In this article, a neutral modeled tracheobronchial fluid was formulated by partial replacement of some constituents in recipes of previously used lung epithelium fluids with local materials and was used in in vitro bioaccessibility extraction of elements-bound to airborne particulates. Dataset of particulate matters-bound trace elements collected in selected locations Ado – Ekiti is presented and the deposition of elements in different regions of respiratory tracts is estimated using Multiple-path particle deposition (MPPD) mathematic model. The data reveals that the formulated fluid has physical characteristics with superior properties to the existing fluids. The data also shows that bioaccessibility of elements were generally low (<30%) except for Cd and As with relatively moderate values (between 45 and 50%). Additionally, the lung deposition modeling shows that greater percentage of Cd  deposition in the lower alveolar part of the respiratory tract while tracheobronchial and extra-thoracic had 33% and 27% respectively. The data sets can be used as references to analyze data obtained using other formulation.The data presented in this article are related to our work on development of tracheobronchial fluid,  The dataset in this article describes the bioaccessibility of elements-bound airborne particulates collected in selected locations of Ado – Ekiti, an urban center in Southwest, Nigeria using modeled tracheobronchial fluid. 22.1in vitro extractions and to evaluate bioaccessibility [The recipe of formulated fluid used for this work was described in previous works ,2, wheresibility  of some 2.20E, Latitude 7.1000N. It is a fast growing urban settlement that has witnessed tremendous growth resulting from rural-urban migration into the city since it was named as the capital city of the Ekit state in 1996. According to population census of 2006 report, Ado - Ekiti has about 484,798 inhabitants [The data collection center for this article is Ado – Ekiti, the administrative headquarter of Ekiti state, southwest Nigeria and lies on Longitude 5.150abitants , and by 2.3−1for 2 hours. For each sampling, the filters were equilibrated in a charged desiccator for 24 hours and weighed before and after sampling. After weighing, particulates were stored in a refrigerator at 4 °C until analysis. A total of 15 samples were collected in each site during sampling period of this work. The samples were then prepared for elemental analysis and bioaccessibility test.Samples of airborne particulates were collected in five (5) selected sites of Ado - Ekiti city using a Dichtomous Sampler  at 2-week intervals for 5 months from November 2016 to March 2017 at height of 1.5–2 m. Five sampling sites were carefully chosen within the city, to reflect different human activities impacting the environments. The sampler consists of an omnidirectional aerosol inlet and a virtual impactor assembly along with tripod stand. It collects particles in two size ranges; d < 2.5 μm and 2.5 < d < 10μm, which are collected on two separate PTFE Teflon (37mm diameter) filters. The sampler operates at a flow rate of 16.7Lmin2.4in vitro extraction test. For the purpose of this dataset, we used PM2.5 particle size only. Samples were placed into five different a 15-ml polypropylene centrifuge tube, with 0.25g of particulates per tube. Each sample was mixed with 10 ml of formulated tracheobronchial fluid, and the tube was made airtight and immersed for 2 h in a shaking water bath at 37 °C (1-h shaking followed by 1 h still). The obtained extractants were cooled to room temperature, then centrifuged for 20 min at 3500 rpm and separated for element determination. The bioaccessibility (%) was determined as the soluble fraction of an element in PM that dissolved in tracheobronchial fluid [Bioaccessibile of elements bound to airborne particulates collected at different locations were determined in al fluid .2.5http://www.ara.com/products/mppd.htm. The human airway morphometric model selected for this dataset was the Yeh/Schum symmetric. Other parameters chosen for the dataset include inhalant properties (aerosol), Breathing scenario ; time (1), mass concentration (1 mgm−3), Breathing frequency (120 1/min), and tidal volume (625 ml). The data input includes size of the particulate  [ae (μm) is the aerodynamic particle diameter.The amount of airborne particulates intake via inhalation exposure pathway is computed as described in previous article . Multipler, CMD) .(1)Inhal


The zebrafish is now an important model organism for cancer biology studies and provides unique and complementary opportunities in comparison to the mammalian equivalent. The translucency of zebrafish has allowed in vivo live imaging studies of tumour initiation and progression at the cellular level, providing novel insights into our understanding of cancer. Here we summarise the available transgenic zebrafish tumour models and discuss what we have gleaned from them with respect to cancer inflammation. In particular, we focus on the host inflammatory response towards transformed cells during the pre-neoplastic stage of tumour development. We discuss features of tumour-associated macrophages and neutrophils in mammalian models and present evidence that supports the idea that these inflammatory cells promote early stage tumour development and progression. Direct live imaging of tumour initiation in zebrafish models has shown that the intrinsic inflammation induced by pre-neoplastic cells is tumour promoting. Signals mediating leukocyte recruitment to pre-neoplastic cells in zebrafish correspond to the signals that mediate leukocyte recruitment in mammalian tumours. The activation state of macrophages and neutrophils recruited to pre-neoplastic cells in zebrafish appears to be heterogenous, as seen in mammalian models, which provides an opportunity to study the plasticity of innate immune cells during tumour initiation. Although several potential mechanisms are described that might mediate the trophic function of innate immune cells during tumour initiation in zebrafish, there are several unknowns that are yet to be resolved. Rapid advancement of genetic tools and imaging technologies for zebrafish will facilitate research into the mechanisms that modulate leukocyte function during tumour initiation and identify targets for cancer prevention. It has been well documented by pathologists that tumour masses are often densely packed with cells of both adaptive and innate arms of the immune system. In 1986, Dvorak first drew the comparison between tumour formation and wound healing, famously describing the tumours as ‘wounds that do not heal’ . InflammThe zebrafish has been a major vertebrate model for developmental biology since the 1960s. Several forward genetic screens lead to the identification of mutants affecting almost every organ and cell type, and most of the causative genes identified were found to be conserved in the human . The comIn this review we discuss the benefits of transgenic zebrafish models that facilitate in vivo live imaging studies of tumour initiation. We describe the studies conducted in zebrafish that have allowed the field to establish the significance of early host inflammatory responses in promoting cancer development at the pre-neoplastic stage, with a focus on innate myeloid cells. We highlight the signals required for the recruitment of macrophages and neutrophils to PNCs and the heterogeneous nature of their responses. Furthermore, we summarise evidence from the literature that suggests these inflammatory components are conserved in early mammalian tumourigenesis. To do so, we make comparisons with studies of inflammation within early mouse neoplastic lesions where available, and also draw upon studies of tumour-associated macrophages (TAMs) and tumour-associated neutrophils (TANs) in the later stages of mammalian cancer. Whilst tumour infiltrating lymphocytes are also key components of the tumour microenvironment in mammalian cancer ,14,15, tCasper strain has been created, which remains translucent throughout adulthood [Zebrafish larvae develop most major organs, vasculature and a fully functional innate immune system within the first 5 days post fertilization. Most significantly, they are naturally transparent. This transparency, combined with fluorescent labelling, allows real-time observation of single cells in a live in vivo model. Furthermore, by deleting genes required for pigmentation, the dulthood . The mosdulthood . This modulthood .V600E, NRASQ61K and HRASG12V under the melanocyte-specific mitfa promoter, or HRASG12V under the kita promoter, are all sufficient to drive melanoma tumour formation, either alone or in combination with p53−/− mutation [Since the establishment of the Tol2 transposase transgenesis protocol for zebrafish, generation of transgenic strains has become a routine procedure ,20. Thismutation ,22,23,24mutation , pancreamutation  and braimutation   are the most abundant type of leukocyte found within tumours, comprising up to 50% of the tumour mass in epithelial tumours ,83. The Neutrophils are frequently found within tumours and, following the convention of TAMs, the term ‘tumour-associated neutrophil’ (TAN) has been adopted. However, TANs are less well characterised in comparison to their TAM counterparts and have been shown to have both pro- and anti-tumour effects . NonetheIn addition to mature neutrophils, under pathogenic conditions such as chronic inflammatory disease and cancer, immature myeloid cells, known as myeloid-derived suppressor cells (MDSCs), are recruited from the bone marrow prior to terminal differentiation ,108. Gra2 (PGE2)Whilst there is abundant evidence that TAMs and TANs have tumour-promoting roles within established tumours, relatively little is known about the role of macrophages and neutrophils at the earliest stage of tumour initiation. This is largely because the early stages of tumourigenesis are difficult to detect in humans and mammalian models. The exception to this is colorectal cancer, for which distinctive structures known as ‘adenomatous polyps’ are formed at the early neoplastic stage . These p2 is a potent inflammatory mediator, synthesised by the COX-2 enzyme. Both PGE2 and COX-2 are present at high levels in colorectal cancer, adenomatous polyps and even pre-neoplastic microadenoma [2 receptors, was found to dramatically decrease intestinal polyp formation in numerous mouse models of colorectal cancer, including APC mutant mice and mice exposed to carcinogens [PGEoadenoma ,119. Checinogens ,123,124.cinogens ,126,127.cinogens . Unfortucinogens .2 is also implicated in the promotion of many other types of cancer, including cancer of the breast [PGEe breast , liver [e breast , lung [1e breast , brain [e breast  and pance breast ,135. Thie breast ,138,139.Research in this field has been impeded by a lack of suitable in vivo models. Most mouse models for the study of tumourigenesis rely on read-outs such as tumour size and occurrence, whilst measurements of the early pre-neoplastic stage are largely missing. These models also lack temporal resolution