Voluntary medical male circumcision (VMMC) remains an essential component of combination HIV prevention services, particularly in priority countries in sub-Saharan Africa. As VMMC programs seek to maximize impact and efficiency, and to support World Health Organization guidance, specific uptake-enhancing strategies are critical to identify.We systematically reviewed the literature to evaluate the impact of service delivery interventions  on VMMC uptake among adolescent and adult men. For the main effectiveness review, we searched for publications or conference abstracts that measured VMMC uptake or uptake of HIV testing or risk reduction counselling within VMMC services. We synthesized data by coding categories and outcomes. We also reviewed studies assessing acceptability, values/preferences, costs, and feasibility.Four randomized controlled trials and five observational studies were included in the effectiveness review. Studies took place in South Africa, Tanzania, Uganda, Zambia, and Zimbabwe. They assessed a range of service delivery innovations, including community-, school-, and facility-based interventions. Overall, interventions increased VMMC uptake; some successfully improved uptake among age-specific subpopulations, but urban-rural stratification showed no clear trends. Interventions that increased adult men's uptake included mobile services (compared to static facilities), home-based testing with active referral follow-up, and facility-based HIV testing with enhanced comprehensive sexual education. Six acceptability studies suggested interventions were generally perceived to help men choose to get circumcised. Eleven cost studies suggested interventions create economies-of-scale and efficiencies. Three studies suggested such interventions were feasible, improving facility preparedness, service quality and quantity, and efficiencies.Innovative changes in male-centered VMMC services can improve adult men’s and adolescent boys' VMMC uptake. Limited evidence on interventions that enhance access and acceptability show promising results, but evidence gaps persist due to inconsistent intervention definition and delivery, due in part to contextual relevance and limited age disaggregation. As countries strive to control the HIV epidemic, voluntary medical male circumcision (VMMC) remains an essential component of combination HIV prevention, particularly in sub-Saharan Africa. VMMC reduces the risk of HIV acquisition by approximately 60% and also provides an opportunity to link men and boys to a range of other HIV prevention and treatment services –3.VMMC for HIV prevention was introduced in sub-Saharan Africa after the 2007 recommendation by the World Health Organization (WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS) to scale-up access to this proven intervention in priority countries where circumcision prevalence was low and HIV prevalence was high . BetweenApproaches to improve VMMC uptake often include one or a combination of diverse interventions, some of which have been implemented and adopted as good practices in VMMC programs in East and Southern Africa. Despite this, their impact has not been well-documented . The impWe conducted this systematic review in accordance with Preferred Reporting Items for Systematic review and Meta-Analyses (PRISMA) guidelines .For the purposes of this review, we defined “service delivery interventions” as changes or improvements to the ways VMMC services are provided. Interventions could emphasize additional, restructured, or intensified information, education, or communication services or more structural aspects like improved provider training; changes to physical space; changes to health services structure, such as altering service location or time of day; using home- or community-based services, task-shifting, or restructuring the flow of services; or co-location of VMMC with other health services, such as hypertension or diabetes screening.Population: Uncircumcised adolescent and adult men (ages 10+) who are potential candidates for male circumcision for HIV prevention within public health VMMC programsIntervention: Service delivery interventionsComparison: No such service delivery interventions, or a different or less intensive type of service delivery interventionOutcomes: Primary: 1) Uptake of VMMC for HIV prevention as defined by WHO; Secondary: 2) Uptake of HIV testing within VMMC services, and 3) Uptake of safer sex and risk reduction counselling within VMMC servicesThe main question for our effectiveness review was: among adolescent and adult men who are potential candidates for male circumcision for HIV prevention within public health VMMC programs, do service delivery interventions lead to improved uptake of male circumcision compared to no such interventions, or a different or less intensive type of intervention? We followed the PICO question format to define our review criteria:In addition, to be included in the review, a study must have: 1) had a study design that presented a pre/post or multi-arm comparison by service delivery intervention exposure, 2) measured one or more of the outcomes listed above, and 3) been published in a peer-reviewed journal or conference abstract. No restrictions were placed based on type or scope of service delivery intervention, location of the intervention, or language of the publication. When information was available for the same study from both a peer-reviewed article and a conference abstract, we only used data from the peer-reviewed article.When possible, outcomes were stratified by whether participants (men) were considered high-risk for HIV or not, since recent modeling suggests VMMC may have greater impacts among men at increased risk for HIV . We defiThe search was developed in conjunction with another related review on economic compensation interventions to increase uptake of VMMC developed for the same WHO guidelines . We therWe searched five electronic databases  and four conferences , International AIDS Society (IAS) Conference on HIV Science, Conference on Retroviruses and Opportunistic Infections (CROI), and International Conference on AIDS and STIs in Africa (ICASA)). Electronic databases were searched from January 1, 1990 through May 31, 2018. The IAC, IAS, and ICASA conference abstracts were searched for all available years (through mid-2018). For CROI, only the most recent conferences (2014–2018) were searched as past conference abstracts are inaccessible. We searched the bibliographies of all studies included in the review and of a previous related review [The following search strategy was adapted for entry into all electronic databases: (HIV [tiab] OR “human immunodeficiency virus” [tiab]) AND (circumcision [tiab] or circumcis* [tiab] or VMMC [tiab]). Only the term “circumcision” was used to search conference abstracts because all conferences searched are HIV-related and search functions are limited.Titles, abstracts, citation information, and descriptor terms of citations identified through the search were screened in duplicate. Full-text articles were obtained of all selected abstracts, and two independent reviewers assessed all full-text articles for eligibility to determine final study selection. Differences were resolved through consensus.Study identification: Author(s), type of citation, year of publicationStudy description: Study objectives, location; population characteristics, main intervention description , description of any additional intervention components, study design, sample size, follow-up periods, loss to follow-upOutcomes: Analytic approach, outcome measures, comparison groups, effect measures and sizes, confidence intervals, significance levels, conclusions, limitationsData were extracted independently in duplicate using standardized data extraction forms. Differences in data extraction were resolved through consensus and referral to a senior study team member when necessary. The following information was gathered from each included study:For randomized controlled trials (RCTs), we assessed risk of bias using the Cochrane Collaboration’s tool . We asseWe analyzed data according to coding categories and