COVID-19 has significantly exacerbated negative health and social outcomes forpeople who use drugs (PWUD) around the world. The closure of harm reductionservices, ongoing barriers to employment and housing, and pre-existing physicaland mental health conditions have increased harms for diverse communities ofPWUD. Adapting current models of health and human service delivery to bettermeet the needs of PWUD is essential in minimizing not only COVID-19 but alsodrug-related morbidity and mortality. This article draws on research, practice,and advocacy experiences, and discusses the potential for digital health toolssuch as remote monitoring and telecare to improve the continuum of care forPWUD. We call for a digital health strategy for PWUD and provide recommendationsfor future program development and implementation. To better support people who use drugs (PWUD) during this especiallydifficult time, many healthcare providers have turned to established and emergingtechnologies to engage PWUD, including to provide opioid agonist therapy (OAT),follow-up care, consultation, and counseling. However, pre-existing barriers to accessing health and human services facedby marginalized communities are likely to play an important role in determining whobenefits from this turn to digital care, particularly for PWUD. Lessons gatheredfrom the first wave of COVID-19 demonstrate a need for the adaptation of currentservice delivery measures to better meet the unique needs of PWUD. In this article,we consider the impacts of COVID-19 on harm reduction programing and the possibilityof developing a digital health strategy to prevent treatment interruptions andenable expanded care options. This article is based on the team’s respectiveexperience as substance use and harm reduction researchers, health and human serviceproviders , activists and persons with livedexperience. We have integrated literature and insights from a program of research onthe needs of PWUD during the pandemic, clinical practice experience, and lessonsfrom our networks.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (COVID-19) pandemichas necessitated a digital health care revolution  due to related public health physical distancing requirements toreduce transmission.6Increasing contamination and toxicity of the illicit drug supply has been reportedby PWUD and drug checking services in Canada and the United Kingdom and iscontributing to worsening overdose rates.8 Factors that limited access tohealth and human services prior to the pandemic include stigma and discrimination,poverty, structural racism and the criminalization of drug use.10 PWUD may be at risk forCOVID-19 related complications due to a combination of biopsychosocial factors suchas income, housing, and food insecurity which reduce one’s ability to isolate,substance use related risks , and high ratesof comorbid illnesses ).11 PWUD may be at risk of COVID-19 transmission through sharingsupplies relating to smoking equipment, which has been documented to be more commonthan sharing injection supplies. Additionally, PWUD have experienced increasing opioid-related morbidity andmortality during the pandemic.The COVID-19 pandemic has significantly exacerbated health disparities for PWUDworldwide due to disruptions in drug supplies, drug markets, and access to harmreduction care. Ontario, another Canadian province, reported similar trends, withapproximately 55 people dying every week of suspected overdoses, roughly a 25%increase in overdose deaths from the median in 2019. This represents the highest opioid overdose death rate since monitoring began. Similar findings have been reported across the United States where reportsindicate that there were over 81,230 drug-related deaths in the year leading up toMay 2020, with the highest percentages occurring between March 2020 and May 2020 asa result of COVID-19. Experiences with drug and COVID-19 related morbidities, mirrored with thelack of available services has left many PWUD relying on emergency rooms for care. This lack of availability has escalated drug-related harms internationally,with reports from contexts as diverse as Bangalore and the United Kingdom indicatingan increase in alcohol and substance-related withdrawal symptoms.19Despite being known for its social welfare system and relatively liberal approach tothe management of illicit drug use relative to the United States, Canada has hadconcerning trends of opioid-related deaths since the onset of the pandemic. In theprovince of British Columbia, opioid overdose deaths in November 2020 (n = 153)increased by 89% compared to November 2019. Many addiction treatment programs have experienced similar barriers, alongwith concerns about physician availability, and some have reduced new client intakesand closed treatment programs (e.g. day programs) which reduces the availability of OAT. Many PWUD have experienced problems in accessing sterile equipment, overdoseprevention education, and community support. A further consequence of programclosures are reduced opportunities for peer employment and engagement, disconnectingPWUD from their communities and reducing well-being.10 To develop accessible andeffective harm reduction interventions, the unique challenges faced by both serviceusers and providers because of COVID-19 must be considered.Harm reduction programs, many of which are precariously funded in the best of times,have faced barriers to maintaining adequate service levels during COVID-19. This hasincluded increased physical distancing and infection control requirements , a lack of personal protextiveequipment and infection control, infections among staff and clients which has led totemporary closures and service restrictions, and staffing and financial resourcesbeing redeployed to other areas of public health.21 An innovative approach waslaunched in British Columbia which loosened prescribing guidelines as part of abroad risk mitigation strategy to support PWUD to physically distance by providingthem with a prescription for oral hydromorphone and benzodiazepines and some stimulants. There have been calls for the scale-up of a ‘safe supply’ (prescription ofsafe drugs to offset the risk of illicit street drugs and meet the needs of PWUD),but questions remain about access. Incorporating telemedicine and remote care strategies in both prescribingpractices and delivery of care to PWUD is an effective alternative to supportexisting clients and patients, minimize stigma associated with in person care forPWUD , improve willingness to participate incare , and expand theavailability of care to remote or rural regions.24Key lessons from the first wave of COVID-19 in Canada, and subsequent localizedlockdowns, include the need for accessible and scaled-up OAT, safe supply programs,increased drug checking, and the importance of maintaining the continuity of carefor PWUD (inclusive of HIV and HCV care cascades).Using existing telecare technology to meet the needs of PWUD: In order to provide care for PWUD during the COVID-19 pandemic, somephysicians and pharmacists have used telehealth options (phone and videoconsultation) to offer OAT and ‘safe supply’ prescribing.2528 The integration of telecarefor providing such treatments have been recommended by the Canadian ResearchInitiative in Substance Misuse. Telecare is also being used in the management of acute withdrawal symptomsand the British Columbia Centre on Substance Use has outlined recommendations forphysicians to treat alcohol withdrawal symptoms. When deemed safe, care forwithdrawal may be provided through telecare services, such as conducting regulartelephone or video assessments for clients. This has allowed PWUD and healthcare providers to maintain COVID-19 physicaldistancing protocols while maintaining treatment regimens and connection, andminimizing the risk of alcohol or drug-related morbidity and mortality. As telecare develops, team-based care options inclusive of physicians, nursepractitioners, social workers, pharmacists, and harm reduction workers withlived/living expertise of drug use should be offered to PWUD to ensure theirpsychological and material needs are also met. Many people living in poverty do not have basic phone coverage to make orreceive a telehealth call let alone the data to videoconference with a provider. Bruneau and colleagues identified the following factors which physicians must consider when adaptingthe provision of drug-related care to telecare options: the accessibility oftelecare options must be assessed on a case-by-case basis, and platforms used fortelecare implementation must be secure and should be explained prior to everyconsultation to ensure informed consent. Advancing telecare should includediscussions about access and in-person options. In the meantime, we are aware ofhealth and human services which have remained open serving as brokers to assist PWUDto access care providers and make and receive phone and video calls. Before thepandemic, many harm reduction programs offered a critical service by letting PWUDaccess Wi-Fi networks. With other services like public libraries and coffee shopsclosed, this basic service is more important than ever.Despite the advantages telecare presents for PWUD, the challenges faced in developingand implementing these programs are significant. Common barriers include the lack ofacceptance or limited availability of technical resources for prescribers or patients.Beyond telecare to complement the care of PWUD with digitaloptions: In addition to telecare, a range of digital healthapplications and services have implications for PWUD. The availability of apps andother technology platforms to support safer drug use are limited, but comparablemodels are available to support managed alcohol consumption and smoking cessation. Technology-based tools or apps are typically used to promote behaviour changeand mitigate risk behaviours among PWUD. However we can draw upon tools thatcurrently exist, and some under development, to support PWUD to use drugs safely andreduce the risk of overdose. For example, the Overdose Risk InfOrmatioN tool (ORION) is an innovativee-health tool implemented in clinical settings across Europe, which incorporatesoverdose risk assessments and provides individuals with information on future harmreduction practices and behaviours which aim at reducing overdose rates (i.e.testing drugs prior to use). An evaluation of ORION (n = 194) demonstrated that over half of theindividuals who engaged with the tool developed some sense of increased overdoseprevention education.33 However, many PWUD experience barriers to engaging withtreatments programs .33 Alternative approaches needto be considered for persons who are not ready or interested in treatment andabstinence. To this end, mobile apps are being developed and implemented to assistin providing remote care for PWUD. Apps aimed at minimizing drug-related harms include services such as remotesupervision  andguidance about safer drug use and overdose prevention. The use of digital services and apps must also be considered in relation tonaloxone distribution. Services which foster delivery of naloxone kits have been developed and should be integrated within