and the means to dissect complex mechanisms involving interactions between multiple cell types. For these reasons, the zebrafish is a uniquely advantageous model for the study of inflammation during early tumourigenesis.G12V, KRASG12V, NRASQ61K, Src, Xmrk, Myc and Akt [Studies utilising transparent zebrafish larvae to model tumour initiation were the first to report a trophic inflammatory response at the pre-neoplastic stage. Feng et al. 2010 first showed that leukocytes are rapidly recruited following the expression of a single oncogene . Subsequ and Akt ,142,143.G12V)Tg, a potent chemoattractant at wounds, was identified as the local chemoattractant guiding neutrophils towards PNCs. This discovery was facilitated by the use of live imaging, which allowed a direct comparison of neutrophil dynamics between tumour initiation and wounding, and the use of a fluorescent H2O2 probe to visualise secretion from PNCs. Although both neutrophils and macrophages were observed to engulf material from PNCs, pan-leukocyte depletion (by Gcsfr + PU.1 morpholino) lead to a greatly decreased number of PNCs demonstrating their pro-tumour function [2 was found to be one of the trophic factors released by leukocytes, directly promoting PNC proliferation through the EP1 receptor [2 only partially rescued PNC numbers in pan-leukocyte-depleted larvae, which suggests that leukocytes produce additional, non-redundant trophic signals.In vivo live imaging with fluorescent leukocyte markers in the e clones . Retentifunction ,7. NeutrG12V. Here, neutrophils were recruited within 8 “hours post induction” (hpi) and leukocyte depletion resulted in a 50% reduction of PNC number at 48 hpi [G12V-transformed keratinocytes expressed the invasion-related genes Mmp9 and Slug [Q61K melanoma model, showed upregulation of genes that correspond with growth- and invasion- promoting properties, e.g. FGF-1 and -6, cathepsin-H and galectin1 -and -3 [A more precise temporal resolution of the inflammatory response to tumour initiation was observed by tamoxifen-inducible transformation of keratinocytes with GFP-tagged HRASt 48 hpi . By usinand Slug . PNCs al -and -3 .G12V expression in the hepatocytes of zebrafish larvae triggered a trophic inflammatory response in the liver [G12V, both PNCs and neutrophils expressed high levels of IL-1β and TGF-β. Abrogation of TGF-β signalling reduced liver size, decreased the number of infiltrating neutrophils, and triggered the upregulation of pro-inflammatory genes , indicating that TGF-β is a key modulator of neutrophil function in the pre-neoplastic microenvironment of the liver.Using the Tet-On system to induce KRAShe liver . As seenhe liver . FollowiThe Tet-On inducible system also allowed Yan et al. (2017) to explore the role of both neutrophils and macrophages in response to tumour initiation in the adult fish liver . PNCs exG12V. β-catenin overexpression also caused oncogenic transformation in hepatocytes, with associated recruitment of macrophages and neutrophils [Transformation with other common HCC oncogenes, Xmrk and Myc, also elevated the expression of TGF-β in PNCs and triggered neutrophil and macrophage recruitment to the liver . This detrophils . Whilst trophils . Here to−/− or Cxcr4−/− resulted in even greater suppression of PNC proliferation. This demonstrates that macrophages/microglia have a potent trophic role during glioblastoma initiation and Cxcr4 is required for their recruitment.Glioblastoma can be modelled in the zebrafish by transgenic overexpression of Akt in neural cells, a gene commonly upregulated in human glioblastoma . Oncogen2+ reporter, Chia et al. (2019) discovered that PNC-microglia interactions are governed by Ca2+-dependent ATP release from PNCs and detection of extracellular ATP by the P2y12 receptor on microglia [2+ levels, inhibition of ATP release, or inhibition of the P2y12 receptor all significantly abrogated PNC-microglia interactions and greatly reduced the rate of PNC proliferation, equivalent to that of macrophage/microglia depletion described above [Interestingly, microglia-mediated trophic support to PNCs in the brain might be cell-contact dependent. By live imaging with a transgenic Caicroglia . This meicroglia ,149,150.ed above ,143. TheG12V transformation of astrocytes [G12V-expressing astrocytes also expressed high levels of IL-8, and blocking neutrophil recruitment by abrogation of Rac2 or Cxcr1 significantly reduced the proliferation of pre-neoplastic astrocytes [Whilst Chia et al. (2018) reported an absence of neutrophils in the brain during Akt-dependent tumour initiation, in contrast, neutrophil recruitment into the brain was observed by Powell et al. (2018) following KRAStrocytes ,142. Alttrocytes . In thistrocytes . This suOverall, the above zebrafish studies have identified numerous signals involved in the activation and recruitment of inflammatory leukocytes in response to tumour initiation see , in addiZebrafish skin and liver cancer models showed an upregulation of the pro-inflammatory cytokines IL-1β and TNFα in response to tumour initiation ,140,146.In mouse models of skin and colon cancer knock-out of IL-1β/IL-1R or downstream signal transducer, MyD88, lead to a dramatic and sustained abrogation of leukocyte recruitment, in addition to a reduced occurrence of colon polyps and skin papillomas ,153,154.Knock-out of TNFα or TNFR1 also reduced leukocyte infiltration and increased resistance to tumour development in mouse models of skin and liver cancer ,163,164.Zebrafish models of both brain and liver cancer have indicated that CSF-1 contributes to the recruitment of macrophages in response to tumour initiation ,142. Likmin model of colorectal cancer, Cxcr4−/− mice exhibited reduced macrophage infiltration and polyp formation [Chia et al. (2018) showed that, whilst CSF-1 played a role in macrophage recruitment, signalling to Cxcr4 was absolutely required for the recruitment of macrophages to the Akt-transformed brain in zebrafish tumour initiation, and that PNC-derived Cxcl12 was the major ligand for this interaction . Cxcr4 sormation . Furtherormation . Thus, tormation ,185,186.IL-8 was expressed in zebrafish PNCs in response to oncogenic Ras expression ,187 and The expression of pro-inflammatory cytokines by macrophages and neutrophils in response to tumour initiation in zebrafish studies corresponds with the hypothesis that tumour-associated inflammation begins with a pro-inflammatory response, which becomes progressively more anti-inflammatory throughout the course of tumourigenesis ,200,201.2 is a central component of pro-tumour inflammation in humans and mice, and has been identified as a potent leukocyte-derived trophic signal at the pre-neoplastic stage in zebrafish [2 is also secreted by macrophages within neoplastic polyps in humans and mice [2 signalling has a direct trophic function in mouse models of colon cancer, for example, through activation of β-catenin [2 also increases COX-2 expression in neoplastic cells, creating a positive feedback cycle, which greatly increases the local level of PGE2 in the colon, driving tumourigenesis [As previously discussed, PGEebrafish . In suppand mice ,206,207.-catenin . Macrophigenesis .2 itself is a central inflammatory mediator governing both positive and negative regulation of inflammation through alternative receptors [2 enhances early inflammation, it later exerts an immunosuppressive effect on both macrophages and neutrophils [2 may also contribute to shaping the inflammatory response at the pre-neoplastic stage. For example, the immunosuppressive effect of PGE2 could prevent neutrophils from performing anti-tumour activities and may contribute to the M2/N2 phenotype of pro-tumour leukocytes. Indeed, COX-2 inhibitors have been shown to repolarize M2 TAMs [2 has been implicated in the induction of MDSCs in tumour-bearing mice [Within mammalian immune responses and cancer, COX-2 expression is upregulated by both pro-inflammatory cytokines, such as IL-1β, and tumour promoters, such as Ras ,211,212.eceptors ,214. Whitrophils ,218,219. M2 TAMs ,221,222,ing mice ,224,225.In zebrafish models of liver tumour initiation, high levels of TGF-β expression by PNCs are associated with both disease severity and the expression of anti-inflammatory genes by pro-tumour neutrophils ,141. The2+-mediated ATP release from PNCs and signalling through the P2y12 receptor [Chia et al. (2019) showed that the trophic effect of microglia during glioblastoma tumour initiation was dependent upon Careceptor . This rereceptor ,149,150.receptor . Althougreceptor , extracereceptor ,233,234.Zebrafish cancer models have shown that PNC-induced inflammation is an important player with respect to tumour initiation. Hitherto, the role of inflammatory leukocytes at the pre-neoplastic stage was largely unknown. Imaging of transparent zebrafish larvae has demonstrated that neutrophils and macrophages are recruited in response to tumour initiation and both have a trophic effect upon PNCs. The ever-expanding collection of transgenic reporters, combined with efficient methods for genetic manipulation in the zebrafish, provide a toolbox for the future analysis of cellular and signalling responses within the developing PNC niche. As such, the zebrafish model will provide much needed insight to support the development of novel strategies for cancer prevention.2 is not the sole leukocyte-derived factor influencing PNC proliferation at this stage [Thus far, zebrafish studies have identified some of the chemoattractants, inflammatory mediators and trophic factors that contribute to PNC-induced inflammation. Whilst these signalling molecules correspond with pro-tumour responses in mouse and man, further research is required to confirm their mechanisms of action at the pre-neoplastic stage. There also remain additional signals that are yet to be discovered, for example, it is apparent that PGEis stage . Furtheris stage . Consideis stage ,235,236,In addition to promoting PNC proliferation, zebrafish cancer models have also provided evidence that genes involved in angiogenesis and invasion are expressed at the pre-neoplastic stage by both PNCs and inflammatory cells ,140,144.+ helper T cells [Tg(lck:eGFP) [+ T cell reporter, Tg(CD4-1:mCherry) [Tg(foxp3a:eGFP) [Alongside innate inflammatory cells, tumour-infiltrating lymphocytes also play key roles in the tumour microenvironment ,14,15. F T cells ,248, T r T cells ,249,250  T cells . The chack:eGFP) ; CD4+ T mCherry) ; and regOther common components of the tumour microenvironment include extracellular vesicles (EVs) and stromal cells, which may also play a role at the pre-neoplastic stage. EVs are known to transmit signals that modulate the behaviour of recipient cells. Cancer-derived EVs have been shown to promote M2 polarisation of TAMs in some cases, and the expression of pro-inflammatory cytokines in others ,253,254.As well as detailed mechanistic studies, the zebrafish model is also amenable to high-throughput screening. The small size of zebrafish embryos allows imaging of the whole organism and several individuals can be screened simultaneously. For example, Precazzini et al. (2019) developed an automated, imaging-based screen with 384-well plates to detect modifiers of melanocyte hyper-proliferation in a zebrafish melanoma model . Taking


