung ultrasound study of 31-year-old spouse 6 months prior to patients COVID-19 Diagnosis.Additional file 5:  Video of first follow-up self-performed lung ultrasound study of 31-year-old spouse 6 months prior to patients COVID-19 Diagnosis.


The magnitude of the host immune response can be regulated by either stimulatory or inhibitory immune checkpoint molecules. Receptor-ligand binding between inhibitory molecules is often exploited by tumours to suppress anti-tumour immune responses. Immune checkpoint inhibitors that block these inhibitory interactions can relieve T-cells from negative regulation, and have yielded remarkable activity in the clinic. Despite this success, clinical data reveal that durable responses are limited to a minority of patients and malignancies, indicating the presence of underlying resistance mechanisms. Accumulating evidence suggests that tumour hypoxia, a pervasive feature of many solid cancers, is a critical phenomenon involved in suppressing the anti-tumour immune response generated by checkpoint inhibitors. In this review, we discuss the mechanisms associated with hypoxia-mediate immunosuppression and focus on modulating tumour hypoxia as an approach to improve immunotherapy responsiveness. Immune checkpoint inhibitors (ICIs) sit at the forefront of cancer immunotherapy. They are emerging as the primary treatment option for many advanced stage cancer patients as a result of their clinical success. Within the last decade, seven ICIs targeting the immune checkpoint receptors cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death protein 1 (PD-1), and the ligand to the checkpoint receptor PD-1 programme death protein ligand 1 (PD-L1) have been approved by the FDA for the treatment of a variety of malignancies ,3,4,5,6.In general, immunotherapies are designed to increase the number and functionality of anti-tumour effector cells. The majority of such treatments target activating antigen-specific T-cells to selectively eliminate tumours through the recognition of unique or aberrantly expressed antigens presented as peptides by major histocompatibility complex (MHC) molecules on the tumour cell surface. ICIs such as anti-CTLA-4, anti-PD-1 and anti-PD-L1 monoclonal antibodies relieve T-cells from negative regulation governed by immune checkpoints by blocking the interactions between immune checkpoint receptors and their respective ligands on tumour cells, infiltrating myeloid cells or T-cells themselves . The immTumours with high expression of PD-L1 respond better to ICIs targeting the PD-1/PD-L1 pathway and are also found to carry more tumour somatic mutations ,13. ThisTumour resistance to ICIs can be classified into three broad categories: (i) primary resistance, in which an anti-tumour immune response cannot be elicited in the patient; (ii) adaptive immune resistance, in which an active anti-tumour immune response is present and can recognise cancerous cells but is unable to eliminate them due to immune evasion mechanisms established within the tumour milieu; and (iii) acquired resistance, in which the tumour initially responds to immunotherapy but subsequently becomes resistant and progresses on therapy ,11,15. TImmunotherapy functions by stimulating the host immune system; an effective immunotherapy must be capable of generating a robust T-cell response that can overcome tumour and TME induced immunosuppression. Apart from co-stimulatory molecules, immune cells also express surface inhibitory receptors that are a part of their intrinsic regulatory mechanism, sanctioned to act as checkpoints that prevent over-activation of the immune response . Such ovIpilimumab (Yervoy™) is a monoclonal antibody that targets the CTLA-4 inhibitory receptor. It was the first checkpoint inhibitor to be approved by the FDA in 2011 for the treatment of patients with unresectable or advanced stage metastatic melanoma . CTLA-4 Since the FDA approval of ipilimumab, six more ICIs targeting the PD-1/PD-L1 inhibitory pathway have been approved for clinical use . Nivolum+ T-cells. The TME can also lead to the upregulation of PD-1 on T-cells themselves, which further impairs the anti-tumour immune response . T. T144]. To date, a limited series of bespoke HAPs have been evaluated in clinical trials, including porfiromycin, banoxantrone, tirapazamine, evofosfamide, PR-104 and tarloxotinib ,147,148.Evofosfamide (TH-302) is a 2-nitroimidazole triggered HAP of a DNA-crosslinking phosphoramidate mustard, which demonstrated considerable hypoxia-selective cytotoxicity in vitro across a range of human cancer cell lines and in vivo anti-tumour efficacy in multiple tumour xenograft models ,150,151.