regularly provided digital care for PWUD tominimize risks of overdose as a result of using alone. In order for the effectivedistribution of naloxone through digital methods, the various distribution policiesbetween jurisdictions must be consolidated. Additional features of harm reduction based apps include information onsocial services available to individuals or a wearable component  that detexts lack of movement associated with overdoses. The integration of mobile apps for the delivery of health and human servicesmay also improve the development of relationships between PWUD and serviceproviders, and mitigate fear, distrust, or discomfort associated with in personcare.Several examples demonstrate how online services can be used to provide individualswith addiction treatment. The initiative is a partnership between the Provincial Health ServicesAuthority, regional health authorities and Lifeguard Digital Health. The COVID-19 pandemic has highlighted urban/rural health disparities andcreated opportunities to assess such supports for PWUD in rural and remote areas.The COVID-19 pandemic has furthered policy discussions about the digitaldivide39  and how to scale-up broadband coveragein underserved communities. As well, whilst we recognize the life-saving potentialof these apps, and that public private partnerships may speed up their development, we call for these technologies to be user controlled and that data not beshared with law enforcement. The need to ensure privacy of personal health information is essential inminimizing the utilization of data by private corporations involved in thedevelopment of technologies.In light of the COVID-19 pandemic, British Columbia has implemented the LifeguardApp, which allows PWUD to communicate with emergency services while they are usingsubstances to minimize risk of fatal overdoses. Previous work has identified the potential advantage that incorporatingtechnology has for PWUD, such as allowing for personalized care and cost effectiveways to reach a wide variety of individuals. Regions such as Australia have developed a nationwide telecare strategy,which focuses on delivering guidance to health care providers on how to integratetelecare in their respective practices . The Australian example has important implications for Canada and the UnitedStates, but differences in how health and public goods are funded remain importantconsiderations. As noted previously, despite Canada having universal access tohealthcare, considerable barriers and disparities exist between rural and urbansettings and among those without health insurance due to housing insecurity orprecarious immigration status. In the United States, access to healthcare withconsiderable variation between urban/rural and on a state-by-state basis, anddraconian drug-polices will need to be considered and overcome. Both countries haveongoing racial disparities with Black, Indigenous, and people of color, includingthose who use drugs, experiencing stigma, marginalization, and exclusion in thehealthcare system. Sustainability plans should be developed with equitableco-leadership from directly impacted communities of PWUD, especially those facingintersecting barriers to accessing healthcare as a result of structural violence andthose facing higher rates of criminalization due to systemic anti-Indigenous andanti-Black racism.Despite the need to simultaneously overcome long-term disparities and deliver rapidimplementation and scale-up during COVID-19, the use of technology mediated care ispromising for PWUD. However, this digital turn requires additional consideration forthose who do not have access to health, social, and harm reduction services,particularly PWUD who reside in remote or rural regions and who lack access tosupervised consumption sites, needle and syringe distribution programs and othercommon harm reduction practices.The path towards scale-up and sustainability will require research in, and evaluationof, the development of technology-based interventions for PWUD. A factor that mustbe considered in the scaling-up of technology-based interventions is the low-barriernature of harm reduction programs. This may result in many interventions offering awide variety of technology (i.e. smart phone or Wi-Fi), considering how the COVID-19response has limited access to Wi-Fi and other technology for PWUD, and ensuringthat the lack of continuous access to technology is not used to discharge or dismissclients. To effectively implement digital care for PWUD, research must be done withmembers of the community and be attentive to potential ethical, legal, and safetyconcerns. Above increased attention to research and program development, we call fora systems level change with integration of strong legal protextions for PWUD and aapproach led by and for PWUD.COVID-19 has exacerbated existing inequities among PWUD while creating uniquechallenges for healthcare providers working with PWUD. To effectively develop andprovide health care to PWUD during and after the COVID-19 pandemic, primary care,mental health, addiction, and harm reduction services should continue to integrateand advance digital health care strategies. As the overdose crisis continues tomerge with the COVID-19 pandemic we need to utilize all available methods to reducedrug related harms and overdose fatalities. Scale up of these services is necessarynow and long past the containment of COVID-19 as a strategy for addressing thedecades long overdose crisis. Meaningful involvement of those with lived and livingexpertise of drug use is needed to ensure that these strategies do not increaseharm.


They resemble the natural formations of organs, cells have to be correctly located relative to each other. It has been shown in cocultures that cells could show spontaneous tissue-like organization when seeded concurrently into the scaffold. The ideal scaffolds should have an interconnected porous structure, well-designed pore size, and adequate porosity not only to allow cell attachment, proliferation, and differentiation, butalso the effective bioactive agents and nutrient exchange during new tissue development. A three-dimensional scaffold is seeded with desired cell types, while an individual organ cell is a specific mechanism for the construction or regeneration of the cells. Particularly, bone organ development at the affected place eventually reduces in vitro practically every tissue and organ in the body. Work is proceeding in creating tissue-engineered liver, nerve, kidney, intestine, pancreas, and even heart muscle and valves. In connective tissues, work has been ongoing worldwide for many years in the engineering of tendon, ligament, bone, and cartilage. Recently, there have many reports of success in skin, bladder, airway, and bone, where tissue-engineered constructs have been used successfully in patients. As the subject of this special issue is the collection of body organ regeneration materials and their Cell Adhesion and Migration for the development and regeneration of the tissues, in this regard biomaterials are significantly involved in the development of the repair of bone tissues. The collected articles report the use and efficacy of different materials like scaffold, composite, 3 days materials to the repair and development of osteoblast, cartilage repair, chondrocytes, and etc. Meanwhile, a review by Tang et al. discusses the recent trends in the development of bone regenerative biomaterials and the use of traditional and modern bone defect repair biomaterials for bone tissue regeneration .Artificial scaffolds have been applied and used as a supporting structure for cell cultures and the domination of cell growth in repairing impaired tissues or organs. During cell regeneration, the scaffold temporarily helps in cell regeneration. It gradually biodegrades either in the healing process or after, producing a new tissue with a desired shape and properties. The challenge of tissue engineering is to mimic what happens in nature. Attempts are being made to engineer Zhu et al. presented the potential of biomimetic intrinsically disordered proteins as bone graft materials . Two biomimetic peptides (P2 and P6) are incorporated into the SmartBonePepR composite to increase the bioactivity of the bone regeneration ability. The SmartBonePepR composite proved multimodal biological effects as good viability, proliferation rate on human MSC cells in-vitro cell analysis, and gene expression analysis. Diwu et al. reported a perfect mimic of human bone material such as selenium substituted Hydroxyapatite (HAP-Se) covered by lactic acid (LA)—Polyethylene glycol (PEG)—Aspartic acid (AS) composite with the loading of vincristine sulfate (VCR) drug (HAP-Se/LA-PEG-AS/VCR) was fabricated for twin purposes of bone regenerations . The HAP-Se/LA-PEG-AS/VCR composite was coated on a titanium implant through electrophoretic deposition (EPD). The porous nature of the composite has expressed the more exceptional biocompatibility in bone cells and toxicity with the cancer cells of prepared composites. The outcome of the investigation proposed the biomaterial suitable for implantation and helps accelerate bone regeneration on osteoporosis and osteosarcoma affected hard tissue.in situ . Autogenous cartilage transplantation, allogenic cartilage transplantation, and artificial substitutes are therapeutic options. An acellular matrix (AM) as a natural biomaterial is gaining increasing attention in tissue engineering applications. An acellular cartilaginous matrix (ACM) and acellular dermal matrix (ADM) are two kinds of the most widely used AMs in cartilage tissue engineering. However, there is still debate over which of these AMs achieves optimal cartilage regeneration, especially in large immunocompetent animals. Wang et al. fabricated porous ADM and ACM scaffolds by a freeze-drying method and confirmed that ADM had a larger pore size than ACM . By recolonizing with goat auricular chondrocytes and in vitro culture, ADM scaffolds exhibited a higher cell adhesion rate, more homogeneous chondrocyte distribution, and neocartilage formation than ACM. Additionally, quantitative polymerase chain reaction (qPCR) indicated that expression of cartilage-related genes, including ACAN, COLIIA1, and SOX9, was significantly higher in the ADM group than the ACM group. In summary, the ADM is appropriate for cartilage regeneration, which can be used for cartilage regeneration in large immunocompetent animals.Cartilaginous defect repair is difficult because of the avascular nature and limited regeneration of the ability of cartilage Ci et al.reported cartilage sheet was prepared into engineered Cartilage gel (ECG) and combined with DBM to explore the feasibility of regenerating 3D cartilage with controlled shape and mechanical strength . The authors introduced the new concept for cartilage regenerations by analogous steel reinforced composite packed ECG and DBM materials. A Biomimetic Biphasic Scaffold Consisting of Decellularized Cartilage and Decalcified Bone Matrixes for Osteochondral Defect Repair was reported by Cao et al. and Bosch-Rué et al. This study developed a biomimsubchondraletic biphasic scaffold for OCD repair via an iterative layered lyophilization technique that controlled the composition, substrate stiffness, and pore size in each phase of the scaffold. The biphasic scaffold consisted of a superficial decellularized cartilage matrix (DCM) and underlying decalcified bone matrix (DBM) materials used for osteochondral tissue regenerations. The results demonstrate that the biomimetic biphasic scaffold has a good osteochondral repair effect.Cao et al.). A biomimetic scaffold with a separated-ring structure—a polycaprolactone (PCL) scaffold with a ring-hollow alternating structure—was three-dimensionally printed as a framework. The collagen sponge was embedded in the hollows amid the PCL rings by pouring, followed by lyophilization. The experimental results showed substantial deposition of tracheal cartilage and formation of a biomimetic trachea mimicking the native trachea both structurally and mechanically. The investigation highlights the advantage of a biomimetic trachea with a separated-ring structure that mimics the native trachea both structurally and mechanically and demonstrates its promise in repairing long-segment tracheal defects.The recent advances in bone regenerations by 3D printed biomimetic scaffold materials are a rapid development of tissue engineering technology, and have provided new methods for tracheal replacement (