Angiogenesis is a term that describes the formation of new blood and lymphatic vessels from a pre-existing vasculature. This allows tumour cells to acquire sustenance in the form of nutrients and oxygen and the ability to evacuate metabolic waste. As one of the hallmarks of cancer, angiogenesis has been studied extensively in animal and human models to enable better understanding of cancer biology and the development of new anti-cancer treatments. Angiogenesis plays a crucial role in the process of tumour genesis, because solid tumour need a blood supply if they are to grow beyond a few millimeters in size. On the other hand, there is growing evidence that some solid tumour exploit existing normal blood supply and do not require a new vessel formation to grow and to undergo metastasis. This review of the literature will present the current understanding of this intricate process and the latest advances in the use of angiogenesis-targeting therapies in the fight against cancer. Under physiological conditions, angiogenesis is a highly regulated process. It plays crucial roles in embryogenesis, wound healing and the menstrual cycle . Angioge3 in diameter before they stopped growing and died, whilst some tumour cells could grow beyond 2 mm3 in size in angiogenesis-rich cell culture. The continued growth of cancer cells in angiogenesis-rich cell culture is explained by reproducing physiological properties in a three-dimensional cell culture model that provides controlled fluid perfusion that permits the regulation of oxygen intake, promoting a circulatory environment that is controlled by computer hardware [In addition to providing nutrients and oxygen to the tumour and the removal of metabolic waste, new vessel formation also enables cancer cells to metastasize and proliferate to distant sites through entry into the newly formed blood and lymphatic system and subsequent extravasation . A lack hardware .The structure of the blood vessels depends on their size; small blood vessels are comprised of endothelial cells (EC), whereas in medium and large blood vessels, ECs are surrounded by pericytes  . In normAfter this strictly controlled vessel formation, the normal vasculature becomes largely quiescent . AngiogeIn cancer, a switch to angiogenesis seems to be an imbalance between stimulatory and inhibitory factors that leads to a pro-angiogenic state . This reJudah Folkman coined the phrase tumour angiogenesis and studied this process extensively . He led Disturbance to this equilibrium results in increased angiogenesis, and thus uncontrollable tumour overgrowth . SeveralMoreover, in hypoxic tumour, tumour-associated macrophages (TAMs), known for their protumour functions, secrete VEGF. VEGF interacts with key immune cells in the tumour micro-environment (TME), namely CD4+ forkhead box protein P3 (FOXP3) + regulatory T cells, which a strong suppressor of anticancer immunity. VEGF is able to attract these regulatory T cells to the TME using the chemoattractant neuropilin 1 (NRP1). Animal studies showed that removing NRP1 was associated with increased infiltration of TME with antitumoural CD8 T cells, with a reduction in tumour growth. Fibroblasts, that are present in abundance in TME and known to support tumour growth, also secrete VEGF. VEGF, in turn, stimulates fibronectin fibril assembly; the latter has a potent protumour effect within the TME .Other angiogenesis promotors include platelet-derived growth factor (PDGF)-B and C and fibroblast growth factor (FGF)-1 and -2 . Both grAs seen in Other important regulators of angiogenesis are angiopoietins. Angiopoietins interact with Tie-2 TK receptor found on EC. Through cooperating with other angiogenesis factors, angiopoietins modulate the activity of the EC ,30. AngiOther promotors of angiogenesis include a wide range of polypeptides, metabolites and hormones that contribute to new blood vessels’ formation in both physiological and disease state . On the The activities of both angiogenesis promoters and inhibitors are regulated through a complex interaction of different pathways. The proangiogenic imbalance often occurs at the gene level due to the activation of oncogenes or inactivation of tumour suppressor genes, all the way to cell environmental factors such as hypoxia, hypoglycaemia, cellular nutrient deficiency and metabolic acidosis . As partIn a seminal paper, Judah Folkman, Doug Hanahan and colleagues presented angiogenic switch in a transgenic Rip1Tag2 mouse module of pancreatic beta-cell carcinogenesis, during the progression from hyperplasia to heavily vascularised cancer . Rip1TagAnother important element of angiogenic switch is stromal cells of tumour microenvironment . ThroughEndothelial progenitor cells (EPC), are also believed to play a role in angiogenesis . Tumour-Importantly, not all tumours rely on new blood vessel formation to survive and grow , and theIn addition there is also evidence from preclinical studies that show that some tumors, such NSCLC and gliomas, never undergo angiogenic switch and rely only on vessel co-option ,57. In cThis approach is presumed to work though improving the delivery of cytotoxic agent(s) to the tumour .An example of this is the use of Cilengitide and Verapamil in conjunction with Gemcitabine or Cisplatin to treat pancreatic ductal adenocarcinoma . In highThe impact of the therapy on the tumour blood flow was assessed by flow cytometry, imaging techniques and the concentration of the drugs in vital organs, tumour, and blood levels. The studies showed increased functional (less leaky) vessel formation, leading to an improved tumour blood supply to both highly and poorly vascularized tumors. This effect translated into tumour regression and improved survival in vivo models. The authors showed that vascular promotion increased the cell uptake of Gemcitabine with reduced side effects. The authors also argued that the promotion of vascularization improved the efficacy of Cisplatin due to better tumour blood perfusion, which improved cytotoxic delivery, leading to tumour regression in mice model. Future studies should deal with the impact of this approach in different tumour sites and their secondaries, address the wide variations in tumour behavior caused by intratumor heterogeneity and focus on the potential complications of promoting neovascularization, such as the risk of significant/life threatening bleeding, and its safety in vascular diseases.As mentioned, the infiltration of tumour microenvironment with immune cells, importantly TAM, is associated with pro-angiogenic factor secretions by these cells. Several experiments studied the inhibition of TAM function or their complete removal from the tumour microenvironment. A study showed that treating K14-HPV/E2 mice with Zoledronic acid (ZA), a bisphosphonate used for skeletal metastasis with anti-inflammatory and anti-osteoclast properties, resulted in the suppressed mobilization of VEGF-A and, consequently, the inhibition of angiogenesis and tumourigenesis . Other sThe Food and Drug Administration (FDA) approved biological therapies in the form of tyrosine kinase inhibitors (TKIs), monoclonal antibodies and fusion peptides in non-small cell lung cancer, metastatic colorectal cancer, medullary thyroid cancer and renal cell cancer . More spMonoclonal antibodies such as Bevacizumab, which blocks the VEGF receptor, or small molecules such as Lapatinib, which inhibits TK downstream of VEGF, are examples of anti-VEGF treatment. Phase 1 trial of Bevacizumab showed that the drug was well tolerated and had good pharmacokinetic properties . A phaseCombining Bevacizumab with chemotherapy, in the first and second line settings of mCRC, improved OS . The AVF+ monocytes [+ Gr1+ and the overexpression of alternative angiogenic signaling molecules [Given the results of animal trials, this modest benefit of anti–VEGF-A/VEGFR therapy against human cancers has been relatively disappointing. One explanation for this modest effectiveness is that most cancer patients are elderly, frail and cannot tolerate high doses, in contrast to relatively healthy tumour-bearing mice that can be given higher doses . Anotheronocytes , myeloidonocytes , pro-angolecules , includiolecules , interleolecules , IL-17 [olecules , and angolecules .Vessels’ co-option as a mechanism to attain blood supply by cancer cells is another resistant mechanism to anti-angiogenic treatment. Preclinical models demonstrated a switch from angiogenesis to vessel co-option during anti-angiogenic treatment ,99. The Targeting angiogenesis has shown limited effectiveness to date, but affirms Folkman’s postulations. This limited success is likely caused by the heterogeneity of blood vessels, as some vessels are susceptible, whilst others are resistant, to the inhibition of VEGF/VEGFR. Furthermore, genomic instability would enable cancer cells to bypass the VEGF/VEGFR axis and stimulate new blood vessel growth using alternative signaling pathways. Future therapy should focus on targeting molecules, as well as VEGF, that are present on large blood vessels’ EC lining. Targeting large vessels could stop the blood perfusion to the entire mass of the tumour, hence this would enhance the pruning of microvessels that are sensitive to the inhibition of VEGF/VEGFR. This concept was tested and supported by the findings of a study that utilised photodynamic energy to thrombose and subsequently blocked the main arteries and draining veins of a mouse ear tumour .Another novel strategy is the use of nano-particles to deliver specific anti-angiogenic agents . For exaTwo independent studies by Chen et al.  and Hu eAs explained above, the vessel co-option acts as an important mechanism of resistance to anti-angiogenesis as well as an important source of blood supply that supports the growth of tumors. Therefore, the inhibition of the vessel co-option is the focus of many research groups, through targeting cell motility or adhesion pathways in tumour stroma. In in a mouse model of liver metastases, Frentzas et al.  showed tExcessive, insufficient or abnormal angiogenesis contributes to tumour survival, growth invasion and metastasis. Targeting single angiogenic (pro or inhibitory) molecules showed promising results in animal trials, but has been of limited success in human cancer. To date, despite their modest impact, anti VEGF continues to be one of the treatment lines of several solid malignancies. Nevertheless, it is believed that antiangiogenic monotherapy aiming at single molecule activity is insufficient to combat the myriad of angiogenic factors produced by cancer cells and its microenvironment and this would explain, at least partly, the modest effect of anti VEGF strategies. Future challenges include a detailed understanding of the many angio-modulating pathways in a more integrated manner to identify more holistic therapeutic approaches to improve survival rate in cancer patients.