outcomes. Given the diverse intervention approaches and different ways of reporting outcomes, we were unable to combine effect sizes in meta-analysis. The terms 'effectiveness' and 'impact' are used throughout to refer to findings from the entire included body of literature, including observational studies which do not prove causality, to match the programmatic use of the terms in the WHO data collection process for which this review was performed.In addition to the effectiveness review, we conducted additional reviews assessing acceptability, values and preferences, costs, and feasibility of service delivery interventions. These reviews added important, complementary evidence for the purpose of developing holistic WHO guidelines . We usedWe included studies in the acceptability review if they presented primary data examining people’s feelings about the acceptability of the interventions included in the main effectiveness review. We focused on studies examining the perspectives of men who have used or potentially would use VMMC, but we also included studies examining the acceptability of the interventions among providers and other stakeholders .We included studies in the values and preferences review if they presented primary data examining people’s feelings about the three PICO outcomes: 1) uptake of medical male circumcision, 2) uptake of HIV testing within VMMC services, and 3) uptake of safer sex and risk reduction counselling within VMMC services. We assessed values and preferences around population-level uptake of circumcision as an outcome, rather than feelings about circumcision itself as a procedure. We were primarily interested in studies examining the perspectives of people who have used or potentially would use VMMC, but we also included studies examining values and preferences among providers and other stakeholders .We included studies in the costs review if they presented primary data examining cost or cost-effectiveness of the service delivery interventions, or otherwise discussed resource use in relation to these interventions. We considered any measure of cost .We included studies in the feasibility review if they presented primary data examining issues with provision of the service delivery interventions or how they fit within health systems . This was intended to complement the effectiveness review by identifying issues related to implementation of these interventions that might be relevant for others seeking to replicate them.All data came from published journal articles or conference abstracts.Electronic database searching retrieved 3,940 results, and an additional 131 were identified through searching conference databases, trial registries, contacting experts in the field, and secondary searching . After rOf the nine included studies in the effectiveness review, four were RCTs ,25,29,30Quality assessment of included studies are summarized in Results regarding VMMC uptake from community-, facility-, and school-based service delivery interventions are summarized in Evidence from two community- (including home)-based RCTs showed increased VMMC among participants in service delivery interventions ,29. One One facility-based RCT examining VMMC uptake at 12-month follow-up found increased likelihood of VMMC uptake among men receiving the Spear and Shield intervention, a four-session HIV/sexual risk reduction education and VMMC promotion program with specialized training for healthcare providers  compared to those who participated in time-matched educational sessions on tuberculosis, malaria, cholera, and water-borne disease prevention and received condoms .Finally, one RCT and two observational studies reported data on VMMC uptake following school-based interventions ,27,28 taMultiple studies included adolescents and young men but did not stratify outcome data by age. Several school-based studies included older men, but participants were predominantly less than 20 years old. Exposure to a community-based demand-generation intervention was associated with greater mean number of clients aged 20–34 and a greater proportion of clients aged ≥20 over a one-month period . The proThree included studies took place in urban areas and six in rural settings; however, there were no clear trends in VMMC uptake by location. One study presented additional data comparing uptake before and after rollout of GIS prioritization of VMMC services; after data-driven programmatic prioritization, VMMC procedures shifted from those primarily conducted in urban settings (around 60%) before GIS to almost all in rural settings (around 90%) afterward .Two studies in Tanzania  measured uptake of HIV testing in VMMC services, comparing service delivery approaches ,29. AuthNo data were found comparing exposure versus non-exposure to service delivery interventions on uptake of safer sex and risk reduction counseling within VMMC services.We identified six studies examining acceptability of service delivery interventions  27,31–3–331–35, At the community level and in schools, discussions with VMMC advisers and coaches were considered particularly helpful for VMMC clients or potential clients. One study surveyed South African men during VMMC services and found that 83% of men said they would not have been circumcised had they not received the intervention of motivational interviews with a VMMC adviser . SimilarNo studies were identified for the review on end users’ and providers' values and preferences for the outcomes of interest.Eleven studies presented cost data ,25,36–44Generally, cost studies showed that service delivery interventions may create economies of scale and efficiencies when implemented widely. Though activities to enhance uptake increase total costs, linkage to VMMC services that use task-shifting/sharing providers, pre-established facilities (both fixed site VMMC clinics and other clinics temporarily repurposed for VMMC services), and integrated HIV, sexual, and reproductive health services may save costs. Five studies assessing community-based interventions compared costs between various service delivery modalities, including mobile vs fixed, outreach vs fixed, and horizontal vs diagonal ,40–43. TOverall, age prioritization interventions increased demand generation costs but resulted in sufficient demand to decrease overall VMMC unit costs and lower costs per HIV infection averted ,25,39. FFinally, one study assessed cost effectiveness of three facility-based quality improvement strategies, which increased in intensity from providing a VMMC training manual to providing a training package including a manual, handover meeting, and individualized guidance and mentoring . QualityThree studies with non-comparative designs were considered feasibility studies, all assessing facility-based interventions –47. DateService delivery interventions were considered feasible, particularly regarding facility preparedness and health care workers. Facility preparedness and service quality improved as the number of VMMC facilities increased. Task-shifting and task-sharing enabled more facilities to offer VMMC , and scale-up increased both surgical and non-surgical efficiencies.One study assessed facility preparedness and VMMC service quality in Kenya, South Africa, Tanzania, and Zimbabwe over the first year of VMMC scale-up . It repoTwo studies highlighted human resource shortages as a potential barrier to the feasibility of VMMC scale-up in Kenya, Tanzania, and eSwatini; both studies reported that task-shifting and sharing were feasible strategies to mitigate these ,47.While these studies addressed general scale-up of VMMC, no studies were identified that assessed feasibility specifically of the interventions that sought to enhance uptake in settings where a higher level of VMMC scale-up had already occurred and more specific person-centered innovations were required.A recent systematic review of barriers and facilitators to VMMC identified a number of personal, community, and service-level issues that could be addressed to improve VMMC uptake through service delivery interventions  but did While service delivery interventions generally increased VMMC uptake, overall