Novel immunotherapies are increasingly being employed in pediatric oncology, both in the upfront and relapsed/refractory settings. Through various mechanisms of action, engagement and activation of the immune system can cause both generalized and disease site-specific inflammation, leading to immune-related adverse events (irAEs). One of the most worrisome irAEs is that of neurotoxicity. This can present as a large spectrum of neurological toxicities, including confusion, aphasia, neuropathies, seizures, and/or death, with variable onset and severity. Earlier identification and treatment, generally with corticosteroids, remains the mainstay of neurotoxicity management to optimize patient outcomes. The pathophysiology of neurotoxicity varies across the different therapeutic strategies and remains to be elucidated in most cases. Furthermore, little is known about long-term neurologic sequelae. This review will focus on neurotoxicity seen with the most common immunotherapies used in pediatric oncology, including CAR T cell therapy, alternative forms of adoptive cell therapy, antibody therapies, immune checkpoint inhibitors, and tumor vaccines. Herein we will discuss the incidence, pathophysiology, symptomatology, diagnosis, and management strategies currently being utilized for immunotherapy-associated neurotoxicity with a focus on pediatric specific considerations. The rapid emergence of novel immunotherapies has dramatically shifted the treatment paradigm in oncology, particularly in those with relapsed/refractory disease where standard therapies have failed. These novel therapies strive to overcome chemotherapeutic and radiotherapy resistance by harnessing the immune system to treat malignancies. Approaches include direct engagement of the immune system, indirect activation of the intrinsic immune response, or targeted delivery of anti-cancer therapeutics. We also include select non-immunotherapy based targeted antibody therapies in this review, such as bevacizumab, alongside other immunotherapeutic agents, given the side effect profiles and indications for use significantly overlaps with that of immunotherapies. With novel mechanisms of action, a host of unique adverse events have also materialized. Generally categorized as immune-related adverse events (irAEs), the constellation of toxicities is broad and includes inflammation-mediated manifestations, components of autoimmunity, and/or on-target/off-tumor effects amongst other etiologies Table 1 Utilization of immunotherapy in pediatric oncology continues to expand, necessitating both an enhanced understanding of how irAEs may manifest and how management strategies may differ in younger children. While generalized irAE or immune effector cell related toxicity management guidelines are increasingly available –14, expeThe most important clinical consideration in children is the difference in approach toward recognition of neurotoxicity. Some tools that are used for detecting neurotoxicity in adults rely largely on language function and cannot be used for young children . Mild orApart from behavioral changes, several other signs and symptoms deserve special consideration in the pediatric population. Headache alone may or may not be considered a true neurotoxicity, yet it can be a harbinger of more serious CNS pathology. Headache can be difficult to ascertain in very young children, who may present instead with irritability. Changes in motor strength often go unrecognized in ill young children because of their limited ability to participate in a direct confrontational exam. Other unique considerations in pediatrics include the type of workup required for neurotoxicity, as children may require sedation for MRI, which can delay diagnosis, and may not be able to tolerate prolonged electroencephalography (EEG) monitoring or nerve conduction studies, hampering our ability to detect subtle toxicities which may be underrecognized.Involvement of neurology consult services should occur prospectively, particularly in patients who have preexisting neurologic comorbidities, those with neurodevelopmental differences , or thosThere is no evidence currently to suggest differences in the mechanism of neurotoxicity-associated with immunotherapy between children and adults. Indeed, toxicities of CD19-directed CAR T cell therapy have been well studied in both children and adults with ALL, with similar rates of neurotoxicity and CRS Table 2.Hematologic malignancies in pediatrics are the most diagnosed pediatric cancers and include acute and chronic leukemias, and both Hodgkin and Non-Hodgkin lymphomas . In geneDespite the remarkable success rates in treating pediatric hematologic malignancies, these patients are at high risk of neurologic sequelae as a result of multi-modal therapy with systemic chemotherapy, repetitive intrathecal therapy, radiation therapy , and hematopoietic stem cell transplantation. Neurocognitive deficits are common, especially if therapy is administered early in childhood , 36. AddGiven that immunotherapy is currently used in relapsed/refractory disease, the impact of prior therapy is important to note, as patients may already have neurologic comorbidities that may impact the patients’ ability to tolerate additional neurologic insults or leave them more vulnerable to neurotoxicity from additional therapy. As timing of immunotherapy continues to evolve with more trials moving it into the upfront treatment setting, immunotherapy may represent an attractive opportunity to decrease neurologic sequelae in this patient population.Pediatric solid tumors located outside the central nervous system (CNS) comprise 20% of all newly diagnosed pediatric cancers. This broad category is composed of sarcomas, neuroblastoma, Wilms tumor, hepatoblastoma, germ cell tumors and other rare malignancies . While tPrimary tumors of the CNS are the second most diagnosed pediatric malignancy, and the leading cause of cancer death in the pediatric population , 44. AppImmunotherapy for pediatric brain tumors is evolving rapidly as a promising new treatment that can potentially improve both morbidity and neurocognitive outcomes in survivors –51. ImpoAnother major challenge in CNS tumor immunotherapy is how to distinguish irAEs  from tumor progression, which is important for ongoing medical treatment management. There are currently no imaging techniques that can reliably distinguish immune-related pseudoprogression from tumor growth in the acute setting . In 2015In addition to primary CNS tumors, brain metastases from non-CNS primary solid tumors and hematologic malignancies also cause significant morbidity. Brain metastases occur in approximately 1.4-4.9% of children with solid tumors –60, and Adoptive cell therapy uses immune cells to target cancer epitopes, either by selecting tumor-targeting immune cell subsets, or by genetically modifying them. The current most successful modality, chimeric antigen receptor (CAR) T cells, uses the latter approach. CARs are synthetic molecules that combine the antibody recognition properties (non-MHC restricted) of a B cell and the effector functions of a T cell. They consist of two domains, an extracellular binder  against a specific cell surface tumor epitope, and an intracellular signaling domain that induces target killing and T cell proliferation –66. The CAR T cell therapy has revolutionized cancer treatment by inducing durable remissions in pediatric patients with relapsed/refractory B cell leukemia (B-ALL). Several studies have been published using CAR T cells for treatment of pediatric patients with relapsed/refractory B cell hematologic malignancies and have demonstrated impressive complete remission rates of 60-90% –72. The Neurotoxicity, now known as immune effector cell-associated neurotoxicity syndrome (ICANS) , is a coAcute neurocognitive functioning and patient reported outcomes measures have also been evaluated in pediatric patients receiving CAR therapy. Data collection in this patient population is feasible, with stable neurocognitive function and clinically meaningful health-related quality of life improvements demonstrated over time , 81.As the application for CAR T cells continue to broaden, so too may the toxicity profile. Several hematologic CAR T cell trials in pediatrics are ongoing including those targeting CD5 and CD7 in T-ALL, and CD33 and CD123 in AML. Additionally, combinatorial CAR constructs with CD19, CD22, and/or CD20 are currently in clinical trials.The pathophysiology of ICANS post-CAR therapy remains under active investigation. Endothelial cell activation, vascular and blood brain barrier disruption, vascular leak, systemic inflammatory response with CRS, and off-tumor/on-target toxicity are all possible mechanisms by which neurotoxicity can occur in pediatric patients , 82, 83.The presence and severity of cytokine release syndrome is the strongest and most consistent risk factor for ICANS in pediatric CAR T cell patients , 77, 91.The American Society for Transplantation and Cellular Therapy (ASTCT) grading criteria are now used to grade ICANS . ICANS gBased on the clinical data that has emerged from CD19-directed therapy in pediatric and young adult patients receiving CAR T cells, management of ICANS remains largely supportive, with administration of anti-pyretics and/or antiseizure medications. Although tocilizumab has demonstrated remarkable results in abrogating CRS severity, it has not proven beneficial in treatment of ICANS , 92, 94.There are many ongoing adoptive cell therapy trials for solid tumors utilizing a patient’s own T cells or NK cells to directly target tumor cells. There are currently published results from clinical trials of CAR modified T cells or NK cells targeting CD171/L1CAM, GD2, and HER2 in pediatric patients with solid tumors –99, 102.Multiple pediatric CNS tumor CAR T cell trials are