Background and Objectives: For using appropriate goal-directed fluid therapy during the surgical conditions of pneumoperitoneum in the reverse Trendelenburg position, we investigated the predictability of various hemodynamic parameters for fluid responsiveness by using a mini-volume challenge test. Materials and Methods: 42 adult patients scheduled for laparoscopic cholecystectomy were enrolled. After general anesthesia was induced, CO2 pneumoperitoneum was applied and the patient was placed in the reverse Trendelenburg position. The mini-volume challenge test was carried out with crystalloid 4 mL/kg over 10 min. Hemodynamic parameters, including stroke volume variation (SVV), cardiac index (CI), stroke volume index (SVI), mean arterial pressure (MAP), and heart rate (HR), were measured before and after the mini-volume challenge test. The positive fluid responsiveness was defined as an increase in stroke volume index ≥10% after the mini-volume challenge. For statistical analysis, a Shapiro–Wilk test was used to test the normality of the data. Continuous variables were compared using an unpaired t-test or the Mann–Whitney rank-sum test. Categorical data were compared using the chi-square test. A receiver operating characteristic curve analysis was used to assess the predictability of fluid responsiveness after the mini-volume challenge. Results: 31 patients were fluid responders. Compared with the MAP and HR, the SVV, CI, and SVI showed good predictability for fluid responsiveness after the mini-volume challenge test . Conclusions: SVV and SVI effectively predicted fluid responsiveness after the mini-volume challenge test in patients placed under pneumoperitoneum and in the reverse Trendelenburg position. Laparoscopic surgery is widely performed because of its advantages with smaller surgical wounds, attenuated pain, and early recovery ,2. CurreGoal-directed fluid therapy (GDFT) aims to maintain central euvolemia based on hemodynamic parameters. During GDT, stroke volume variation (SVV), which is a variation in stroke volume (SV) induced by cyclic intrathoracic pressure changes during mechanical ventilation , can be http://cris.nih.go.kr (KCT0002962). This study adhered to the applicable STROBE guidelines. A total of 42 adult patients scheduled for laparoscopic cholecystectomy were enrolled, and written informed consent was obtained from them. The exclusion criteria included body mass index (<18.5 kg/m2 or >35 kg/m2); patients with any gross deformity of the rib cage or spine; patients with heart disease ; patients showing serum creatinine (>1.4 mg/dL); and patients receiving hemodialysis or peritoneal dialysis.This study was approved by the Kyung Hee University Hospital at the Gangdong Institutional Review Board , and written informed consent was obtained from all individual participants included in the study. The trial was registered before patient enrollment at A pneumoperitoneum was created using the Veress needle technique, and a 5 mm trocar was placed in the umbilical region to facilitate the insertion of a 5 mm video scope. Intra-abdominal pressure (IAP) was set at 12 mmHg, and rate of insufflations was increased to 2 to 3 L/min. The patient was placed in a supine position with a 15–20 degrees reverse Trendelenburg tilt and 10 degrees lift on the right side. The epigastric 12 mm port was made, the third 5 mm port was inserted at the mid-clavicular line, and the fourth 5 mm port was cannulated at the right mid-axillary line at the level of the umbilicus.® Base Primea; Fresenius Kabi, Bad Homburg, Germany) with effect-site concentrations of 3 ng/mL. Rocuronium bromide (0.6 mg/kg) was given to facilitate tracheal intubation. Anesthesia was maintained with 3–6% desflurane and 50% oxygen in the medical air. The effect-site concentrations of remifentanil target-controlled infusions were adjusted between 2 and 5 ng/mL. The depth of anesthesia was monitored using the bispectral index  and maintained between 40 and 60. Desflurane administrations and remifentanil target-controlled infusions were intermittently adjusted during surgeries according to the bispectral index or hemodynamic parameters but were not changed during fluid challenges or hemodynamic measurements. Volume-controlled mechanical ventilation using an anesthesia machine  was performed and set with a fixed tidal volume of 8 mL for the ideal body weight and a respiratory rate of 10–16 per minute to maintain an end-tidal carbon dioxide concentration between 35–40 mmHg. A positive end-expiratory pressure of 5 cmH2O was applied in all patients. During the main procedure, all patients were placed in the reverse Trendelenburg with their right side raised. Pneumoperitoneum was achieved by continuous carbon dioxide insufflation, maintaining a 12 cmH2O of IAP.General anesthesia and patient monitoring were performed according to our institutional standards. Intraoperative monitoring included electrocardiography, intra-arterial blood pressure, end-tidal carbon dioxide concentration, and peripheral oxygen saturation. Anesthesia was induced with propofol (2 mg/kg) and target-controlled infusions of remifentanil  via FloTrac™ (Edwards Lifesciences Corp.) sensors. Cardiac output and SV were calculated by real-time arterial waveform analysis. The SVV was computed over 20 s using the following formula:max, SVmin, and SVmean indicate the maximal, minimal, and mean stoke volume in a respiratory cycle, respectively.After zeroing the atmosphere, cardiac output, SV, and SVV values were obtained continually by arterial waveform analysis. SVThe intraoperative basal fluid was administered at a rate of 2 mL/kg/h of crystalloid solution. Hemodynamic parameters were measured just before and 1 min after the fluid challenge. The fluid challenge was carried out with 4 mL/kg of crystalloid solution  over 10 min via an infusion pump. During the fluid challenge, the respiratory rate and anesthetic concentration was not changed. Patients who achieved a stroke volume index (SVI) increase ≥10% after fluid challenge were classified as fluid responders and the others as fluid nonresponders. At each timepoint, the cardiac index (CI), mean arterial pressure (MAP), heart rate (HR), SVI, and SVV were recorded. Our primary outcome was the predictability of each hemodynamic parameter for fluid responsiveness after the mini-volume challenge. For the secondary outcome, we investigated the hemodynamic changes before and after the mini-volume challenge.t-test or the Mann–Whitney rank-sum test. Categorical data between the two groups were compared using the chi-square test or Fisher’s exact test. A paired t-test was used to compare the changes before and after the fluid challenge. In order to compare the changes in hemodynamic variables simultaneously at each timepoint and the differences between responders and nonresponders, the two-way repeated measure analysis of variance was used. The receiver operating characteristic curve (ROC) analysis was performed to assess the predictability of fluid responsiveness after the fluid challenge for each hemodynamic variable . The optimal cut-off value was determined using a value based on the Youden index, which was calculated at the maximum (sensitivity + specificity -1). All results were expressed as mean ± standard deviations, medians (interquartile range), or numbers (%). A p-value <0.05 was regarded as statistically significant. Statistical analysis was performed using MedCalc® version 13.2.0  or SigmaPlot 10.0 .The sample size was determined using the difference between the area under the curve at 0.75  and 0.5 (null hypothesis). Assuming a type I error of 0.05 and the desired power of 0.80, 38 patients were required for the analysis. Expecting a dropout rate of 10%, we enrolled 42 patients. A Shapiro–Wilk test was used to test the normality of the data. Continuous variables were compared using an unpaired Among 42 patients, 31 patients were fluid responders (73.8%). Demographics and perioperative data of patients are shown in p = 0.0001, 0.0012, and 0.0001, respectively).The serial changes in hemodynamic parameters during surgery are shown in p <0.0001). The optimal cut-off values for predicting an SVI increase of ≥10% after the mini-volume challenge in each parameter was ≥11% for the SVV ; ≤2.7 mL/min/m2 for the CI ; and ≤40 mL/m2 for the SVI .The ROC curve of each parameter is shown in In the present study, the SVV, as well as the SVI and CI, showed good predictability for fluid responsiveness after the mini-volume challenge test in patients undergoing laparoscopic cholecystectomy under pneumoperitoneum and being placed in the reverse Trendelenburg position.The GDFT is composed of minimal maintenance fluid therapy and additional volume administrations guided by fluid responsiveness . ExcessiThe SVV has been proven to be a reliable dynamic indicator for predicting fluid responsiveness without the implementation of the fluid challenge ,8,14. ThIn the present study, a relatively small volume of crystalloid (4 mL/kg) was administered to perform fluid challenge. Conventionally, the fluid challenge technique for investigating fluid responsiveness uses 500 mL of colloid  or 7–10 Our results showed that the SVV, CI, and SVI showed good predictability of the SVI increase . Considering CI is derived from the SVI and HR, both the CI and SVI are closely associated with preload-dependent states anticipated during pneumoperitoneum and in the reverse Trendelenburg position. The SVV, which has the diagnostic threshold for fluid responsiveness between 11% and 13%, with a sensitivity of 0.82 and specificity of 0.86 , also shThe present study had several limitations. First, in the present study, we used an uncalibrated system for the SV measurement technique. The uncalibrated SV measurement using the pulse contour analysis of the arterial waveform could be limited in hemodynamically stable patients, thereby being less valuable than using echocardiography or pulmonary arterial catheters. Although pulmonary arterial catheterization or transesophageal echocardiography can provide more reliable hemodynamic parameters, it is clinically too invasive and requires a high medical cost to use in daily practice or anticipated hemodynamically stable surgery. Second, the SVV measurements using the pulse contour analysis technique may be inaccurate in patients with arrhythmia from abnormal chest or abdomen diseases. Thus, it should be a clinical concern when interpreting the SVV for fluid management. Third, we enrolled patients undergoing laparoscopic cholecystectomy. Since laparoscopic cholecystectomy requires a relatively short procedure time and is a low-risk surgery with less hemodynamic changes, a zero-fluid balance strategy would be appropriate, and GDT may not be necessary. Even invasive arterial monitoring with FloTrac™ sensors is not required during laparoscopic cholecystectomy, in contrast with other major surgeries, such as esophagectomy or hepatectomy. However, it requires both pneumoperitoneum and the reverse Trendelenburg position. The results from these conditions would be helpful to apply in other major surgeries requiring GDT. Fourth, although SVV and SVI were useful for predicting fluid responsiveness after the mini-volume challenge test in laparoscopic cholecystectomy during our study, the small sample size could limit its clinical implications, and physicians should be careful when applying the present results to other clinical situations.In conclusion, by using a mini-volume challenge test, the SVV, CI, and SVI can be useful predictors of fluid responsiveness in patients placed under pneumoperitoneum and in the reverse Trendelenburg position.