uptake following some interventions remained low. This may reflect the time it takes for those exposed to interventions to decide to pursue VMMC and undergo the procedure. For example, a qualitative study of behavior change pathways to VMMC in Zimbabwe highlighted the dynamic nature of demand for VMMC, which involves a man’s progression through multiple stages of change over time . Indeed,Although we grouped interventions into three categories (community- (including home-), school-, and facility-based) for this review, we found a wide range of intervention approaches within these categories. The diversity of interventions and limited replication of intervention strategies in multiple settings made it difficult to compare interventions or to assess the true effect and feasibility of interventions that appear to be promising. Four similar studies were undertaken in Tanzania; all found that providing periodic outreach campaigns increased uptake. Though VMMC programmes and implementing partners in priority countries have employed diverse service delivery interventions to achieve targets and improve VMMC coverage, we were only able to find published studies evaluating such interventions in five countries: South Africa, Tanzania, Uganda, Zambia, and Zimbabwe. This may partially reflect the challenges of creating locally relevant, people-centered service delivery approaches or the infrastructure needed to undertake research and publish peer-reviewed intervention evaluations.The majority of interventions identified in the effectiveness review were community-based and included changes to VMMC referral approaches with enhanced follow up, intensified demand creation efforts with community-based services closer to work or home, campaigns, mobile services, and use of technology to strategically plan service delivery for hard-to-reach locations. Campaigns were more effective in increasing VMMC uptake among adolescents than adult men, who responded more to interpersonal communication-based approaches. Because VMMC uptake is generally higher among adolescents, it could be that older men feel less comfortable accessing VMMC in high-volume settings or have unique concerns that are better addressed by tailored communication. For example, a qualitative study of social and individual factors influencing VMMC decisions among adult men in Tanzania found that men often expressed shame at seeking services with younger boys . This stHowever, we only identified five studies that focused on reaching specific age groups with VMMC. Two of these, both in Tanzania, aimed to reach adult clientele (age >20) with VMMC; these studies had somewhat mixed results but showed that periodic mobile service delivery and interpersonal motivational advisors/mentors were promising strategies for enhancing uptake among adult men. The remaining three studies with age-targeting focused on school-based interventions, which unsurprisingly yielded greater uptake among adolescents 10–19 years) compared with adult men; these approaches relied on trusted mentors and facilitated access to clinics. In addition, two studies of VMMC services in the general male population  provided age-disaggregated uptake data; consistent with the literature –9, both  years coWhile we found data for the effectiveness review on VMMC uptake, we found limited data on uptake of HIV testing within VMMC services, and no comparative data on uptake of safer sex and risk reduction counseling within VMMC services. HIV testing and safer sex and risk reduction counseling are considered standard parts of HIV testing services, so researchers may not have measured these components of VMMC separately. While these measures are a potential measure of service quality, this is also challenging, as individual clients may opt-out of these services, and 100% uptake on either of these measures might indicate coercive or non-optional uptake. Further, despite the lack of studies quantifying uptake of counseling during VMMC, relationships between VMMC, counseling, and risk compensation have been assessed substantially in the literature; evidence indicates that VMMC leads to little or no risk compensation in sub-Saharan Africa –60.Reviews of VMMC acceptability, cost, and feasibility identified studies from seven countries and found promising results. Findings from the acceptability review suggest that service delivery approaches were generally considered acceptable and were perceived to help men choose to get circumcised. However, service delivery interventions that do not adequately consult or partner with community members were met with less acceptance.Findings from the cost review indicate that mobile and outreach services may reduce VMMC unit cost, compared with fixed services. Further, while age prioritization interventions were shown to increase demand generation costs, they also resulted in sufficient demand to decrease overall VMMC unit costs and lower costs per HIV infection averted. While we found few studies assessing the feasibility of VMMC service delivery interventions, findings from our feasibility review suggest that task-shifting or sharing and facility preparedness play an important role in intervention success. These are important considerations for future service delivery interventions.Strengths of this review include our attention to a range of factors relevant to policy and programmatic decision-making beyond intervention effectiveness, including values and preferences, acceptability, cost, and feasibility. We also used rigorous search, screening, data extraction, and analysis methods to identify all possible articles and conference abstracts that met our inclusion criteria. However, our search did not include grey literature or program reports, which may have provided valuable additional information. Many VMMC programs may be implementing service delivery strategies without rigorous or published evaluations. We organized our findings for the effectiveness review by study design to distinguish between the quality of evidence of RCTs compared with observational studies, but the diversity of included articles, populations, study designs, and outcomes made it difficult to synthesize results. Further, because several included articles used a combination of intervention approaches—and some of these included a control or comparison group also receiving some element of the intervention—we were limited in our ability to precisely conclude which intervention approaches were responsible for reported effects. Additional research should consider further assessing the differential impacts as well as the impact of age- or HIV risk-prioritization interventions on uptake among subgroups.Findings from this systematic review indicate that changes to service delivery to increase VMMC uptake among a target population, specifically adult men or adolescent boys, can be effective, cost-efficient, and acceptable. These various intervention strategies, including interpersonal mentorship/advisor support, targeted demand-generation activities to increase awareness and intent, with improved service access by periodic VMMC campaigns, mobile VMMC service delivery, facilitated transportation, and community (and parent for adolescents) engagement seem to increase uptake of VMMC. But their full impact is unclear due to a lack of published, high-quality evaluations of such interventions. As VMMC programs continue striving to meet targets and better engage men, there is increased need to identify cost-effective and efficient strategies that are accessible and acceptable to those at highest risk and harder to reach. While further evaluations are needed, given the time to undertake such evaluations, approaches using program data should be improved to guide changes in interventions, partnering with men and communities. Given the challenges other HIV services have had reaching men in sub-Saharan Africa, the diverse and creative approaches to offering services presented in this review represent promising opportunities for VMMC and other HIV service provision.S1 Table(DOCX)Click here for additional data file.S1 File(DOC)Click here for additional data file.