underway, as well as trials using tumor specific T cells, but toxicity data is just beginning to emerge . Common Immune checkpoint proteins are a part of the adaptive immune system and their role is to prevent the immune system from over-engaging and destroying healthy cells . Under nUse of checkpoint inhibitors in pediatric clinical trials often includes patients with different oncologic diagnoses including hematologic (Hodgkin’s and Non-Hodgkin’s lymphoma) and solid tumor malignancies, with few cases reported in leukemia –121.The overall reported incidence of neurotoxicity across trials ranges from 20-53%, with headaches being the most common neurologic symptom reported , 121. OtWith ICIs, two types of neurologic toxicities can be expected: systemic immune-mediated neurologic disorders, which can occur with ICI use for any indication, and local immune activation at the tumor site, which is unique to brain tumors. Although these presentations are typically easy to distinguish, there may be overlap, and both will likely respond to steroids.No randomized controlled trial results have yet been published on the use of ICI for treatment of pediatric brain tumors. Efficacy against malignant gliomas in pediatric patients with congenital mismatch repair deficiency (CMMRD) has been described in case reports, with clinical trials ongoing. Two siblings with CMMRD and glioblastoma responded to nivolumab, with their courses complicated by focal seizures and reversible radiographic pseudoprogression . In chilThe mechanisms of checkpoint-blockade induced neurotoxicity are incompletely understood. Since most ICI toxicities have a high degree of overlap with known autoimmune or inflammatory conditions, failure of self-tolerance  and contWork up for neurotoxicity may include neuro-imaging, CSF studies, ophthalmologic exam, neurophysiologic testing for peripheral nerve pathologies, and antibody titers , 132. AnTreatment of neurologic conditions related to ICI treatment is generally similar to interventions for when the same syndromes occur without ICI use. There is no evidence beyond expert opinion to support this approach. Depending on the severity of irAEs, immunotherapy may need to be paused or discontinued. If this is not sufficient, immunomodulators, primarily corticosteroids, may be indicated, as laid out in the Society for Immunotherapy of Cancer (SITC) and American Society of Clinical Oncology (ASCO) consensus guidelines of care for ICIs , 12, 134Antibody-based therapy in cancer therapeutics utilizes the recognition properties of an antibody to bind to a specific antigen on a malignant cell. Upon binding, the immune system triggers downstream processes that promote cell destruction and apoptosis either through direct cytotoxicity, non-restricted activation of cytotoxic T cells, and/or Fc-mediated immune effector engagement . The toxAntibody-based therapy in pediatric hematologic malignancies has demonstrated great clinical success in treating patients with relapsed/refractory acute lymphoblastic leukemia. Blinatumomab is a 55 kD-fusion protein that is a bispecific T cell engager that binds to and activates CD3+ T cells and links these T cells to the CD19 surface antigen on leukemia cells. Blinatumomab creates a synapse between the T cells and CD19+ cells, which results in upregulation of cell adhesion molecules, production of cytolytic proteins, release of inflammatory cytokines, and causes proliferation of T cells which results in lysis of CD19+ cells . SeveralIn pediatric patients with ALL receiving blinatumomab, neurologic toxicities have occurred in approximately 32% of patients in published reports –148. MosThe pathophysiology of neurotoxicity post-blinatumomab is elusive and remains an active area of research. T cell adhesion to blood vessel endothelium, subsequent endothelial activation, and T cell migration into the perivascular space has been described as a potential mechanism for neurotoxicity . AdditioRisk factors in pediatric patients for development of neurotoxicity with blinatumomab treatment are not well defined, however higher doses of blinatumomab have correlated with higher incidence of neurotoxicity . In adulIn patients who develop treatment-associated neurotoxicity, work up should be performed based on symptoms present and severity, with neuroimaging and EEG performed in those with suspected seizures or severe nAEs. The majority of events resolve with a short interruption in the infusion, dose de-escalation, or complete discontinuation of therapy, and corticosteroids can be used in the presence of severe neurotoxicity. Very few fatalities in pediatric and young adult patients have occurred post blinatumomab , 149.Targeted therapy using monoclonal, conjugated, and unconjugated antibodies has been utilized in early phase clinical trials, with some drugs now included in upfront treatment in hematologic malignancies. These include antibodies targeting CD20 , CD22 , and CD30  with specific clinical trials discussed in Clinical trials with BITEs in pediatric solid tumor are ongoing with no toxicity data available.Many unmodified and modified monoclonal antibodies have been evaluated for high-risk, relapsed, and refractory solid tumors. Reported neurotoxicity from published clinical trials on specific agents is summarized in Adie’s pupil is an ocular abnormality associated with anti-GD2 antibody therapy. It is characterized by mydriasis and accommodation deficits and is thought to be due to binding of antibody to neural structures in the ciliary and sphincter muscles of the eye. Most vision abnormalities can be corrected with prescription glasses and symptoms often improve or completely resolve after completion of therapy .Posterior reversible encephalopathy syndrome (PRES) is also associated with GD2 targeting antibody therapy and can present with hypertension, seizures, headache, visual disturbance, and/or altered mentation. Characteristic radiographic findings of PRES on MRI of the brain are edematous changes most often seen in the parietal and occipital lobes. Prior brain irradiation is significantly associated with incidence of PRES in patients who received the anti-GD2 antibody, 3F8. Most patients have complete resolution of symptoms over weeks .Bispecific T cell engagers are in clinical trials for pediatric CNS tumors however toxicity data is not yet available.via neutralizing free circulating VEGF-A. Since it reduces contrast enhancement of tumors, interpretation of tumor response by imaging criteria alone is problematic. In pediatric trials, bevacizumab has been used together with chemotherapy and/or radiation. While safety profiles are favorable, efficacy has not been well established due to small trial size. Serious nAEs are rare; the most common of these are intracranial hemorrhages (ICH), which affect 1-4% of patients and are typically intratumoral  . Some vaTumor vaccines have been the most extensively studied immunotherapy in the pediatric brain tumor population . PeptidePediatric cancer immunotherapy comprises a diverse set of approaches for heterogeneous diseases, with distinct biology and mechanism of action of immunotherapy approaches. This makes it inherently challenging to draw conclusions on risks and benefits given small sample sizes. Current efforts are focused primarily on creation of novel immunotherapies to target tumors more effectively, and limit broad, more systemic toxicities seen with chemotherapy and radiation therapy. However, the emergence of neurotoxicity as a key adverse event in immune cell engaging therapies for hematologic malignancies has invigorated interest in this topic in the pediatric population.in vitro and in vivo models to help evaluate these toxicities. Although the mechanisms of neurotoxicity remain unsolved, efforts at understanding the pathophysiology of this toxicity must be boosted to engineer safer novel therapies and inform diagnostic and therapeutic toxicity guidelines. For instance, based on pre-clinical models noting increased IL-1 in xenograft models post-CAR therapy, several clinical trials are now investigating use of anakinra as a therapeutic strategy to mitigate neurotoxicity. Future directions should focus on collaborative larger scale trials to enhance data sharing and to develop algorithms for early detection and effective treatment strategies for neurotoxicity.Compared with the ability to create novel targeted therapies and our level of understanding on efficacy and target choice of immunotherapies, the mechanistic understanding of these novel toxicity profiles is lagging, at least in part due to lack of suitable Additionally, through multicenter trials, prospective evaluation of biomarkers and ancillary studies such as long-term neurocognitive assessments can be performed and may help validate biomarkers and enhance clinicians ability to identify patients at risk of developing acute and long-term neurotoxicity after novel immunotherapies.All authors planned and wrote the manuscript and contributed to the article and approved of the submitted version.This work was supported in part by the Intramural Research Program, Center of Cancer Research, National Cancer Institute and NIH Clinical Center, National Institutes of Health , and the National Institute of Neurological Disorders and Stroke Mentored Clinical Scientist Research Career Development Award .The content of this publication does not necessarily reflect the views of policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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The introduction of monoclonal antibodies (mAbs) to the treatment of inflammatory bowel disease (IBD) was an important medical milestone. MAbs have been demonstrated as safe and efficacious treatments of IBD. However, a large percentage of patients either fail to respond initially or lose response to therapy after a period of treatment. Although there are factors associated with poor treatment outcomes in IBD, one cause for treatment failure may be low mAb exposure. Consequently, gastroenterologists have begun using therapeutic drug monitoring (TDM) to guide dose adjustment. However, while beneficial, TDM does not provide sufficient information to effectively adjust doses. The pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs are complex, with numerous factors impacting on mAb PK and PD. The concept of dashboard-guided dosing based on Bayesian PK models allows physicians to combine TDM with factors influencing mAb PK to individualize therapy more effectively. One issue with TDM has been the slow turnaround of assay results, either necessitating an additional clinic visit for a sample or reacting to TDM results at a subsequent, rather than the current, dose. New point-of-care (POC) assays for mAbs are being developed that would potentially allow physicians to determine drug concentration quickly. However, work remains to understand how to determine what target exposure is needed for an individual patient, and whether the combination of POC assays and dashboards presents a safe approach with substantial outcome benefit over the current standard of care. Inflammatory bowel disease (IBD) includes two types of auto-immune disorders that