Radiation-induced glioma (RIG) is a highly aggressive brain cancer arising as a consequence of radiation therapy, for which there is currently no effective treatment. In order to test new drugs in the hope of finding more effective therapies, we need mouse models that faithfully replicate human RIG. Our laboratory collected tumour cells at autopsy from a paediatric patient with RIG following treatment for a different brain tumour. Using these cells, we created a mouse brain tumour model that retains all the characteristics and features of the original patient tumour from which it was derived. This unique model allowed us to study the progression of RIG in the brain, and to identify drugs that may be effective in the treatment of this disease. This mouse model will also allow us to test the efficacy of new treatments, with the hope of improving the prognosis for patients diagnosed with this disease.PIK3CA, upregulation of PDGFRA and AKT2, inactivating mutations in NF1, and a gain-of-function mutation in PTPN11. Additionally, deletion of CDKN2A/B, increased IDH1 expression, and decreased ARID1A expression were observed. Detection of phosphorylated S6, 4EBP1 and ERK via immunohistochemistry confirmed PI3K pathway and ERK activation. Here, we report one of the first PDX models for RIG, which recapitulates the patient disease and is molecularly distinct from primary brain stem glioma. Genetic interrogation of this model has enabled the identification of potential therapeutic vulnerabilities in this currently incurable disease.Radiation-induced glioma (RIG) is a highly aggressive brain cancer arising as a consequence of radiation therapy. We report a case of RIG that arose in the brain stem following treatment for paediatric medulloblastoma, and the development and characterisation of a matched orthotopic patient-derived xenograft (PDX) model (TK-RIG915). Patient and PDX tumours were analysed using DNA methylation profiling, whole genome sequencing (WGS) and RNA sequencing. While initially thought to be a diffuse intrinsic pontine glioma (DIPG) based on disease location, results from methylation profiling and WGS were not consistent with this diagnosis. Furthermore, clustering analyses based on RNA expression suggested the tumours were distinct from primary DIPG. Additional gene expression analysis demonstrated concordance with a published RIG expression profile. Multiple genetic alterations that enhance PI3K/AKT and Ras/Raf/MEK/ERK signalling were discovered in TK-RIG915 including an activating mutation in  Tumours arising in the brain stem, including diffuse midline glioma (DMG) with histone H3 K27M mutation, often referred to as diffuse intrinsic pontine glioma (DIPG), are among the most aggressive types of paediatric brain cancer. Recent molecular characterisation of paediatric high grade gliomas ,2,3 led A small study of five RIGs reported that these tumours are histologically and cytogenetically indistinguishable from de novo paediatric high-grade gliomas . Indeed,ERBB3, SOX10 and PDGFRA has been reported in paediatric RIG [TP53, PDGFRA and PIK3CA, homozygous deletion of CDKN2A and MTAP, and alterations in multiple receptor tyrosine kinase and Ras-Raf-MAPK genes [IDH1, IDH2, H3F3A, HIST1H3B HIST1H3C, ACVR1 or TERT [EGFR amplifications and mutations or deletions in PTEN have been reported in a small number of cases [Studies investigating molecular alterations in paediatric RIGs are few and report small numbers of patient samples. These studies suggest that paediatric RIGs demonstrate more overlap with pilocytic astrocytomas (PAs)  and adultric RIG ,14, as wPK genes ,18,19,20 or TERT ,22, althof cases ,18.The limited molecular data available precludes any real ability of researchers and clinicians to definitively characterise and understand these tumours. In order to discover more effective treatments and to improve survival rates for these patients, we need models through which we can identify potential molecular targets and then test appropriate therapeutics preclinically. Whilst in vitro models are useful, there is a limit to their translational utility, and indeed variation in culture methods can have a significant impact on gene expression and drug responses . The mosPDX models are the gold-standard in cancer research for understanding disease progression and for preclinical testing of new therapies. Although they can be challenging to establish, PDX models reward researchers by closely recapitulating the heterogeneity of different individual cancers ,25, partA previously well four-year-old male presented with a one-week history of vomiting and headache. Magnetic resonance imaging (MRI) of the brain revealed the presence of a large posterior fossa mass with accompanying hydrocephalus a. There The patient underwent resection of the majority of the mass; however, residual disease was evident within the left cerebello-pontine angle on the post-operative MRI c. HistopThe patient was treated as per the Children’s Oncology Group (COG) protocol CCG-99701 Arm B, with radiation and chemotherapy . RadiatiAt nearly 16 years of age, and 11 years after the initial diagnosis of medulloblastoma, the patient presented with a two-week history of left hand and left foot numbness. Clinical examination revealed a new left-sided seventh cranial nerve palsy and mild left upper limb weakness with reduced sensations in a glove and stocking distribution. An MRI showed an enlarging mass within the pons and upper medulla with an expanding lesion showing an area of cavitation and rim enhancement at its posteroinferior margin a. These A PDX model (TK-RIG915) of this disease was successfully generated by implanting tumour cells isolated post-mortem into the brains of immunodeficient mice. Upon tumour-related morbidity, tumours were removed, dissociated and reimplanted into the brains of successive recipient animals a. InitiaHistological assessment of the patient tumour and matched PDX tumours was performed in order to characterise the model and ensure that the PDX faithfully recapitulated the original patient tumour c,d. H&E We sought to fully molecularly characterise TK-RIG915 and the matched patient tumour by performing methylation profiling, RNA sequencing (RNAseq), and whole genome sequencing (WGS) analysis. Using methylation arrays, we attempted to determine the best-fit tumour subclass using the Molecular Neuropathology (MNP) 2.0 classifier . Neithern = 229) [In the absence of a clear CNS tumour classification based on methylation profiling, we performed clustering analysis using RNA expression data to determine which brain tumour subtype our samples most closely related to. We compared the expression profile of the patient and the PDX model against a cohort obtained through the ZERO childhood cancer precision medicine program of high-risk paediatric cancers  . Both thn = 229) ,8.We compared the patient’s clinical history with the criteria defined by Cahan et al. , which iAt the time of diagnosis, the patient tumour from which TK-RIG915 was derived was diagnosed and treated as DIPG; however, our analysis of DNA methylation and RNA expression suggested that this disease, and the subsequent derived PDX model, is a RIG and molecularly distinct from primary DIPG. Therefore, we sought to characterise the molecular mechanisms driving tumour growth in TK-RIG915 by interrogating the DNA methylation and RNAseq data further, as well as performing WGS analysis on the tumour and matched germline DNA from this patient.CDKN2A/B, MTAP and multiple interferon genes on genes a–d. WGS on genes . The varEGFR, TP53 and PTEN [EGFR was not amplified  for H3 K27 tri-methylation (H3K27me3) confirmed this, with both the patient and PDX tumours staining positively a, supporand PTEN ,36. EGFRmplified a,b, and mplified . Copy nu tumours a,b, and  tumours b. These  tumours a.PIK3CA [A number of mutations known to drive PI3K/AKT and Ras/Raf/MEK/ERK signalling were observed in both the patient tumour and TK-RIG915. A known pathogenic activating mutation in PIK3CA  was obsePIK3CA ,39,40 anPIK3CA ,41. ThisPIK3CA . The remPIK3CA  were eitNF1 gene) negatively regulates cellular proliferation by downregulating RAS activity [NF1 was found to harbour a pathogenic stop-gain truncation mutation in exon 26 (p.Glu1123Ter) as well as a frameshift mutation in exon 3 (p.Asn78LysfsTer29), neither of which were previously reported from exome sequencing [PTPN11) (p. Phe285Ser) [CDKN2A/B tumour suppressor locus, drove the growth of this tumour.Pathogenic mutations in two key genes that drive the Ras/Raf/MEK/ERK pathway were identified in both the patient and PDX tumours. Neurofibromin  , which ee285Ser)  and GOF e285Ser) ,49. WhilNF1 transcript levels were significantly lower in both the primary and matched TK-RIG915 PDX tumours compared to both reference sets, consistent with other tumours in the reference cohorts that had homozygous deletion of NF1  transcripts in TK-RIG915 compared to both reference sets . n of NF1 a. In addnce sets a. ARID1A complex  and can  complex ,52,53. AAKT2 and IDH1 transcripts compared to both reference sets , compared to both reference sets were also detected in TK-RIG915 , further supporting that this tumour is distinct from the original diagnosis of primary DIPG.The patient