Many different drugs and agents may cause nephrotoxic acute kidney injury (AKI) in children. Predisposing factors such as age, pharmacogenetics, underlying disease, the dosage of the toxin, and concomitant medication determine and influence the severity of nephrotoxic insult. In childhood AKI, incidence, prevalence, and etiology are not well defined. Pediatric retrospective studies have reported incidences of AKI in pediatric intensive care units (PICU) of between 8% and 30%. It is widely recognized that neonates have higher rates of AKI, especially following cardiac surgery, severe asphyxia, or premature birth. The only two prospective studies in children found incidence rates of 4.5% and 2.5% of AKI in children admitted to PICU, respectively. Nephrotoxic drugs account for about 16% of all AKIs most commonly associated with AKI in older children and adolescents. Nonsteroidal anti-inflammatory drugs (NSAIDs), antibiotics, amphotericin B, antiviral agents, angiotensin-converting enzyme (ACE) inhibitors, calcineurin inhibitors, radiocontrast media, and cytostatics are the most important drugs to indicate AKI as significant risk factor in children. Direct pathophysiological mechanisms of nephrotoxicity include constriction of intrarenal vessels, acute tubular necrosis, acute interstitial nephritis, and—more infrequently—tubular obstruction. Furthermore, AKI may also be caused indirectly by rhabdomyolysis. Frequent therapeutic measures consist of avoiding dehydration and concomitant nephrotoxic medication, especially in children with preexisting impaired renal function. Many different drugs and agents are currently being taken into consideration as the causality of nephrotoxic acute kidney injury (AKI) in children. Predisposing factors such as age, pharmacogenetics, underlying disease, dosage of the toxin, and concomitant medication determine and influence the severity of nephrotoxic insult. The culprit toxins are predominantly drugs, but exogenous  and endogenous  substances and toxins from animals play a role as well. Throughout this teaching article, incidence, pathophysiological mechanisms, and treatment options are discussed in general followed by characteristics of problematic drugs. Because of the paucity of multicenter studies exploring AKI in children, previously conceived literature of AKI in adults ought to be considered.Tumor lysis syndrome is not discussed. However, calcineurin inhibitor toxicity is briefly mentioned, as it has been respectively inclusive to the topic throughout multiple publications –8.Most authors define AKI as a sudden decline in glomerular filtration rate (GFR) mirrored by doublings of serum creatinine and azotemia. Because a precise clinical definition remains elusive, studies comparing epidemiology and outcome can be problematic . This figure was most likely generated by hypoxic/ischemic and nephrotoxic insults , 12. OthCommunity-based statistics estimate the incidence of AKI attributed to drug nephrotoxicity as being between 0% and 7% , 18 and In childhood AKI, incidence, prevalence, and etiology are not well defined. Pediatric retrospective studies have reported incidences of AKI in pediatric intensive care units (PICU) of between 8% and 30% –39. It iTable NSAIDs possess antipyretic, analgesic, and anti-inflammatory effects. They are frequently used in children and have numerous therapeutic indications; for example, in the treatment of fever, postoperative pain, and inflammatory disorders such as juvenile idiopathic arthritis and Kawasaki disease. Their major mechanism of action is through inhibition of prostaglandin biosynthesis by blockade of cyclooxygenase (COX). In euvolemic states, prostaglandins have a negligible effect on renal hemodynamics. In states of volume depletion, there is upregulation of the renin–angiotensin system as well as increased catecholamine release that, in addition to producing renal vasoconstriction, stimulates renal prostaglandin production. These prostaglandins counteract the vasoconstrictor effect by decreasing preglomerular resistance and thereby maintaining renal perfusion and glomerular filtration. This protective effect is inhibited by the blockade of prostaglandin synthesis by NSAIDs. The disposition of most NSAIDs has been mainly studied in infants older than 2 years of age . Next toAlthough AKI is a well-known adverse effect of these drugs, the incidence of nephrotoxicity due to NSAIDs, especially in children, is unknown. It is probable that these substances carry a good risk–benefit ratio when taken under adequate supervision. However, when taken continually by the elderly or individuals with comorbid conditions (dehydrated children), the frequency of adverse reaction rises dramatically.The vast majority of healthy children who ingest therapeutic doses of NSAIDs for a limited duration tolerate them without any significant adverse effects. However, the risk of renal toxicity potentially increases in situations where there is stimulation of the renin–angiotensin system; for example, with volume depletion or preexisting chronic renal disease. Some case reports of AKI following NSAID administration have been published. In most of these cases, dehydration played an important role –54. But In neonates the therapeutic and/or preventive use of ibuprofen and indomethacin for closure of patent ductus arteriosus (PDA) in preterm and/or low birth weight infants was analyzed in a Cochrane review. It showed that ibuprofen reduced the risk of oliguria. It also demonstrated that a prolonged course of indomethacin was associated with a decreased incidence of renal function impairment, as evidenced by a lower proportion of infants having diminished urine output –62. CurrIn adults treated with Mesalazine because of inflammatory bowel disease, nephrotoxicity is reportedly evident in the treatment of one out of every 150 adult patients. Although more than 30 cases in adults have been described in the literature, only three cases of pediatric patients younger than 16 years have been published -lactam antibiotics comprise penicillins, cephalosporins, monobactams, clavulanic acid, and carbapenems. Cephalosporins and carbapenems have been associated with nephrotoxicity in humans , 78. NepMacrolide antibiotics such as erythromycin, clarithromycin, azithromycin, and dirithromycin may indirectly cause AKI via enhancing calcineurin levels by inhibiting cytochrome P-450 isoenzyme CYP 3A4 . This neA number of additional anti-infective drugs that may cause AKI in children include sulfonamides, trimethoprim-sulfamethoxazole (TMP-SMX) and quinolones. Some of these are of interest because of their use in treating infectious complications in patients with HIV infections. Systematic evaluation of renal side effects of these substances in children are lacking, though aspects of nephrotoxicity in adults are discussed in detail by Vaamonde et al. .Nephrotoxicity of amphotericin B has been a matter of research for a long time. Despite its significant renal side effects, amphotericin B is still the drug of choice in the treatment of severe systemic fungal infections, especially in immunocompromised children. Its nephrotoxicity and measures of prevention have been recently reviewed by Koren and coworkers , 89 and Antiviral agents  have become increasingly available for clinical use, especially to treat HIV infections. AKI is a significant and potentially therapy-limiting side effect to some of them. Antiviral agents have also been known to cause irreversible renal damage in adults . AntivirFoscarnet causes a reversible decrease in GFR in 20–60% in adults –106. TwoPublished data regarding renal failure in antiretroviral agents is scarce, especially in children. Some patients have been reported with AKI attributed to ritonavir, but the risk of AKI in this group of drugs appears low.The use of ACE inhibitors and angiotensin II receptor type 1 antagonists (AT2-receptor antagonists) has increased dramatically over the last decade –115. ACEThe presence of AKI due to ACE-inhibitor-induced interstitial nephritis has been described in few adult cases, but an occurrence of this kind in children has yet to be documented . In contIt is generally accepted to be true, though not exactly proven, that the renal hemodynamic effects of ACE inhibitors are due to the blockade of a renin–angiotensin system. In a significant percentage of renal patients  and in patients with congestive heart failure, especially when volume depleted , the maintenance of GFR is highly dependent on angiotensin-II-mediated efferent vasoconstriction. In the case of instituting ACE inhibitors and/or AT2-receptor antagonist treatment, close surveillance of renal function and blood pressure is needed, at least during the first 2 weeks, because a decline in renal function may be transient. The treatment should be started at low doses then gradually increased. The concomitant use of NSAIDs should be avoided, because in these situations, the afferent vasoconstriction (by prostaglandin synthesis inhibition) in combination with efferent vasodilatation (due to ACE inhibition) may result in a marked decline of GFR. The sometimes inevitable combination of ACE inhibitors and calcineurin inhibitors needs close monitoring