cause prolonged inflammation of the digestive tract: ulcerative colitis (UC) and Crohn’s disease (CD). One difference between these two types of IBD is the location of inflammation. In CD, inflammation can occur throughout the entire gastrointestinal tract, from mouth to anus. In UC, the colon and rectum are primarily affected. Although CD and UC are different diseases, differentiation based on the clinical picture can be difficult. Both UC and CD may present with abdominal pain, diarrhea, rectal blood loss, weight loss, vomiting, or signs of anemia. Diagnosis is usually made based on endoscopic evaluation and biopsies taken for histopathological assessment. Not only are the locations of these two diseases different, associated complications also differ. CD can be characterized by late complications such as a stenosing and/or penetrating phenotype which can result in fibrostenotic strictures,  fistula, or abscesses; UC does not have late complications. Although the exact etiology is unknown, IBD is likely caused by a combination of genetic and environmental factors leading to an immunological response and intestinal inflammation ,2. HistoIn 1998, approval of the first monoclonal antibody (mAb), infliximab (IFX), for CD, provided an exciting new treatment option. Since then, multiple mAbs with diverse mechanisms of action have been marketed for IBD. Currently available drugs for treatment of IBD include chemical agents such as steroids, immunomodulators, Janus kinase (JAK) inhibitors, and mAbs such as the anti-integrins, anti-interleukin-12/23p40, and anti-TNF agents (The treatment paradigm for CD and UC involves two regimens: (1) initial treatment period (induction) and (2) second regimen (maintenance), which are supported by evaluations during clinical development programs. Maintenance regimens are typically less intensive either in the dose or frequency than for induction . Knowledin vivo post-translational modifications (PTM), such as deamidation  behavior, whereby disposition involves neonatal-Fc receptor (FcRn)-antibody binding, which can be influenced by FcRn genetic polymorphism. Recently, the VNTR2/3 genotype in the FcRn gene was shown to be associated with lower mAb exposure during induction in IBD patients ,6. Intramidation –10 and nmidation , contribmidation ; hyperglmidation . Whethermidation ,15. In amidation . Numeroumidation –18, whicmidation ,20, whicPediatric patients present unique issues, given both growth and maturation effects  and the Two mAbs often used for treatment of IBD are IFX and adalimumab (ADL), administered intravenously and subcutaneously, respectively. Both anti-TNF agents have proven to be successful in inducing and maintaining clinical and endoscopic remission –32. HoweOnly a portion of patients achieve the predefined treatment response at the end of induction and continue into maintenance treatment.Only a fraction of patients who receive maintenance treatment achieve treatment response.Patients can lose treatment response after being in stable response for a period of time.Individualized dosing for mAb drugs began to acquire clinical attention many years after biological drugs had made a substantial, positive impact on patients with CD, UC, and other disease conditions. Impetus to investigate dose individualization of mAb drugs stems from findings of IBD trials and clinical practice. Some important common findings are as follows:Incomplete response to induction treatment may be attributable to inadequate exposure or patient factors that preclude treatment response. Similarly, loss of response (LOR) to maintenance treatment may be due to inadequate exposure in general or secondary treatment failure due to immunogenicity against the mAb drug or non-immune-mediated processes.Factors associated with primary non-response to anti-TNF agents are smoking, disease duration of more than two years, small bowel disease, and underdosing during the induction phase, presumably due to fecal loss. Brandse and colleagues demonstrated that IFX could be measured in feces of patients with acute severe colitis starting with IFX induction treatment . The higDevelopment of immunogenicity against the mAb is a key concern because of the potential association with reduced systemic drug exposure and treatment effectiveness. ADA formation, i.e., immunogenicity, can lead to secondary LOR in IBD patients –42. WhilADA can form complexes with the mAb, either neutralizating biologic effect and/or accelerating clearance. Since immunogenicity is often accompanied by subtherapeutic serum drug levels, maintaining adequate mAb concentrations is crucial in the treatment with anti-TNF agents. Current strategies to reduce the immunogenicity include scheduled instead of episodic treatment with anti-TNF agents and combination treatment with an immunomodulator, such as methotrexate or a thiopurine. However, if ADA does occur in patients treated with anti-TNF monotherapy, addition of immunomodulators during treatment can be used to suppress ADA. Research in IBD patients showed that addition of immunomodulators to anti-TNF monotherapy in patients with LOR due to immunogenicity led to increased serum drug levels, accompanied by ADA decrease to undetectable levels –50. The Potential subtherapeutic exposure is another key area of clinical investigations. Most IBD trials evaluate efficacy and safety at one or two dose regimens, which produce a range of systemic exposure as measured by the trough concentrations. In some trials, dose escalation based on clinical response may be an option; however, trial design often hampers robust evaluation of the benefit of dose escalation. Consequently, clinicians continue to investigate various dosing paradigms to benefit as many patients as possible with existing medicines, a quest universally applicable to chemical drugs and biological drugs. Literature reports have revealed potential reasons for IBD treatment failure as shown in Research has indicated that dosing according to a “one size fits all” principle is no longer optimal in IBD treatment. The observation that not all patients respond the same way to a certain dosing regimen emphasizes the need for adequate, more personalized dosing using therapeutic drug monitoring (TDM). There is a shift toward individualized dosing in which TDM plays an important role and is increasingly utilized by gastroenterologists.TDM is defined as measuring patient responses  and adjusting the therapy  accordingly, where ADA is relevant to mAbs for IBD treatment. This is particularly true for treatment with mAbs and thiopurines, with the former agents exhibiting highly variable clearance and the latter agents having a narrow therapeutic range. Patient factors influencing the PK of a drug should also be taken into account. For example, multiple analyses of IFX population PK data derived from IBD patients identified multiple factors influencing the clearance of IFX; the presence of ADA, low serum albumin, high serum CRP, and high body weight are the most important factors associated with increasing clearance of anti-TNF agents ,39, and et al. compared dashboard-driven dosing with actual administered dosing regimens in the standard-of-care setting  to achieve the predefined clinical outcome, patient factors driving PK exposure, patient factors driving the treatment response (including characteristics of patients who will not respond to treatment), immunogenicity, and enabling technical tools. The latter covers those for measuring patient factors, biomarkers, clinical outcomes, drug concentrations, and ADA, and for identifying patient factors through PK modeling, PD modeling, or integrated PKPD modeling.Desired treatment outcomes are defined by clinicians and reflected in clinical trials as study endpoints, which may evolve over time with increasing knowledge of the disease and the advancement of technology. For example, clinical response, clinical remission, mucosal healing, and endoscopic remission are some of the endpoints used in IBD trials in addition to exploratory biomarkers ,58. WhilTo move beyond empirical experimentation, proactive identification of patient factors that influence treatment response is very important as illustrated in et al.  and approved in 2008 for the treatment of CD. It was subsequently withdrawn from the market by its manufacturer after being linked with three cases of progressive multifocal leukoencephalopathy (PML). PML is an often-fatal opportunistic brain infection caused by the John Cunningham (JC) virus. Patients taking natalizumab are at risk of PML if they are JC virus antibody positive . It is hypothesized that high concentrations of natalizumab may affect immune cells’ ability to traffic to the brain and control the JC virus ,65. In MImmunogenicity was shown to negatively affect the systemic exposure and clinical outcomes; however, the role of ADA data in clinical management of IBD patients is not well defined. The proportion of patients who achieved desired clinical outcome is smaller in ADA-positive patients as compared to ADA-negative patients, and an ADA-positive finding has been associated with lower systemic drug concentration –71. HoweIt is worth noting that immune-mediated reduction in systemic exposures is one of several possible mechanisms for LOR . Other pThe use of TDM for IBD has several issues, including the influence of disease activity on PK and the wide range of target trough values in the literature . TDM utiAn important issue that should be addressed is the benefit from proactive TDM compared to reactive TDM. A recent review of TDM  suggesteMost IBD treatment regimens contain a higher-intensity dosing for induction followed by a lower-intensity dosing for maintenance, indicating that achieving adequate serum drug levels during induction is as important as during maintenance. Studies have illustrated that trough concentrations during induction are higher than the trough concentrations during maintenance . Howeveret al. showed substantially lower (34%) costs for those randomized to the TDM arm compared to IFX dose intensification to 5 mg/kg every 4 weeks  in IBD patients with secondary LOR , and cover only a small portion of the approved biologics (mostly those indicated to treat IBD). Most TDM LDTs utilize a sandwich or bridging enzyme-linked immunosorbent assay (ELISA). In this format, either a recombinant antigen or an anti-idiotype antibody is used as a capture reagent. A recombinant antigen, anti-idiotype antibody, or anti-isotype antibody is used as a labeled detection reagent. While tests for the same therapeutic mAbs are available from several different diagnostic companies, there is a lack of standardization for the different assays . Cell-baThe current mAb TDM protocol involves two strategies: either the patient must have blood drawn approximately 1–2 weeks before treatment to allow time for sample shipment and running the assay, or the patient has a sample drawn at the clinic visit, and that data is utilized at the subsequent visit. Taking a sample prior to a clinic visit requires an additional medical appointment. Also, because the TDM measurement is not taken at trough, which is when most publications established