was diagnosed as harbouring a DIPG, according to the WHO-based criteria of the time and based on the MRI characteristics of the tumour. However, our analyses have revealed that the patient tumour and corresponding PDX are molecularly distinct from typical diffuse brain stem gliomas. Specifically, a number of recurrent genetic alterations have been reported for primary DMG including the hallmark H3 K27M mutation, as well as mutations in d PDGFRA ,59,60,61SOX10, ERBB3, PDGFRA, OLIG2, NKX2.2 and BRINP3 . That sutations ,18,21,22ns [TP53 ,18,19,20DNA methylation-based classification has become a widely-accepted method to aid in molecular identification of CNS tumours . InteresPIK3CA observed in TK-RIG915 occurs at a cancer-associated hotspot known to upregulate kinase activity [PIK3CA mutation in TK-RIG915 was mutually exclusive with PTEN mutation/deletion [AKT2 and PDGFRA over-expression, both also frequent aberrations observed in glioma [In the case of the RIG we describe here, there were multiple molecular alterations identified that are targetable and may have therapeutic benefit. Firstly, increased activation of the PI3K/AKT/mTOR pathway was identified as a driver of TK-RIG915 pathogenesis. The mutation in activity ,41. Consdeletion . Other an glioma ,73. Althn glioma .IDH1 was observed in TK-RIG915 and is also consistent with observations in primary GBM [IDH1 have a worse prognosis than IDH1-mutant GBM [IDH1 overexpression, and the subsequent increase in α-ketoglutarate (α-KG), is associated with increased migration of GBM cells and enhanced PI3K/AKT/mTOR pathway activity [IDH1 expression may sensitise GBM cells to radiotherapy, a core clinical treatment for paediatric brain tumours [Overexpression of mary GBM . GBM wittant GBM , and it activity . Encoura tumours .ARID1A. Inactivating mutations or reduced ARID1A expression is common in multiple cancer types including glioma where it is associated with poorer prognosis [ARID1A and activating mutations in PIK3CA [ARID1A in TK-RIG915 may contribute to the network of genetic alterations already driving activation of PI3K signalling. Of note, the regulation of PIK3IP1 is dependent on EZH2 methyltransferase and inhibition of EZH2 activity combined with loss of ARID1A expression is synthetically lethal [TK-RIG915 also demonstrated low RNA expression of the SWI/SNF complex component rognosis ,77. Thern PIK3CA , and ARIn PIK3CA ,52,53. ANF1 loss together with a PTPN11/SHP2 GOF mutation was observed with concomitant upregulation of ERK1/2 phosphorylation. In terms of disease initiation, inherited germline mutations in PTPN11 are associated with Noonan syndrome, where patients have increased risk of cancer development [PTPN11 mutations can increase susceptibility to DNA-damage-induced malignancies [Lastly, hyperactivation of the Ras/Raf/MEK/ERK pathway was identified in TK-RIG915, where elopment . As it hgnancies , it is cgnancies . Given tCDKN2A deletion) may have been effective in slowing or reducing tumour progression [The patient from which TK-RIG915 was derived was offered treatment as part of a clinical trial for DIPG; however, this was ultimately ineffective. Through genomic and transcriptomic analyses we understand now that this tumour was a RIG and is molecularly distinct from typical DIPGs; moreover, this study identified genetic drivers and potential therapeutic targets. The cooperative nature of the genetic alterations driving the PI3K and MEK signalling pathways in TK-RIG915 is summarised in gression ,83,84,85gression ,13,86.This case has uncovered novel insights into radiation-induced brain cancer secondary to medulloblastoma and highlights the importance of enhancing our understanding of these cancers. Most importantly, we have developed and characterised one of the first PDX models of paediatric RIG. The only other reported PDX mouse models of paediatric RIG were derived from a single patient , highligThe parents/guardians of the patient gave their informed consent before donation of the autopsy tissue for research purposes, and for retrospective research access to relevant medical records and previously obtained pathology samples for the same patient. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Child and Adolescent Health Service, Western Australia (HREC: 1769/EP (PRN 0000002372) A Perth Children’s Hospital Oncology Protocol for Collecting and Banking Paediatric Research Specimens; approved 21/08/2003).6 per mouse) were implanted into the brains of four 8-week-old mice using a Hamilton syringe. Upon tumour-related morbidity, the brain was bisected sagittally at the implantation site and one half of the brain containing the tumour was kept for histology. The remaining tumour was removed, dissociated and reimplanted into the brains of successive recipients as described above.Animal experiments were approved by the Animal Ethics Committee of the Telethon Kids Institute and performed in accordance with Australia’s Code for the Care and Use of Animals for Scientific Purposes . Immunodeficient BALB/c nude mice were obtained from the Animal Resources Centre . Approximately 17 h post-mortem, tumour tissue from the patient (ID 738889) was mechanically dissociated, filtered through a 100 μm cell strainer, and suspended in matrigel . Cells , Ki67 , β-catenin  and synaptophysin  and patient autopsy RIG tissue was stained with H&E, GFAP, Ki67 and p53  using the Benchmark Ultra Immunostainer using DAB as a substrate . Reticulin staining was performed as per Gordon and Sweet’s reticulin staining method. All other tissue sections (5 µm) underwent microwave antigen retrieval in a citrate buffer before immunostaining with the following antibodies and dilutions: Olig2 , Nestin , Vimentin , Tri-methyl-histone H3 (K27) , EGFR , PTEN , PDGFRα , phosphorylated S6 (S235/S236) , phosphorylated S6 (S204/S244) , phosphorylated 4EBP1 (T37/T46) , phosphorylated ERK1/2 (T202/Y204) . Additionally, mouse PDX tumour samples were stained with GFAP , Ki67 , and p53 . Sections were incubated with species-specific biotinylated goat anti-IgG secondary antibodies, followed by detection with an Elite ABC kit and NovaRED peroxidase substrate, then counterstained with Gill’s haematoxylin according to manufacturer’s instructions . H&E staining was performed as per standard protocols using a Leica Autostainer XL.Genomic germline DNA was prepared from peripheral blood mononuclear cells using a QIAamp DNA Mini Kit  as per the manufacturer’s instructions for DNA extraction from lymphocytes. Genomic tumour DNA and RNA were prepared from fresh frozen patient and PDX tumour samples using an All Prep DNA/RNA Mini Kit  as per the manufacturer’s instructions. DNA quality was determined by gel electrophoresis and spectrophotometry , and quantified using fluorometry . RNA quality and quantity were determined using the LabChip GX nucleic acid analyser  .DNA from patient tumour and PDX tumours were compared using STR analysis performed at the Genetics Resources Core Facility at Johns Hopkins University  using a PowerPlex 18D kit , PCR product electrophoresed on an ABI Prism 3730xl Genetic Analyzer and data analysed using GeneMapper v4.0 software  as per their standard protocols.www.molecularneuropathology.org, version 11b4 v2.1) [Genomic DNA (500–1000 ng) was treated with sodium bisulphite using the EZ DNA methylation kit  and bisulphite conversion was confirmed by methylation specific PCR. Quantification of DNA methylation was performed at the Australian Genome Research Facility  using the Human Methylation EPIC (EPIC) BeadChip run on an Illumina iScan System using the manufacturer’s standard protocol . Raw idat files were uploaded to an online DNA methylation-based classification of CNS tumours platform (b4 v2.1)  and basiWGS data obtained from the patient germline and tumour DNA samples were analysed as reported in . For theRNAseq analysis and expression profiling was performed as reported in . ClusterTo date, the genetic and molecular alterations underpinning RIG have not been well-characterised. We have added to this limited body of knowledge through extensive characterisation of one of the first PDX models of paediatric RIG. The case report we describe here and TK-RIG915 were molecularly distinct from typical paediatric brain stem glioma and instead showed some genetic overlap with adult primary GBM, concordant with other reports. Whilst some similarities between these tumour types exists, there remain key differences between patterns of genomic and transcriptomic alterations between adult primary GBM and TK-RIG915, and indeed the majority of RIGs previously reported, suggesting that perhaps RIGs may form their own CNS tumour subtype. Extensive characterisation of an increased number of RIGs is crucial to definitively address this possibility and increase our understanding of these tumours. Interrogation of the genetic alterations present in this case revealed a number of potential therapeutic targets that may be effective against this disease including inhibitors of MEK, PI3K, AKT, mTOR, IDH1, EZH2 or CDK4/6. Through the development of a rare RIG PDX, we have created an important tool to help facilitate both drug discovery and novel therapeutic preclinical testing for this highly aggressive and currently fatal disease.