and expertise.AKI has not been seen after the administration of other antihypertensive agents that do not interfere with the renin–angiotensin system.The therapeutic potential and renal side effects of CyA and tacrolimus are well known to pediatric nephrologists. The introduction of calcineurin inhibitors has improved the outcome of renal and other organ transplants dramatically, but cyclosporine-related nephrotoxicity has become a significant side effect to manage . Indeed,GFR impairment is caused by intrarenal vasoconstriction that induces a decrease in renal blood flow. A great deal of effort has been taken to unravel the mechanism of CyA-induced vasoconstriction. Some mechanisms and mediators have been found. The renin–angiotensin–aldosterone system is involved , as are Especially in renal transplant patients, nephrotoxicity caused by calcineurin inhibitors may be difficult to distinguish from transplant rejection. In this situation, a renal biopsy may be indicated. Otherwise, in extrarenal organ transplantation and nonrenal autoimmune disease patients, a creatinine rise is very likely to be caused by CyA. As mentioned above, interaction of concomitant drugs enhancing CyA levels via inhibition of cytochrome P450 system  needs to be considered, as this combination can cause AKI. Drugs that decrease renal blood flow should also be avoided whenever possible. Especially in bone marrow transplantation (BMT), CyA can cause acute HUS, usually with severe AKI, carrying a poor prognosis.The acute nephrotoxicity profile of tacrolimus is very similar to that of CyA, as it induces reversible functional changes with the same frequency and intensity . There aThe incidence of AKI after administration of tri-iodinated radiocontrast media has been found to be between 1% and 20% in adults . IncidenRadiographic contrast-agent-induced nephropathy is caused by vasoconstriction-mediated renal medullary ischemia and direct toxic damage to renal tubular epithelial cells. These effects may be partially mediated by the generation of reactive oxygen species. Most cases present as nonoliguric AKI, although oliguric renal failure is possible as well . Maximum2), particularly in patients concurrently treated with cisplatin   has become available in the treatment of MTX-induced renal dysfunction. CPDG(2) administration has been well tolerated and results in consistent and rapid reductions in plasma MTX concentrations by a median of 98.7%. Early administration of CPDG(2) in combination with leucovorin may be beneficial for patients with MTX-induced renal dysfunction and significantly elevated plasma MTX concentrations .The combination of nephrotoxic drugs in the setting of HSCT intensifies nephrotoxic probability. Renal aspects of this specific therapeutic maneuver have been reviewed elsewhere , so hereA growing number of case reports have been published in adult medicine regarding the nephrotoxicity of bisphosphonates. With regard to zoledronate, the incidence of AKI has been found to be between 9% and 13%. The pathology showed diffuse tubular atrophy and injury without interstitial nephritis. Bisphosphonate-induced AKI in children remains undocumented , 164.Whereas therapy with intravenous immunoglobulin has been known to carry a 1–15% risk of AKI in adults –167, onlToxin-induced AKI by prescription of herbal and alternative medicines is uncommon in Europe but has become a common cause of mortality and morbidity in Africa . AniClinical management and supportive therapy of children with suspected or established AKI caused by nephrotoxicity is not unlike other forms. A child’s clinical history may help reveal which drugs had been administered within the previous 5–7 days. Postrenal obstruction needs to be excluded by ultrasound, and infective or immunological causes need to be addressed by laboratory investigations. Hemolysis and rhabdomyolysis need to be excluded, especially in cases of dark urine (which is sometimes misinterpreted as macrohematuria).It is of great importance to assess the exact status of hydration  and to decide whether a load of fluid or diuretics may be necessary. Correction of volume depletion and/or congestive heart failure and reversing diminished renal perfusion are of primary importance. Especially in patients with dehydration, special attention has to be paid to good fluid management and replacement to establish adequate renal blood flow and perfusion. The administration of renal-dose dopamine (1–3 µg/kg min) was shown to be ineffective on patient survival or outcome in both adults –174 and Diuretics have also been used in patients with oliguric AKI to improve urine output, which may decrease the concentration of toxins and improve clearance of solute and medications. It should be noted, however, that the increase in urine flow provided by diuretic therapy does not reflect an improvement in GFR, nor has it been demonstrated to prevent or facilitate recovery from acute tubular necrosis . In addiAs children with nephrotoxic insults are more likely to suffer nonoliguric renal failure, serum chemistry needs to be supervised very carefully in high-risk children. It has also to be considered that in children with reduced muscle mass , GFR is overestimated by assessment of serum creatinine (or Schwartz formula). These two aspects may delay the diagnosis of AKI or may result in GFR overestimation. When medications are prescribed in AKI, the mechanism of drug elimination, metabolic pathway, and possible further nephrotoxic effects must be considered and adjustments made for the degree of renal failure.In case of suspected or definite nephrotoxic agents being the cause of AKI, drug withdrawal is the most important measure physicians can take, as avoidance of further possible nephrotoxic agents needs to be strictly considered. In all children, the basic principles of managing AKI are fluid maintenance, electrolyte homeostasis, and adequate nutrition. Only few patients will require specific treatment as acetylcysteine in radiocontrast nephropathy. In interstitial nephritis, corticosteroids may be given, but their benefits are not proven by RCTs.Regarding indication and optimal choice of renal replacement therapies, we refer to published reviews and guidelines –184.The mortality of children with AKI was found to be between 8% and 89% in retrospective studies , 40. In In the setting of HSCT, the doubling of serum creatinine is associated with a twofold increase in mortality in adults, and the need for dialysis predicts a mortality rate of 80–90% , 188. InThe true incidence of nephrotoxicity as a cause of AKI in children is unknown. In general, drugs are an infrequent cause of community-acquired AKI, especially in children. However, drugs and hypoxia are the leading etiologic factors for hospital-acquired AKI, and it remains a significant cause of morbidity and mortality in children. Despite advances in understanding the pathophysiology of AKI, little progress has been made in its treatment. The judicious use of all nephrotoxic drugs and their combinations, together with adequate hydration, are still the most important measures to take in the minimization of nephrotoxicity. Children with AKI caused by nephrotoxic agents have a significant risk for chronic renal injury.Questions (answers appear following reference list)AIn most children with NSAIDs-induced AKI, dehydration played an important role.BNSAIDs-induced AKI is mostly caused by interstitial nephritis.CMesalazine-induced AKI is always caused by interstitial nephritis.DGlucocorticoids play an important role in treatment of NSAIDs-induced AKI.Which one of the following statements is true of NSAIDs-induced AKI?AHave higher rates of nephrotoxicity-induced AKI compared with older children.BHave generally a poor long-term prognosis in relation to nephrotoxicity-induced AKI.CBenefit from a “pharmacological dosing” of gentamicin regarding nephrotoxicity.DBenefit generally from ibuprofen compared with indomethacin regarding the closue of PDA.Which one of the following statements is true? Neonates:Aβ-lactam antibiotics need tubular transport for nephrotoxicity.BRenal replacement therapy always shortens the time of renal insufficiency by removing toxins.CCeftriaxone may induce AKI indirectly by hemolysis and nephrolithiasis.DMacrolide antibiotics may cause indirect AKI by enhancing calcineurin levels by inhibiting cytochrome P-450 isoenzymes.Which one of the following answers is not true?AACE inhibitors are forbidden in children with steroid-resistant nephrotic syndrome because of their risk of AKI.BThe concomitant use of NSAIDs and ACE inhibitors should be avoided.CAKI caused by ACE inhibitors is always characterized by interstitial nephritis.DConcomitant therapy with AT2-receptor antagonists enhances side effects of ACE inhibitors.Which one of the following answers is true?ANephrotoxicity is the most significant and limiting adverse effect caused by calcineurin inhibitors.BThe acute nephrotoxicity of CyA is a hemodynamically mediated phenomenon.CA kidney biopsy is often necessary in renal transplant children to distinguish between CyA nephrotoxicity and rejection.DCyA-induced HUS in BMT carries a rather good prognosis.Which one of the following answers is not true?