for target drug levels, translating the resulting TDM measurement into dose adjustment is more difficult. In the second case, dose adjustments or treatment changes would not be made until the patient’s next dose, generally 2–8 weeks later, depending on the drug.Point-of-care (POC) TDM may be particularly valuable in acute settings such as severe ulcerative colitis. Such patients often clear mAb more rapidly due to drug loss through the gut (protein-losing enteropathy)  and dosePOC TDM is currently unavailable for mAb therapeutics in the USA. CE-marked tests for IFX are commercially available in Europe ,101. BotVeritope selection is rapid and does not require immunization of animals.Veritopes are easily synthesized and inexpensive to produce.Veritope peptides are easily modified by traditional methods .MAb TDM tests that utilize mimetope peptides as capture or detection reagents in ELISA and lateral flow immunoassay (POC device) are also being developed. Peptides specific for rituximab and alemtuzumab were originally described ,103, andIn contrast, recombinant protein expression is not always feasible and anti-idiotype antibody identification is tedious and not always successful. Large-scale production of recombinant proteins and anti-idiotype antibodies are also costly and stability may be an issue with the former. Unlike cell-based assays, mass spectrometry, and HMSA, Veritopes are amendable to multiple immunoassay formats that do not require highly trained users or expensive equipment. Finally, Veritopes are surrogate ligands that only recognize active (unbound) drug, unlike mass spectrometry, which quantitates all drugs  in the sample.Laboratory results for ADA are highly dependent on the assay used to measure ADA. Historically, ADA assays are prone to interference by the presence of drug in samples and sensitive assays only detect ADA in the presence of low amounts of drug . HoweverE–R relationship for mAbs, although this is more often when the data come from a wide dose range and include data from numerous patients such as phase 3 studies. The E–R curves may suggest that a higher responder rate is associated with higher exposure, , and steeper E–R curves may suggest greater potential benefit with dose intensification. However, whether intensified dosing regimens will lead to greater clinical efficacy with acceptable safety profiles remains to be experimentally demonstrated because extrapolation of efficacy and safety is challenging. Moreover, understanding patient factors that would drive the treatment response remains an area for future research.MAbs such as IFX and ADL have proven to be effective in the treatment of auto-immune diseases. However, a significant proportion of patients do not respond to induction treatment or lose response during maintenance, presumably because of inadequate drug exposure. TDM, with the use of dashboard systems, can contribute to adequate dosing by taking into account specific patient factors influencing mAb clearance. Clinical trial data can often illustrate the In summary, TDM is a growing part of the precision medicine approach. Evidence supporting correlation of interpatient PK variation with treatment outcomes is extensive and growing. As combination therapy use increases in the treatment of many diseases, PK variability and factors influencing individual patient exposure are generally different between drugs, and therefore, exposure to different drugs in a combination setting may ultimately need to be addressed. While not currently available for all biologics, POC TDM is warranted in certain circumstances and enables personalized, precision dosing. Most current methods for TDM are not amenable for rapid POC testing, but alternative technologies are being developed to make POC TDM reliable and practical for patients treated with biologic therapies. An unmet clinical need exists to mitigate the risks of interpatient PK variability among patients treated with mAbs.


Kidney transplantation is the best treatment modality for end-stage kidney disease, leading to improvement in a patient’s quality and quantity of life. With significant improvements in short-term outcomes, prolonging long-term allograft and patient survival remain ongoing challenges. The ability to monitor allograft function, immune tolerance and predict rejection accurately would enable personalization and better prognostication during post-transplant care. Though kidney biopsy remains the backbone of transplant diagnostics, emerging biomarkers can help detecting kidney allograft injury early enough to prevent permanent damage and detect injury before it is clinically apparent. In this review, we summarize the recent biomarkers that have shown promise in the prediction of acute rejection with a focus on antibody-mediated rejection in kidney transplantation. In the current era of kidney transplantation, one-year patient and allograft survival exceed 95% . Though The presence of pre-existing or de novo donor-specific antibodies (DSA), histologic evidence of glomerular injury and microcirculatory inflammation that have been implicated in development of transplant glomerulopathy and/or late kidney allograft failure are antibody-mediated processes ,7,8,9,10Though we are making major advances in understanding the pathophysiology and immunology of ABMR, we continue to rely on traditional markers like creatinine, proteinuria and kidney biopsy for screening and definitive diagnosis. Kidney biopsy, which is the gold standard to diagnose and differentiate between types of rejection and pathologic processes, is invasive, can have complications and is costly. Kidney biopsy is also subject to sampling error and interobserver variability between pathologists and different transplant centers . MarkersPrior reviews have comprehensively described how the expansion of the “omics” field has led to a new era of transplant biomarkers which could be predictive, diagnostic or prognostic ,18. BiomHistologic evaluation via a kidney biopsy remains the gold standard of differentiating T cell-mediated rejection (TCMR) and ABMR. In 1993, the Banff schema was introduced with numeric coding of histologic features to allow for objectivity and definition of grades of acute rejection . ABMR isThe “molecular microscope diagnostic system” (MMDx) is a microarray-based system that examines messenger RNA (mRNA) expression patterns in transplant biopsy tissue to predict the diagnosis of acute TCMR or ABMR . HalloraInvasive biomarkers, though crucial in determining an accurate diagnosis, are subject to variability in sampling and interpretation and more often are utilized when the disease process may have reached an irreversible state.The presence of DSAs remains one of the most important biomarkers in the diagnosis of ABMR as defined by the Banff criteria . Many stNew biomarkers, such as dd-cfDNA, are being developed to overcome the difficulties of detecting the earliest stages of kidney transplant dysfunction and rejection. Although histology obtained via needle biopsy remains the gold standard for diagnosis of rejection, dd-cfDNA serves as an alternative to the invasive renal allograft biopsy and has evolved to a frequently used tool for surveillance and diagnosing of kidney transplant rejection, as well as monitoring of therapy ,45,46,47Routine surveillance (protocol) biopsies have been used in some centers to monitor patients with stable renal function following kidney transplantation, but these are expensive, invasive, pose significant logistical issues and are subject to variability of interpretation, thus limiting wider application. The current standard of care in monitoring patients following kidney transplantation ranges from not using surveillance biopsies at all, using them only in patients at high immunologic risk, to routine use (protocol biopsies) in all patients. Subclinical acute rejection (subAR) is the presence of histological features of acute rejection on renal biopsy in the absence of a decline in renal function. SubAR is present in approximately 25% of surveillance biopsies in renal transplant recipients with stable renal function . TherefoThe TruGraf assay was developed to rule out subclinical rejection in patients with otherwise stable allograft function. Unlike surveillance biopsies which are invasive, it is a DNA microarray-based gene expression blood test that can be obtained easily . The tesNon-HLA antibodies directed against endothelial receptors play an important role in allograft vascular injury. Lefaucheur et al. showed in a prospective cohort study of 1845 patients that participants with anti-angiotensin II type 1-receptor antibodies (AT1R-Abs)-associated rejection had a higher prevalence of hypertension and more vascular rejection with arterial inflammation . Other sLike AT1R-Ab, Banasik et al. have shown that anti-ETAR (endothelin 1 type A receptor) antibody is associated with worse graft function at 12 months post-transplant and with more cases of vasculopathy or arteritis in a series of 116 patients . There iWith the arrival of high throughput sequencing and microarray technologies, the ability to perform molecular assays from peripheral blood has created new possibilities for non-invasive biomarkers in transplantation. The authors of the multicenter prospective BIOMARGIN study identified an eight gene assay with a ROC AUC of 79.9%, to diagnose acute antibody-mediated rejection , which hThe authors of the AART study  developed a 17-gene panel using quantitative PCR from peripheral blood samples called kSORT (Kidney Solid Organ Response Test). kSORT accurately predicted the risk and prevalence of acute rejection with an AUC (area under curve) of 0.92 and a PPV of 93.21% . Howeverγ expression. Rabant et al. showed that combining urinary CXCL10: Cr ratio with detectable DSA significantly improved the accuracy of diagnosing ABMR non-invasively [Non-invasive and effortless to obtain serially, the urine sample as a source of biomarker has endless potential. The chemokines CXCL9 and CXCL10, when measured in urine, have been shown to serve as a surrogate for non-invasively measuring intra-graft IFNvasively . Urinaryvasively .Utilizing urinary transcriptomics has led to the identification and profiling of distinct mRNA signatures in the urine that are associated with allograft injury . MeasureThe onset of active or chronic ABMR plays a major role in determining the long-term survival of an allograft. It is an exciting era to have a diverse array of predictive and diagnostic biomarkers currently under investigation. Although currently the majority of these tools are not widely available or validated, it is possible that a search for a better diagnostic tool may uncover novel therapeutic options as well. For now, histological examination of the kidney remains the current gold standard for diagnosis of acute rejection, especially in the setting of elevated creatinine. The emergence of a non-invasive, highly sensitive and specific biomarker, or combination of biomarkers, that is independently validated in randomized controlled trials and/or serves as a surrogate for long-term graft survival is eagerly awaited by the transplant community.