Varicella-zoster virus (VZV), a common and ubiquitous human-restricted pathogen, causes a primary infection (varicella or chickenpox) followed by establishment of latency in sensory ganglia. The virus can reactivate, causing herpes zoster  and leading to significant morbidity but rarely mortality, although in immunocompromised hosts, VZV can cause severe disseminated and occasionally fatal disease. We discuss VZV diseases and the decrease in their incidence due to the introduction of live-attenuated vaccines to prevent varicella or HZ. We also focus on acyclovir, valacyclovir, and famciclovir (FDA approved drugs to treat VZV infections), brivudine (used in some European countries) and amenamevir  that augur the beginning of a new era of anti-VZV therapy. Valnivudine hydrochloride (FV-100) and valomaciclovir stearate (in advanced stage of development) and several new molecules potentially good as anti-VZV candidates described during the last year are examined. We reflect on the role of antiviral agents in the treatment of VZV-associated diseases, as a large percentage of the at-risk population is not immunized, and on the limitations of currently FDA-approved anti-VZV drugs. Their low efficacy in controlling HZ pain and post-herpetic neuralgia development, and the need of multiple dosing regimens requiring daily dose adaptation for patients with renal failure urges the development of novel anti-VZV drugs. Varicella-zoster virus (VZV), also known as human herpesvirus 3 (HHV-3) has a double-stranded DNA genome of 125 kb, encoding for approximately 71 open reading frames (ORFs). The herpesvirus family includes three subfamilies, α, β, and γ-herpesvirinae. VZV together with herpes simplex virus 1 and 2 (HSV-1 and HSV-2) belong to the α-herpesvirinae, characterized by establishment of latency in neurons.VZV is the causative agent of chickenpox (varicella), a common infantile illness. Like all herpesviruses, VZV undergoes a lifelong latent state following primary infection. During latency, the viral DNA persists in the dorsal root ganglia and cranial root ganglia. VZV reactivation produces skin lesions characteristic of herpes zoster (shingles), causing significant morbidity, but rarely mortality . Herpes VZV is highly infectious and enters the body via the respiratory tract, followed by rapid spread from the pharyngeal lymphoid tissue to circulating T lymphocytes . After 1The narrow host range (VZV infection is highly restricted to humans) coupled with the highly cell-associated nature of the virus, has hampered the development of a reliable animal model that mimics VZV diseases in human, thereby hindering VZV pathogenesis studies.Primary VZV infection generally occurs in childhood and is usually mild but complications can occur in adults and in immunocompromised patients. Disseminated varicella infections with multi-organ failure have been reported in both immunocompetent ,4,5 and Similar to other α-herpesviruses, VZV is able to infect the central nervous system (CNS) to cause encephalitis, either as a complication of varicella or HZ, but also in the absence of rash. About 50% of VZV encephalitis cases occur in individuals with immunosuppressive conditions, in whom viral reactivation is more common. A high viral diversity and mixed infections with different clades in the cerebrospinal fluid (CSF) from patients with VZV encephalitis, which can be explained by viral reactivation from multiple neurons, may contribute to the pathogenesis of VZV encephalitis .VZV infections among immunocompromised patients can be more severe, of longer duration than in immunocompetent individuals and may have unusual clinical presentation with multidermal involvement and hyperkeratotic skin lesions ,15. VZV ® , for immunization against VZV infections in 1998. To prevent mild breakthrough infections among vaccinated children following a single dose of vOka, a recommendation for a 2-dose schedule was given in 2006 to improve immunity to VZV. The live-attenuated heterogeneous vaccine preparation vOka is used routinely in many countries worldwide, which resulted in a significant decline in the incidence of varicella disease, with a decrease risk of developing zoster , a structural analogue of the natural compound 2′-deoxyguanosine guanine, Zoviraxuanosine , is a pouanosine . ACV-TP,uanosine  is a safl-valine  due to a®, Vectavir®], a 2′-deoxyguanosine analog, resembles acyclovir in chemical structure, mechanism of action and spectrum of antiviral activity guanine, Denaviractivity  27,28].,28.®, VeE)-5-(2-bromovinyl)-2′-deoxyuridine, bromovinyldeoxyuridine Zostex®, Zonavir®, Zerpex® (Brivudine (BVDU), -N-[2-[4-anilino]-2-oxoethyl]-1,1-dioxothiane-4-carboxamide} (The helicase-primase inhibitor (HPIs) amenamevir {ASP2151, oxamide}  was apprAmenamevir is an oxadiazole phenyl derivative with potent activity against both VZV and HSV , while tCidofovir, the first FDA-approved acyclic nucleoside phosphonate (ANP) for intravenous treatment of HCMV retinitis in AIDS patients in 1996, is mostly used off-label for the intravenous or topical treatment of severe infections caused by various DNA viruses, including various herpesviruses other than HCMV, polyoma-, adeno-, pox- (molluscum contagiosum virus and orf virus) and human papillomaviruses (HPV). In the case of VZV, the drug is used off label for therapy of acyclovir and/or foscarnet resistant infections. Cidofovir has a phosphonate group bypassing the first phosphorylation step by viral kinases. Cellular kinases convert the drug to the active diphosphate form (CDVpp), which acts as a competitive inhibitor of the viral DNA polymerase, causing slow down elongation and premature chain termination during viral DNA synthesis . CDVpp i®), a direct inhibitor of viral DNA polymerases, is independent of activation by the viral TK  have been performed. Therefore, antiviral agents with a higher potency may achieve a more rapid decrease in viral replication consequently reducing neural damage and both acute and chronic symptoms of HZ. In addition, currently available antivirals require 3–5 times daily dosing regimens that need to be modified for patients with renal impairment. The medications used to treat the pain associated with PHN are only palliative, are often insufficient in terms of relief for the patients and do not provide a cure for HZ. Hence, drugs with superior anti-VZV activity, with the ability to prevent PHN, able to provide better pain relief, and with a more simplified dosing regimen are indeed needed. These drugs would also be very useful for the management of disseminated VZV primary infections and complications of VZV reactivation, including VZV vasculopathy, meningoradiculitis, cerebellitis, myelopathy, ocular disease, and zoster sine herpete .The varicella vaccine in infants diminishes the consequences of chickenpox in terms of both healthcare and economic burden, while the zoster vaccine protects immunocompetent adults from HZ and reduces disease severity in those who develop HZ. Adverse events were not seen in clinical trials performed with the VZV vaccines. However, rare but important side effects are being increasingly described most likely due to the increased administration of VZV life vaccines worldwide. A few cases of disseminated varicella infections due to the VZV vaccine strain were described in immunocompromised patients, requiring treatment with antiviral agents ,46. VZV Clinicians should also be aware that very rarely, the varicella vaccine can be transmitted and cause invasive disease as shown by a case report showing that the VZV vaccine was transmitted within a family from a child with shingles resulting in varicella meningitis in an immunocompetent adult .Therefore, antiviral therapy will be needed for treatment of rare VZV diseases even after widespread implementation of vaccination programs.Treatment of VZV infections with acyclovir or valacyclovir in the immunocompetent hosts is not associated with emergence of. In the immunocompromised hosts, VZV infection tends to be severe and persistent, requiring prolonged therapy with antivirals and ACV-R mutants have been isolated after long-term treatment with acyclovir ,56,57,58Resistance to acyclovir in VZV appears because of mutations in either the TK or the DNA polymerase genes. The most frequent mutants isolated both in cell culture and in the clinic are TK mutants ,63. MutaEmergence of ACV-R in the course of a chronic infection caused by the Oka vaccine VZV strain was reported in an immunosuppressed child vaccinated prior a tumor diagnosis requiring intensive antitumor therapy . ClinicaThe gold standard for VZV therapy remains acyclovir and its prodrug valacyclovir. Other nucleoside analogues such as penciclovir, its prodrug famciclovir and brivudine can also be used. These antiviral agents rely on the viral TK for their first phosphorylation and have as target the viral DNA polymerase. VZV mutants arising under pressure with these nucleoside analogues bearing mutations in the viral TK can be treated with foscarnet, a direct inhibitor of viral DNA polymerases. However, foscarnet can be associated with significant renal toxicity and cannot be used for VZV DNA polymerase mutants emerging under acyclovir as most of them show cross-resistance to foscarnet. As cidofovir is a nucleotide analogue that bypass the activation by the viral TK and usually DNA polymerase mutants that are resistant to acyclovir and/or foscarnet remain sensitive to cidofovir, this drug can be used for VZV infections refractory to acyclovir, penciclovir and/or foscarnet. Unfortunately, cidofovir can also cause renal toxicity. Amenamevir, an helicase-primase inhibitor, has only been approved in Japan and its development was discontinued in United States due to toxicity concerns. The drawbacks of the currently available treatments for VZV-associated diseases highpoint the necessity for new, safe and highly effective anti-VZV agents. Drugs able to inhibit virus growth by targeting different steps of the VZV replicative cycle will be very useful for the management of drug-resistance infections in the clinic as well as for limiting the probability of emergence of antiviral drug-resistance and could also form the base for combination therapy.Research should focus on the discovery and development of new anti-herpesvirus compounds having more potent activity than the currently available VZV antivirals. Besides, the search of new lead compounds able to block viral enzymes other than the viral DNA polymerase should be favored. In the last decade, only a few anti-VZV drugs were compared to valacyclovir in clinical trials to evaluate their efficacy in diminishing HZ associated pain and severity. In 1999, the potent and selective anti-VZV activity of some unusual bicyclic nucleoside analogues (BCNAs) was reported by Mc Guigan and collaborators . For BCN50) values as low as 0.0001–0.0005 µM against reference VZV strains as well as clinical isolates in vitro .,87.86,87BCNAs have also different catabolic pathways than BVDU and BVaraU , providi50 for approximately 24 h, supporting the potential for once-a-day dosing in phase II trials. FV-100 was well tolerated at all doses and no serious adverse events were reported.The safety of the oral prodrug of Cf-1743, i.e., FV-100 , was evaluated in three randomized, double-blind, placebo-controlled clinical trials: (i) a single-ascending-dose study in 32 healthy subjects aged 18 to 55 years ; (ii) a multiple-ascending-dose study including 48 subjects (18 to 55 years-old) that received 100 mg once daily (QD), 200 mg QD, 400 mg QD, 400 mg twice a day, and 800 mg QD for 7 days); (iii) a two-part study in subjects aged 65 years and older with a single 400-mg dose in 15 subjects and a 400-mg QD dosing regimen for 7 days in 12 subjects . FV-100 n = 117), FV-100 400 mg 1× per day (n = 116), or valacyclovir 1000 mg 3× per day (n = 117). Burden of illness (BOI), incidence and duration of clinically significant pain (CSP), pain scores, incidence and severity of PHN, and times to full lesion crusting and to lesion healing were used to evaluate efficacy. Safety was evaluated based on adverse event (AE)/ severe adverse events (SAE) profiles, changes in laboratory and vital signs values, and results of electrocardiograms. The BOI scores for pain through 30 days were 114.5 (200 mg FV-100), 110.3 (400 mg FV-100), and 118.0 . The incidences of PHN at 90 days were 17.8%, 12.4%, and 20.2%, respectively, for FV-100 200 mg, FV-100 400 mg, and VACV. Adverse event and SAE profiles were similar in the two FV-100 and the valacyclovir groups. Although this trail missed the primary end-point , a difference between FV-100 400 mg and valacyclovir groups was found in the 90 days data (14% reduction). These results point to a potential effect of FV-100 on subacute and chronic pain and demonstrate the potential utility of FV-100 as an antiviral for the treatment of shingles, diminishing both pain burden of the acute episode and incidence and severity of PHN. Reduced incidence of PHN, the most clinically meaningful endpoint, and reduced use of additional pain medication (opioids) for the management of PHN in the FV-100 arm vs. valacyclovir arm was seen. Compared to the standard of care valacyclovir, FV-100 offers secondary benefits over valacyclovir because FV-100 is once daily dosing vs. valacyclovir three-times dosing.The safety of FV-100 and its efficacy in reducing pain associated with acute HZ compared to valacyclovir was evaluated in a prospective, multicenter, parallel-group, double-blind, randomized study . PatientR)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine, omaciclovir -3methyl]-4[(1-oxooctadecyl)oxo]butylester]   is an orBecause of the limitations associated with the use of cidofovir, the hexadecyloxypropyl (HDP) ester of cidofovir   was syntRegarding the clinical data of brincidofovir against VZV, a retrospective study including 30 HSCT recipients, provided limited-evidence on the potential efficacy of brincidofovir for prophylaxis of HSV and VZV in this group of patients . The sucPhenoxazine  binds strongly to guanine in a duplex and improves TT-TT stacking interactions with adjacent bases. The phenoxazine scaffold is commonly employed to stabilize nucleic acid duplexes and fluorescent probes incorporating a phenoxazine component have been used for the study of nucleic acid structure, recognition, and metabolism. Phenoxazines are well-known in the field of medicinal chemistry because they have various biological activities, i.e., anticancer, antiviral, antidiabetic, antioxidant, anti-Alzheimer, anti-inflammatory, and antibiotic properties .d-ribofuranosyl)-1,3-diaza-2-ox-ophenoxazine (compound 7a)   and 0.56–1.69 µM and 25.9 µM . The PME-5-azaC mono-octadecyl ester (compound 17) was the most effective and selective PME-5-azaC prodrug when evaluated against HSV (EC50 of 0.15–0.35 µM), VZV (EC50’s of 0.32–0.79 µM) and HCMV (EC50’s of 0.24–1.12 µM) compared to a minimal anti-herpesvirus activity of the parent compound PME-5-azaC. Although the bis(hexadecylamido-L-tyrosyl) and the bis(POM) esters of PME-5-azaC proved to be very potent anti-herpesvirus compounds, their selectivity indices were reduced relative to the mono-octadecyl ester prodrug.Novel prodrugs of 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine (PMEO-DAPy) and 1-[2-(phosphonomethoxy)ethyl]-5-azacytosine (PME-5-azaC) prodrugs were synthesized with a pro-moiety consisting of carbonyloxymethyl esters , alkoxyalkyl esters, amino acid phosphoramidates and/or tyrosine  114]. P. P114]. S)-FPMPA amidate prodrug displayed interesting activity against HIV (in the low nanomolar range), HBV and VZV. The (S)-FPMPA amidate prodrug (compound 22a), one of the most active compounds, had 2000-fold better activity than the parent phosphonate against both TK+ and TK− VZV strains (EC50s in the 0.05 μM range). Compound 22a was also active against HCMV but lacked selectivity for HCMV  though it proved selective for VZV. Compounds 25a and 25b (both enantiomers of the guanine containing amidate prodrugs) showed similar activity and selectivity against VZV (EC50’s in the 0.079−0.89 μM range) and HCMV (EC50’s in the 1.98−5.94 μM range), but compound 25b proved more active against HIV and HBV than its diasteromer 25a. Among the pyrimidine analogues, compound 23a (the cytosine containing (S)-Asp-prodrug) was the only congener displaying moderate activity against both VZV TK+ (EC50 = 3.15 µM) and TK− (EC50 = 1.32 µM) viruses and against HCMV [EC50 = 0.76 µM (AD-169 strain) and EC50 = 2.02 μM (Davis strain)].Acyclic nucleosides bearing a 3-fluoro-2-(phosphonomethoxy)propyl (FPMP) side chain have moderate anti-HIV efficacy but are devoid of activity against DNA viruses. Two enantiomeric series of FPMP nucleosides bearing the four natural nucleobases was recently synthesized  Table 3Table 3. S)-cHPMPA bearing different amino acid motifs -3H-pyrazolo1,3,5-triazin-4-one)  in the amino acid sequence of the open reading frame 40 (ORF40), which encodes the major capsid protein, with most of the mutations located in the regions corresponding to the “floor” domain of the major capsid protein of HSV-1. Treatment with 35B2 altered the localization of major capsid protein in vOka-infected fibroblasts but not in fibroblasts infected with the drug-resistant clones. Normal major capsid protein localization in the 35B2-treated infected cells was restored by overexpression of the scaffold proteins. Using electron microscopic analysis, lack of capsid formation could be demonstrated in the 35B2-treated infected cells. These findings indicated a new class of anti-VZV agents targeting the herpesvirus major capsid proteins and inhibiting normal capsid formation.A derivative of pyrazolo1,3,5-triazin-4-one, coded as n-4-one)  was iden45B5 methyl][(4-chlorophenyl)sulfonyl]amine, coded as 45B5 , was ide133G4 [N-(4-chlorophenyl)-5-nitro-3-thienylcarboxamide] (50 = 19.3 µM) but also against HCMV  , which wnst HCMV . The com17a (17a inhibited VZV plaque formation with EC50’s of 1.7–3.6 µM and a selectivity of 10–20 (ratio MCC/ EC50), being inactive against a variety of RNA and DNA viruses. A structure-activity relationship study showed that the substitution on the tryptamine moiety strongly influenced the compounds’ activity/toxicity. The concomitant presence of a biphenyl and methyl(phenyl) carboxy group at the amino group of tryptamine was required for anti-VZV activity.A library of indole-based derivatives was designed, synthesized and evaluated for antiviral activity against a broad spectrum of viruses . The N-b17a  displayeCephalotaxus, harringtonine (HT) and homoharringtonine (HHT)  and 74.71 ng/mL (HTT). The inhibition of VZV replication by HT and HHT was related to down-regulation of VZV lytic genes.The alkaloid esters isolated from the genus ne (HHT) , exhibitGiven that HT and HHT inhibit replication of both RNA and DNA viruses, antiviral activities could be due to effects on cellular rather than viral factor(s) required for viral replication. HT and HHT were suggested to interfere with signal transduction pathways and transcription factors involved in VZV lytic gene expression. Furthermore, HT and HHT were reported to block cell cycle progression at G1/S or G2/M transition and considering that cell cycle regulation affects herpesvirus lytic gene expression and replication, a plausible mechanism to explain the anti-VZV activity of HT and HHT may be through effects on cell cycle in infected cells . This st50 values ranging from 3.6 to 11.3 μM against TK+ and TK− VZV and without measurable cell-growth inhibition. Compound 8a (50 of 3.62 μM (TK+ strain) and 7.85 µM (TK− strain) and CC50 >100 µM, followed by compound 7d [EC50 of ~10–11 µM and CC50 >100 µM]  . None ofDespite the availability of a vaccine for the prevention of pediatric varicella in children and for the prevention of HZ in adults, there will continue to be a need for antiviral drugs. Some people in the elderly category are not able to mount a strong response to the vaccine. In immunocompromised persons, including patients with advanced HIV infection, varicella vaccination should be done exclusively with the subunit HZ vaccine as the life-attenuated vaccine is contraindicated for fear of disseminated vaccine-induced disease. On the other hand, the vaccine coverage is still quite limited and childhood immunization with lower coverage could theoretically shift the epidemiology of the disease leading to an increase in the number of severe cases in older children, teenagers and adults.Infections with VZV are a serious cause of morbidity and mortality among immunosuppressed patients and in the elderly as PHN (the major complication of HZ) can be very difficult to manage. Currently available antiviral agents can shorten the duration of HZ and can promote rash healing, though they are not quite effective in preventing PHN, most likely due to the delay between diagnosis and the start of antiviral treatment. New antiviral chemotherapeutics with a different mechanism of action are under development for the management of HZ, including valnivudine hydrochloride (FV-100), prodrug of the bicyclic nucleoside analogue Cf-1743, the helicase-primase inhibitor amenamevir (ASP2151) that got only approval in Japan because of toxicity issues, and the carbocyclic nucleoside analogue valomaciclovir stearate (EPB-348), the omaciclovir prodrug. Although the development of amenamevir has been halted due to problems of toxicity following a trial in United States, the results with valnivudine hydrochloride and valomaciclovir stearate indicated that these drugs are effective, well-tolerated, with once-daily therapy regimen in the treatment of HZ. Further studies are needed to prove an advantage of valnivudine hydrochloride (FV-100) and valomaciclovir stearate over the standard of care valacyclovir. The development of other helicase-primase inhibitors and new drugs with a different target against VZV would be of value to manage ACV-resistant strains. Furthermore, inhibition of multiple targets in the viral replication cycle has the potential for synergistic effects when used in combination therapy, which would be of particular importance for immunocompromised hosts because it may avoid the need for extended therapeutic regimens and the risk of developing antiviral resistance during prolonged monotherapy. A potential anti-VZV drug candidate needs to be at least as effective and safe as the gold standard of treatment for HZ, i.e., valacyclovir. It will need to have also some advantages over valacyclovir, such as longer intracellular half-life allowing once a day dosing, superior efficacy, independence of TK for activation and/or target another enzyme than the DNA polymerase.Antiviral agents will still have a role in the treatment of VZV-associated diseases. A large percentage of the HZ at-risk population does not receive the vaccine. The currently FDA-approved antiviral drugs, including acyclovir, valacyclovir and famciclovir have low efficacy in the control of pain for HZ patients and a significant proportion of these patients (~20–40%) develops PHN. These antivirals require multiple dosing regimens daily that need to be modified for patients with renal failure. Treatment with currently approved antiviral agents is recommended for VZV-associated infections in immunocompromised individuals, where VZV infections can be very severe. Among immunocompetent patients, antiviral therapy is recommended for adolescents and adults suffering from varicella and for patients  with HZ. Antiviral therapy will also be valuable to treat rare but significant side effects caused by the VZV vaccine. Although development of drug-resistance in considered much less common in VZV than in HSV, emergence of VZV drug-resistance in an emerging concern among immunosuppressed patients who have received prolonged antiviral therapy. Novel antiviral chemotherapeutics with different mode of action than the currently available anti-VZV drugs are required for the treatment of ACV-resistant strains in the immunocompromised host. Furthermore, compounds with a better activity than the currently approved drugs will be extremely useful if they are able to shorten the time to complete crusting of the shingles rash, time to complete pain resolution and prevention of PHN.In the medical literature, cases of coronavirus disease 2019 (COVID-19) and HZ co-infections are being reported. An atypical bilateral acute retinal necrosis was diagnosed in a COVID-19 positive 75-year-old woman, who was immunosuppressed because of chemotherapy for a relapse of diffuse large cell B-cell lymphoma (DLBCL) . Tartari