While the area of plantation forest increased globally between 2010 and 2015, more than twice the area of natural forests was lost over the same period . Consequently, there is an increasing need to understand how plantation land use affects biodiversity. The relative conservation value of plantation forests is context dependent, being influenced by previous land use, management regimes and landscape composition. What is less well understood, and of importance to conservation management, is the consistency of diversity patterns across regions, and the degree to which useful generalisations can be provided within and among bioregions. Here, we analyse forest birds in Ireland, France and Portugal, representing distinct regions across the Atlantic biogeographic area of Europe. We compared taxonomic, functional and phylogenetic diversity of bird communities among conifer plantations and semi-natural oak forests, and assessed correlations between species traits and forest type across these regions. Although bird composition (assessed with NMDS ordination) differed consistently between plantation and oak forests across all three regions, species richness and Shannon diversity did not show a consistent pattern. In Ireland and France, metrics of taxonomic diversity (richness and Shannon diversity), functional diversity, functional dispersion and phylogenetic diversity were greater in oak forests than plantations. However, in Portugal taxonomic and phylogenetic diversity did not differ significantly between forest types, while functional diversity and dispersion were statistically significantly greater in plantations. No single bird trait-forest type association correlated in a consistent direction across the three study regions. Trait associations for the French bird communities appeared intermediate between those in Ireland and Portugal, and when trait correlations were significant in both Ireland and Portugal, the direction of the correlation was always opposite. The variation in response of bird communities to conifer plantations indicates that care is needed when generalising patterns of community diversity and assembly mechanisms across regions. Globally, the area of natural and semi-natural forests , decreased by 6.5 million hectares per year between 2010 and 2015 ) Quercus spp) forests and conifer plantations, and use this spatial variation to evaluate differential bird responses to these two types of forest. To assess whether these responses are consistent among regions, we compiled data from three regions  representing distinct Atlantic biogeographic areas of Europe. Specifically, we examined (a) if bird community composition and diversity  in conifer plantations differed from semi-natural forests in each study region, (b) if these responses were consistent across study regions, and (c) whether consistent trait-environment relationships determined which species responded positively or negatively to plantation forests.To assess if bird community-forest type relationships are consistent between regions, we compare bird communities in semi-natural oak (Quercus robur) and Sessile oak (Q. petraea), but also comprised Downy birch (Betula pubescens) and Holly (Ilex aquifolium). In France, the study area was located in the southwest region of Aquitaine, the largest area of pine plantations in the country. Landscapes are dominated by mosaic plantations of even-aged stands of native Maritime pine (Pinus pinaster) with rotation cycles of 40–50 years; these plantations are interspersed with open habitats and fragments of semi-natural oak forest dominated by Pedunculate oak, Pyrenean oak (Q. pyrenaica) and Silver birch (B. pedula). In Portugal, the study sites were located in the Alto Minho region, in the northwest of the country, in a mosaic landscape of forest, agriculture and scrubland. Surveyed semi-natural oak forests were dominated by Pedunculate oak and Pyrenean oak, but also included Iberian Downy birch (B. celtiberica). Conifer plantations were monocultures of the native Maritime pine with rotation cycles of 20–45 years; this species is one of the most dominant commercial species in Portuguese planted forests, especially in northern regions. Although Maritime pine is a native species to Portugal and France, its natural distribution is unclear as a result of extensive commercial exploitation, which has significantly increased its distribution into areas where it may have not occurred naturally [We recorded forest bird communities from three study regions located in Ireland, France and Portugal, within the Atlantic biogeographical region of Western Europe  using the phylo.d function in the ‘caper’ package [D assesses phylogenetic signal in binary traits (such as community membership) against null models of Brownian motion trait evolution and phylogenetic randomness, and is scaled to be zero under Brownian motion trait evolution and one under phylogenetic randomness [D. We calculated phylogenetic diversity and dispersion for each forest patch and phylogeny, before taking the mean value across phylogenies for each study region.We obtained subsets of a global phylogeny , and sub package . We calc package ; D assesndomness . Only spAll taxonomic, functional and phylogenetic metrics (see above) were calculated at the forest-patch level, before taking the mean value across each forest type and across each study region. As the majority of metrics calculated had non-normal distributions, we used non-parametric means tests (Mann-Whitney U) to identify differences between semi-natural oak forests and conifer plantations.RLQ, the fourth-corner procedure can analyse quantitative variables, qualitative variables or a mixture of both. We used binary coding of the environment matrix (forest type) against a quantitative and binary trait matrix resulting in the generalized statistic being equal to the Pearson correlation coefficient r. Binary coding also allowed for directional correlations (positive or negative) between species traits and forest types. We applied permutation Model 1 (with 9999 permutations) to test the null hypothesis that R is not linked to Q, when examining links between the fixed table of species traits and the fixed table of site attributes, mediated by the observed abundance data of matrix L [a priori [Trait-specific responses to conifer plantations were analysed by means of fourth-corner analysis, using the ‘ade4’ package , 43. Foumatrix L . We optea priori . We useda priori .Significant differences in bird community composition were found between conifer plantations and semi-natural oak