Aspergillus niger. The prediction integrated a diverse range of features, including evolutionary profile, gene expression, transcriptional regulation and biochemical characteristics. Results on both the training and the independent testing dataset showed that our method achieved over 90 % accuracy, and recalled over 60 % of pectinolytic genes. Application of the ML model on the A. niger genome led to the identification of 83 pectinases, covering both previously described pectinases and novel pectinases that do not belong to any known pectinolytic enzyme family. Our study demonstrated the tremendous potential of ML in discovery of new industrial enzymes through integrating heterogeneous (post-) genomimcs data.Pectinolytic enzymes are a variety of enzymes involved in breaking down pectin, a complex and abundant plant cell-wall polysaccharide. In nature, pectinolytic enzymes play an essential role in allowing bacteria and fungi to depolymerize and utilize pectin. In addition, pectinases have been widely applied in various industries, such as the food, wine, textile, paper and pulp industries. Due to their important biological function and increasing industrial potential, discovery of novel pectinolytic enzymes has received global interest. However, traditional enzyme characterization relies heavily on biochemical experiments, which are time consuming, laborious and expensive. To accelerate identification of novel pectinolytic enzymes, an automatic approach is needed. We developed a machine learning (ML) approach for predicting pectinases in the industrial workhorse fungus,  Genome and transcriptome data analysed in this work are available in the Mycocosm and GEO database, respectively. Individual accession numbers are listed in Table S1, available in the online version of the article.Novel pectinolytic enzymes predicted in this study are listed in Table S6.https://github.com/maopeng2018/pectinase_prediction).The curated sequence and expression features and source code for predicting pectinolytic enzymes have been deposited in GitHub genomics data.Pectinolytic enzymes are involved in breaking down pectin, a complex and abundant plant cell-wall polysaccharide. Due to their important biological function and increasing industrial applications, discovery of novel pectinolytic enzymes has received global interest. However, traditional enzyme discovery and characterization is time consuming and expensive. This paper describes a machine learning (ML) approach for predicting pectinases in the industrial workhorse fungus, Pectin is a heteropolymer that is abundantly present in the plant cell wall . It is cAspergillus niger is an industrial workhorse that has been widely exploited as a cell factory to produce organic acids and various enzymes, including pectinases  (Table S6). The identification of these non-pectinolytic enzymes indicated that they were strongly co-expressed or co-regulated with the pectinolytic gens, which is in line with the synergistic interaction among different polysaccharides during fungal plant biomass degradation . Given tAmong the six tested ML models, the RF performed better than the others. This is consistent with the performance of RF in other studies . In bothOur study has demonstrated the tremendous potential of finding industrially important pectinolytic enzymes by integrative analysis of heterogeneous (post-)genomics features using the ML method. With the continuing expansion of (post-)genomics data on enzyme evolution, transcriptional expression and regulation, proteomics data, together with a better understanding of the physicochemical properties of enzymes, the ML-based bioinformatics approach will play an increasing role in mining big biological data, especially for the discovery of novel microbial enzymes.


High-risk neuroblastoma is an aggressive childhood cancer that is characterized by high rates of chemoresistance and frequent metastatic relapse. A number of studies have characterized the genetic and epigenetic landscape of neuroblastoma, but due to a generally low mutational burden and paucity of actionable mutations, there are few options for applying a comprehensive personalized medicine approach through the use of targeted therapies. Therefore, the use of multi-agent chemotherapy remains the current standard of care for neuroblastoma, which also conceptually limits the opportunities for developing an effective and widely applicable personalized medicine approach for this disease. However, in this review we outline potential approaches for tailoring the use of chemotherapy agents to the specific molecular characteristics of individual tumours by performing patient-specific simulations of drug-induced apoptotic signalling. By incorporating multiple layers of information about tumour-specific aberrations, including expression as well as mutation data, these models have the potential to rationalize the selection of chemotherapeutics contained within multi-agent treatment regimens and ensure the optimum response is achieved for each individual patient. Neuroblastoma is a paediatric malignancy of the sympathetic nervous system, accounting for 7–8% of childhood cancers . While iPromisingly, patient outcomes for neuroblastoma have improved substantially since the early 1970s, with 5-year disease-free survival increasing from 50% to 75% in 2005 , and to Most neuroblastomas present with no obvious genetic predisposition or family history, and a generally low somatic mutation profile ,11. TherTherefore, this review will provide a brief overview of the molecular and genetic landscape of neuroblastoma, along the relevant targeted therapies in current use and their potential limitations. Building on this, we will further explore the potential for patient-specific simulations of drug response to be developed into a personalized medicine approach applied to the treatment of all high-risk neuroblastoma patients with rationalized combinations of standard-of-care chemotherapy drugs.ALK mutations and MYCN amplification are the only validated de novo drivers of neuroblastoma [Seminal genomic studies have shed light on the underlying tumour landscape of neuroblastoma, although like many paediatric malignancies, neuroblastomas usually harbour fewer somatic genomic aberrations and mutations than adult tumours ,20. Furtblastoma ,23, althMYCN amplification occurs in ~20% of neuroblastomas and has been established as a key driver of an aggressive and chemoresistant tumour phenotype, frequently observed in high-risk neuroblastoma and predictive of poor patient outcome [The MYC family of transcription factors regulate multiple cellular processes including apoptosis, proliferation, the cell cycle, mitochondrial function and glycolysis . MYCN am outcome . Early i outcome , while t outcome ,27 and z outcome  models wMYCN—with BET inhibitors JQ1 and OTX015 being considered for clinical evaluation [Frustratingly, while overexpression of MYCN is a known major driver of disease, it also represents a potentially difficult avenue for therapeutic targeting in the context of a personalized medicine approach. The lack of targetable surfaces in its DNA binding domain and conserved homology among the MYC family proteins has meant that efforts aimed at direct MYCN inhibition have thus far been ineffective . Alternaaluation . Additioaluation ,36, whilaluation ,37.ALK mutations are the most frequently observed somatic mutations in neuroblastoma, with either point mutations, amplification or fusion events occurring in 7–10% of cases [ALK is a member of the insulin receptor tyrosine kinase (RTK) superfamily, for which high expression levels are usually confined to neurons during nervous system development, although it can also be highly expressed in neuroblastoma tumours . ALK mutof cases ,40.Given mutant ALK has been successfully targeted in other diseases such as in anaplastic large-cell lymphoma, non-small-cell lung cancer and myofibroblastic sarcoma, ALK has been heavily investigated as a therapeutic target in neuroblastoma . ALK inhNTRK1, NTRK2, NTRK3), are RTKs with established spatiotemporal roles in the development and maintenance of the peripheral nervous system [The tropomyosin receptor kinase family, TrkA, TrkB, TrkC . Loss of function mutations, or the deletion of ATRX has also been shown to sensitise neuroblastoma cell lines and PDXs to the current standard-of-care drug irinotecan, in combination with the PARP inhibitor olaparib [The necessary preclinical and clinical investigation of many other aberrant genes is still lacking.  in vivo , and ARI outcome . While tolaparib . HoweverIn line with the low rate of recurrent somatic mutations in neuroblastoma, it is now widely recognized that cancer progression generally requires both genetic and epigenetic involvement ,69,70. ADNA methylation is a master epigenetic regulatory pathway governing genomic programming of a cell modulated by DNA methyltransferases (DNMTs) . A loss Histones are key DNA scaffolding proteins, which undergo post-translational modifications such as, acetylation, methylation, phosphorylation and sumoylation, that individually are able to dictate chromatin conformation and consequently, gene transcription . The addHistone acetylation is another key post-translational modification, which regulates vital cellular mechanisms including cell death, cell cycle progression and differentiation . HistoneGenetic analysis has also demonstrated that the sub-clonal heterogeneity of neuroblastoma tumours is reduced at relapse, suggesting that clonal selection drives the emergence of resistant subclones and is a significant factor in refractory or relapsed disease . NotablyHowever, an emerging theme in clinical oncology is the need to focus efforts upon preventing relapse from occurring through improvements in first-line therapies, given the inherent therapeutic intransigence of relapsed, and often metastatic, tumours. This need is highlighted by Fletcher et al., who proposed that future clinical trials should be tailored to firstly recognise high-risk patients likely to fail standard-of-care therapy and then identify the actionable aberrations that may drive sub-clonal expansion, facilitating the treatment of these patients before relapse, where patient outcome is significantly poorer . ClearlyAs outlined above, high-risk patients that present with unresectable and/or metastatic disease are administered neoadjuvant induction therapy. Induction therapy entails high intensity cycles of combination of anthracyclines, alkylating agents, platinum agents, microtubule destabilisers and topoisomerase II inhibitors. While regimens vary around the