+ T-cells [+ T-cells in the tumour, and allowed evofosfamide, in combination with CTLA-4/PD-1 blockade, to cure most animals bearing TRAMP-C2 tumours  [+ T-cell proliferation and a significant drop in MDSC proliferation [Using a syngeneic murine TRAMP-C2 prostate model, it was demonstrated that the improved blood vessel density observed following evofosfamide administration correlated with a reduction in hypoxic fraction and a simultaneous increase in tumour infiltration of CD3 T-cells . When suurvival) . Evofosfferation . Synergyferation . Overallferation . In immuferation . Promisiferation .2 in gas phase). Under hypoxic conditions, tarloxotinib undergoes controlled fragmentation, leading to the release of a potent, irreversible, pan-human epidermal growth factor receptor (pan-HER) inhibitor (tarloxotinib-TKI) [Tarloxotinib is a molecularly targeted HAP developed from the prototype tyrosine kinase inhibitor prodrug SN29966 ,159. Tarnib-TKI) . The relPreclinical studies have shown the superior anti-tumour activity of tarloxotinib compared to various clinical-stage EGFR-TKIs. Notably, tumour regressions were more profound and durable compared to afatinib (irreversible EGFR inhibitor) and cetuximab  in murine xenograft models of epidermoid carcinoma (A431) and HNSCC (FaDu) . Through+ T-cell/MDSC and CD8+ T-cell/Treg ratios after the administration of tarloxotinib. Further studies showed increased antigen-release to the lymph nodes, increased T-cell function in the tumour and changes in cytokine production profiles after tarloxotinib administration [Tarloxotinib has been demonstrated to enhance the efficacy of ICI. In vivo studies showed that treatment with tarloxotinib reduced the hypoxic fractions within syngeneic murine tumours and delayed tumour growth when combined with ICIs . Investistration ,165. OveCP-506 is a second generation analogue of PR-104 that is resistant to aerobic activation by aldo-keto reductase 1C3 ,167. CP-2 in gas phase). Hypoxia-selective cytotoxicity ratios up to 200-fold were observed in 2D and 3D cell culture models, and CP-506 inhibits the growth of a variety of tumour xenograft models. The observed therapeutic effect was selective for hypoxic cells and causally related to tumour oxygenation. For example, the administration of CP-506 in mice bearing the triple negative breast cancer tumour model MDA-MB-468 is observed to eliminate tumour hypoxia detected by pimonidazole and EF5 binding 24 h later  led to reduced HIF-1-mediated upregulation of CCL28 expression, which can potentially lead to reduced Treg recruitment and Treg-induced tumour angiogenesis . Furthert cancer . The PhaThe novel HIF-2 allosteric inhibitor belzutifan   is curre+ T-cells (increased IFN-γ) [Additional evidence for this hypothesis is illustrated by the targeting of the HIF-1-induced cell surface pH regulatory enzyme, carbonic anhydrase IX (CAIX). CAIX contributes to the acidification of the TME by hydrating carbon dioxide to produce protons, and its expression is associated with poor prognosis . A studyd IFN-γ) .Replicating biological vectors such as viruses or bacteria have been shown to selectively infect, replicate in, and lyse hypoxic tumour tissue, providing an alternative means to eliminate tumour hypoxia. Infection of the tumour microenvironment can often provide an immunostimulatory effect, with the potential for anti-tumour immune responses and synergy with current cancer immunotherapy strategies.Clinicaltrials.gov (accessed 31 March 2021).An oncolytic virus preferentially infects and lyses cancer cells, resulting in the spread of progeny virus particles to adjacent tumour cells where the oncolytic process is repeated . TropismWhilst hypoxia typically inhibits adenoviral replication , other tSalmonella typhimurium (VNP20009) is thought to target tumours due to its preference for hypoxia in tumour cores, in contrast to the more oxygenated outer regions [Many genera of bacteria have been shown to specifically and preferentially target solid tumours and cause tumour cell lysis. Tumour selectivity is usually achieved by utilising the existing characteristics of the tumour microenvironment, such as necrosis and hypoxia, with these areas providing a haven for anaerobic and facultative anaerobic bacteria to germinate ,192. A g regions . FacultaClostridium novyi-NT spores germinate in necrotic regions of tumours and have resulted in objective responses in induced tumours in mouse models and naturally developing neoplasia in companion dogs. When injected intratumourally, C. novyi-NT resulted in increased phagocytosis and NK cell-like function after treatment, while intravenous injection increased LPS-induced TNF-α production, lipoteichoic acid (LTA)-induced IL-10 production and NK cell-like function post-treatment [C. novyi-NT is currently undergoing Phase I evaluation with pembrolizumab (NCT03435952).reatment . C. novyImmune checkpoint inhibitors are undoubtedly a promising treatment for cancer, but their efficacy is often impeded by complex sets of tumour-intrinsic and -extrinsic resistance factors which constrain their broader therapeutic potential. The concept that tumour hypoxia within the TME is one of the major drivers for immunosuppression is becoming well established. Several studies have modulated tumour hypoxia and hypoxia-driven pathways to improve immunotherapy responsiveness with promising results. The hypoxia-targeted therapeutic strategies discussed in this review provide additional therapeutic opportunities to combine with clinical stage ICIs to increase the number of responders amongst cancer patients and improve patient outcome. More preclinical studies are needed to ensure that novel therapies that overcome hypoxia-mediated immunosuppression do so without detrimental effects on the immune system. For example, the strict oxygen-dependence of HAP activation (termed the k-curve) is an important feature given the presence of mild physiological hypoxia in various lymphoid organs ,195,196.