forests in all regions sampled . Community composition in Irish assemblages showed greater separation between forest types, while the overlap in forest type ellipses was more obvious in the Portuguese assemblages, and intermediate in France .Responses of bird communities to conifer plantations were inconsistent between Ireland and Portugal. In Ireland, species richness and Shannon diversity were significantly lower in conifer plantations than in semi-natural oak forests , while bird communities were also less functionally diverse and clustered in functional space  and in France , but did not differ significantly between these forest types in Portugal . PhylogeNo single trait-forest type association correlated in a consistent direction across all three study regions . Again, In Ireland, small-bodied bird species with short life spans, large clutch sizes and small home ranges were associated with conifer plantations . In FranIn this study we examined whether taxonomic, functional and phylogenetic variation in bird assemblages between semi-natural forests and conifer plantations were congruent across a large latitudinal gradient in Western Europe. Although differences in bird communities between conifer plantations and native semi-natural oak forests were evident in all regions, responses to plantation forests were not consistent between Ireland, France and Portugal. In Ireland and France, taxonomic, functional and phylogenetic diversity were lower in conifer plantations than semi-natural oak forests, which is consistent with previous studies reporting significant decreases in species richness  and chanDifferences in bird community responses to conifer plantations could result from broad-scale extrinsic factors such as climate, landscape and regional context  and birdAlthough it remains unclear which factors drive the differences observed between the three study regions, the lack of consistency in responses between regions indicates the importance of ecological contexts on bird community responses to conifer plantations. Therefore, we call for caution when attempting to generalize research findings from one location to another, or when scaling results up from regional to biogeographical scales.Spinus spinus) and coal tit (Periparus ater) [Picus sharpei) and golden oriole (Oriolus oriolus), were positively associated with conifer plantations. This pattern suggests that environmental filters acting in one region can be absent or reversed in another, such that the selection for small species in conifer plantations in Ireland was not observed in Portugal, where larger species were associated with conifer plantations.Functional traits associated with bird species occurrence in conifer plantations differed among regions. For example, migratory bird species were positively associated with conifer plantations in France, while in Ireland it was resident species that were positively associated with conifer plantations. Again, it is not possible to definitively identify why trait associations varied from Ireland to Portugal, but we can propose some potential explanations for some traits. In Ireland, species with traits associated with fast life history strategies  and small home range size were positively associated with conifer plantations. Previous studies have demonstrated the importance of Sitka spruce plantations for bird species sharing this collection of traits , such as siskin (us ater) . Possiblus ater)  affects Picus viridis), common redstart (Phoenicurus phoenicurus) and spotted flycatcher (Muscicapa striata), can nest in oak forest patches while foraging in nearby conifer stands. Conversely, conifer plantations in this region are often close or adjacent to open habitats , which allows typical edge species such as tree pipit , or species associated with young pine plantations such as grasshopper warbler (Locustella naevia) and Dartford warbler (Sylvia undata) to occur in older adjacent conifer plantations, especially along the forest edge [The surrounding landscape context may influence the occurrence of birds in conifer plantations , so diffest edge , 49. In est edge , 53. Theest edge , 49.The responses of bird communities to conifer plantations, and the traits that determine which species were associated with plantations, varied among the three study regions. The inconsistent trait-environmental correlations among the three regions sampled suggest that functional generalizations across large geographical regions should be made cautiously. Although it was not possible to identify formally the contribution of intrinsic differences attributed to forest structure and management versus extrinsic differences attributed to landscape-scale factors, assemblage and climate; our work highlights that these processes can lead to context-dependent responses to conifer plantations. It is therefore important to capture these context-dependent processes if responses of birds to forestry are to be extrapolated confidently to new regions.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Cognitive training has been shown to improve neural plasticity, increase cognitive reserve and reduce the risk of dementia in older adults. Specifically, learning to play the piano has been shown to be an engaging, multimodal form of cognitive training. However, accessing this form of cognitive training can pose a challenge for older adults. Socially assistive robots present a unique opportunity to increase access to user-tailored piano learning cognitive training. The present study utilized a robot-led four-week piano lesson feasibility intervention for older adults with mild cognitive impairment . Cognitive Status was assessed during screening via the Telephone Interview for Cognitive Status, and after screening via the Mini-Mental State Exam and the CNS Vital Signs computerized test suite to measure cognitive domain-specific functioning. Perceptions and acceptance of the robot were measured using the Robotic Social Attributes Scale (RoSAS) and Technology Acceptance Scale. Cognitive function improved after four weeks of training in the verbal memory, executive function, reaction time and cognitive flexibility domains, and in the computed neurocognitive index score (p<0.05). Survey data and qualitative interviews show that participants perceived the robot instructor as socially engaging, competent, useful, and easy to use. These results provide insight into the potential of SARs to facilitate cognitive training in the form of piano lessons, as well as recommendations for creating a suitable robot instructor for this application.