world, the USA-based, Children’s Oncology Group outline six cycles of induction chemotherapy: cycles 1,2- topotecan and cyclophosphamide; cycles 3, 5- cisplatin and etoposide; cycles 4, 6- cyclophosphamide, vincristine and doxorubicin ,104. ProThe most significant challenge for high-risk neuroblastoma and indeed most cancers, remains the treatment of relapsed or refractory disease. Despite recent advances, to date there remains no curative treatment regimen and prognosis for these patients remains dire despite intense multimodal salvage therapy. The camptothecin analogues topotecan and irinotecan are commonly used in salvage therapy, with established activity against refractory ,106,107 Despite the wide-spread use of these chemotherapy agents, outside of their direct cellular targets, the exact mechanism of apoptosis induction remains poorly defined . AlthougTherefore, the development of dynamic mathematical models that encapsulate the apoptotic pathways activated and required by these drugs will then allow the inclusion of individual tumour specific changes and the simulation of patient-level drug response. Further developments will also require more sophisticated approaches that can predict the optimal application of emerging and pre-existing therapies within context of multi-agent treatment regimens. However, many such approaches are already beginning to emerge, which are outlined below.Traditionally, a personalized medicine approach involves tailoring the use of specific targeted therapeutics to individual patients based upon the presence of an actionable genetic mutation within their tumour. However, the low frequency of actionable mutations within neuroblastoma means that there is little scope for a personalized medicine approach that will benefit the entire patient population. Recent advances with ALK and Trk inhibitors have demonstrated a potential to benefit subsets of high-risk neuroblastoma patients. In addition to this, clinical trials are also underway to match targeted therapies to the emergence of actionable mutations within relapsed neuroblastoma, but due to the established decrease in treatment efficacy in relapsed neuroblastoma, a focus on preventing relapse by optimizing induction therapy is desperately needed .To achieve this, what is urgently needed is a paradigm in which standard-of-care chemotherapy drugs, and emerging broad-spectrum drugs, can be deployed as tailored induction therapy regimens based upon individualized predictions of drug response. To develop such a personalized medicine approach, it will firstly be necessary to understand the apoptotic mechanism of each individual drug and the mechanism of synergy between potential drug combinations. By incorporating this mechanistic data into predictive, mathematical models, patient-specific perturbations can then be introduced, allowing the simulation of drug response and predictions of optimal drugs and drug combinations.To achieve this ambitious aim, advances in computational modelling approaches will undoubtedly be necessary. This requirement arises chiefly due to the highly complex nature of signalling networks, which often contain inter-linked pathways and regulatory structures that prevent an intuitive understanding of network states and functional outcomes ,125,126.In the context of personalized medicine, mechanistic modelling approaches are most suitable due to their ability to facilitate the inclusion of the spatiotemporal aspects of intracellular signalling that are typically absent in other modelling approaches. Consequently, mechanistic models can provide more accurate in silico simulations of biological systems and the key regulatory structures that signalling networks utilize to ensure robust decision-making processes . This chNonetheless, the power of mechanistic models for a personalized medicine approach is already becoming apparent. For instance, we have previously demonstrated the prognostic significance and predictive capacity of a patient-specific modelling approach for neuroblastoma patients . In thisALK mutations may be good candidates for PD-1 checkpoint inhibitors such as nivolumab [A similar model was created to predict PD-L1 expression and hence susceptibility to checkpoint inhibitor immunotherapy in neuroblastoma . An inteivolumab .Previous studies from other cancer types have also demonstrated the potential for the development of models capturing variations in the expression level of key network components for personalized predictions of drug response and thus the tailoring of treatment strategies to individual patients. This includes previous models of the acquisition of resistance to endocrine therapy in breast cancer, based upon the expression of EGFR and HER2  and the Another example comes from a model of the interconnected network of BCL-2 family, BH3-only, and other apoptotic regulatory proteins in colorectal cancer, which was used to simulate the influence of tumour-specific changes in expression of these proteins on apoptotic signalling in response to chemotherapy and investigate the association of these predictions with clinical response . An addiClearly, an extensive amount of future work will be required to fully map and model the extended apoptotic pathways required for response to chemotherapy in neuroblastoma cells, along with a focus on the bespoke collection of patient data to facilitate the generation of patient-specific simulations of drug response. In addition to this, determining the functional significance of relevant somatic mutations within these pathways and incorporating these alterations within patient-specific simulations will be a bottleneck that may only be overcome with extensive experimentation. While daunting, the effort required to establish this modelling platform will yield valuable data on strategies to optimize induction therapy regimens for high-risk neuroblastoma patients, thereby improving response rates and reducing the incidence of relapse. However, these approaches may also provide insight into the mechanisms of chemoresistance that arise within relapsed/refractory neuroblastoma tumours, providing further avenues of research aimed at improving outcomes for this young patient population.


However, the limited knowledge base that exists on the gender differences indelta-9-tetrahydrocannabinol (THC) retention inhibits a forensic physician's ability to makerobust assessments of cannabis detection test results. Furthermore, noticeably absent in any gender discussion is trans THC retention . This letter seeks to draw on the current knowledge base in thecis-population to suggest how THC retention may occur in trans individuals.As the usage of medicinal marijuana rises and cannabis decriminalisation becomes moreaccepted globally, Which, due to THC's lipophilic nature, results in women, on average, having a greatercapacity for THC storage and consequently retention. Another study found that women have agreater cannabinoid receptor 1  availability than men. Therefore, women have a greater opportunity to uptake and re-uptake THC (and itsmetabolites) in the body, retaining it for longer. On the other hand, female rodents have beenshown to have a higher number of the liver enzymes that metabolise THC. Therefore, you might expect women to have greater THC metabolism and consequentlyfaster elimination. Women are also more likely to suffer from stress-related disorders which would result in greater hypothalamic-pituitary adrenal (HPA) axis activationfreeing more THC from fat cells.It's held that average women have a higher percentage body fat than average men. Conversely, another study also describes how men are more likely to be resistant toantilipolytic effects. In other words, men are more likely to break down adipose and thus release THC.Research on consumption found that men consume marijuana in greater amounts and at higherrates, meaning men have a greater concentration of THC to eliminate.8 However, if we consider the effect THC has on each of these sex hormones,a difference emerges. Research has shown THC can cause a small rise in testosteroneconcentration which would therefore result in more HPA axis induction. Conversely, THC has been found to inhibit progesterone production, which would lead toless HPA axis activation.Testosterone and progesterone have both been found to potentiate corticotrophin-releasinghormone (CRH) stimulated adrenocorticotropic hormone (ACTH) release. Whilst, due to trans-individuals being more likely to suffer from stress-relateddisorders they may also experience weaker THC retention as a result of increased HPA axis induction. Furthermore, the THC interaction with sex hormones is especially pertinent as itrelates to trans-individuals and the hormone therapy they may be taking.Drawing from the aforementioned evidence, trans-individuals may experience both stronger andweaker THC retention. Stronger THC retention may be seen in transwomen as a result of anincrease in total body fat after hormonal transition, resulting in greater THC storage availability. With the 2014 amendment to the Road Traffic Act 1988 introducing legal limits to THC,and the increasing prevalence of trans-athletes, it is essential for clinical forensicmedicine to understand the context of the results they see. It is not impossible to imagine a scenario where an athlete or driver fails a cannabistest despite being abstinent for days or weeks due to either taking part in a sporting eventthat activates the HPA axis or having strong THC retention factors in their physiologicalmake-up.As part of the regulations set out by The Faculty of Forensic & Legal Medicine of theRoyal College of Physicians, forensic physicians are expected to interpret drug interactions,impairment, and intoxication in a variety of situations.Shifting societal context necessitates forensic physicians and forensic toxicologists tobetter understand the difference between sex and gender, as well as the retention differencesin genders and the trans community. The points highlighted in this letter may help to betterinform forensic practitioners  assessments of cannabisdetection results in the trans population, based on the presence of Body Mass Index (BMI)>30, female gender (cis or trans), stress-related disorders, or on transgender hormonaltherapy. Based on this, future researchers could explore whether these data points may offeran ‘at the point of investigation’ scoring system to assist forensic physicians andtoxicologists. Moreover, the novelty of this domain and the current weak evidence basesurrounding transgender cannabis use, should encourage greater toxicologic research to beperformed.