Osteosarcoma is the most common primary malignant bone tumor mainly occurred in children and adolescence, and chemotherapy is limited for the side effects and development of drug resistance. Advances in nanotechnology and knowledge of cancer biology have led to significant improvements in developing tumor-targeted drug delivery nanocarriers, and some have even entered clinically application. Delivery of chemotherapeutic agents by functionalized smart nanocarriers could protect the drugs from rapid clearance, prolong the circulating time, and increase the drug concentration at tumor sites, thus enhancing the therapeutic efficacy and reducing side effects. Various drug delivery nanocarriers have been designed and tested for osteosarcoma treatment, but most of them are still at experimental stage, and more further studies are needed before clinical application. In this present review, we briefly describe the types of commonly used nanocarriers in osteosarcoma treatment, and discuss the strategies for osteosarcoma-targeted delivery and controlled release of drugs. The application of nanoparticles in the management of metastatic osteosarcoma is also briefly discussed. The purpose of this article is to present an overview of recent progress of nanoscale drug delivery platforms in osteosarcoma, and inspire new ideas to develop more effective therapeutic options. Chemotherapy is an important approach in cancer therapy. Effective treatment of cancers needs accurate delivery of an enough intracellular dose of chemo-drugs to kill the cancer cells Osteosarcoma is the most common primary malignant bone tumor mainly occurred in childhood and adolescence In this review, we retrospectively summarized the recent advances of nanocarriers for targeted drug delivery in osteosarcoma. We discuss the commonly used types of drug delivery nanoparticles, controlled drug release upon different stimuli, and nanocarriers modification strategies for targeted drug delivery. The application of nanoparticles in the management of metastatic osteosarcoma is also briefly discussed. And we hope this review will provide readers a general understanding of current status in osteosarcoma nanomedicine, and inspire further investigations in novel drug delivery nanosystems for osteosarcoma treatment.Nanosized drug delivery systems can be roughly classified into organic and inorganic carriers Liposomes are spherical vesicles with a hydrophilic cavity surrounded by one or several lipid bilayers that allows the encapsulation of drugs with different solubility. Hydrophobic drugs can be entrapped by the lipid bilayer and hydrophilic drugs can be encapsulated in the central aqueous core in vitroA variety of nanoscale liposomes for anti-osteosarcoma agent delivery have been explored in the past years. Clinical trials have demonstrated that inclusion of liposomal muramyl tripeptide phosphatidyl ethanolamine (L-MTP-PE) could clinically and significantly improved the long-term survival of osteosarcoma patients Many different polymers have been widely used for anti-cancer drug delivery and have received increased interest in recent years. Commonly used polymers from synthetic such as poly lactide-co-glycolic acid (PLGA) and PEG, or from natural origin such as hyaluronan and chitosan, demonstrate good biocompatibility and biodegradability. A range of biodegradable polymeric drug delivery systems designed for localized or systemic administration of therapeutic agents has been under clinical trials or approved for cancer treatment in situ crosslinked nanogel based on HA has been synthesized for codelivery of DOX and cisplatin, two of the most widely clinically used chemo-drugs with proved synergistic effects, to osteosarcoma Different biodegradable polymers have been used for designing safe and efficient nanocarriers for anti-osteosarcoma agent delivery. Suksiriworapong et al. Micelles are usually formed by amphiphilic polymers and have attracted considerable attention as promising nanocarriers for drug delivery. Polymeric micelles consist of a core and shell structure. In principle, the micelle core part is usually hydrophobic and can encapsulate poorly water-soluble agent, whereas the outer shell is able to stabilize the micelles in aqueous environment and can be modified with stimuli-responsive or tumor-targeting moieties in vitro and in vivoSeveral studies have reported different kinds of micelles for osteosarcoma treatment Even if micelles are highly stable in aqueous environment due to their low critical micellar concentration, they may also have a tendency to be dissociated in dilution or high ionic strength. A way to overcome this problem is introducing the cross-linking bridges in the hydrophobic core or in the hydrophilic shell, and thus regulating the drug release Dendrimers are nanoscale, globular, radially symmetric, water-soluble macromolecules with well-defined sizes, branched structures, and high density of modifiable functional groups in vitro and in vivo.Dendrimers has been investigated as chemo-drug or gene delivery systems in osteosarcoma. Recently, a new type of nanogels containing DOX was synthesized by incorporating generation 5 (G5) PAMAM dendrimers and DOX into alginate (AG) nanogels Metallic nanocarriers can be pure metallic particles such as gold, silver, and copper; or metallic compound such as oxides and Mxene; or hybrid polymers that consist of metal ions or clusters such as metal organic frameworks (MOFs) Among the pure metallic nanoparticles, gold and silver nanoparticles are the most commonly investigated in osteosarcoma therapeutics. Due to the remarkable properties such as high surface area to volume ratio, stable nature, multi-functionalization, facile synthesis, high permeability and retention effect, and photothermal conversion capability, gold nanoparticles (AuNPs) have long been considered as a potential tool for cancer treatment in vivo were not further explored in these studies. Among the metallic oxide nanoparticles, iron oxide such as ferroferric oxide (Fe3O4) was the most commonly investigated nanomaterials in osteosarcoma. And these nanoparticles were mostly used for thermal therapy due to its ability to convert the energy of magnetic field into heat Metallic compound-based nanoparticles reported in osteosarcoma treatment are mainly metallic oxides, and these metallic oxide nanoparticles can serve as intrinsic therapeutic agents without the need of loading chemotherapy drug. For example, the anti-cancer effect of titanium dioxide (TiO2), terbium oxide (Tb2O3), zinc oxide (ZnO) and cerium oxide (CeO2) nanoparticles has been evaluated verified in osteosarcoma cells Mesoporous silica nanoparticles (MSNs) have attracted considerable attention for drug or gene delivery because of their excellent characteristics including simple fabrication process, uniform morphology, variable particle size, modifiable surface, tunable pore size and volume, and FDA recognized biosafety in vitro doxorubicin delivery rate, compared with unfunctionalized MSNs, antibody-conjugated MSNs or even free DOX. Hartono and colleagues The use of MSNs as drug or gene delivery systems in osteosarcoma have also been widely reported. Shahabi et al. Because of the superior nature such as safety, high drug loading capacity, controllable drug release and modifiable surface, MSNs are considered to have the potential to be a more promising platform for cancer therapy compared to other inorganic nanocarriers such as copper, gold, and silver which exhibit some cytotoxicity. However, there is still a long approach before clinical translation and commercialization Carbon-based nanomaterials such as carbon nanotubes (CNTs), graphene oxide (GO), mesoporous carbon, and carbon dots, have drawn considerable attention and been extensively investigated for cancer therapy because of their good physicochemical properties including easily-modified surface, exellent photo-thermal conversion ability, supramolecular π-π stacking, and high adsorption ability Among these different carbon nanomaterials, GO and CNTs were the mostly reported in osteosarcoma. Tang et al. in vitro. Another research demonstrated that SWCNTs could specifically inhibit the process of TGFβ1-induced osteosarcoma cells dedifferentiation, prevent the stem cell phenotypes acquisition and reduce the osteosarcoma stem cells viability CNTs have also attracted considerable attention for cancer therapy. Yan et al. Though many exciting advances in the field of carbon-based nanomaterials have been made, yet there were relatively few investigations of these nanomaterials as drug delivery systems in osteosarcoma. More focused and systematic research on tumor targeting, toxicity and pharmacokinetics of these nanomaterials is needed in osteosarcoma.Calcium phosphates (CaP) nanoparticles, particularly hydroxyapatite nanoparticles (HANPs), are considered as promising nanocarriers to bone tissues because they are biocompatible, biodegradable, non-immunogenic, PH-sensitive and facilely modifiable, and have shown to be preferentially accumulated in bone tissues in vitro and in vivo, and show less cytotoxicity to normal cells in vitro and in vivo. The selenium released from Se-HANs could induce tumor cell apoptosis through an inherent caspase-dependent apoptosis pathway synergistically orchestrated with ROS generation.Son et al. Targeted drug delivery generally means delivery of the intravenously administered drugs to the target site, e.g. tumors. Targeted drug delivery systems are designed to facilitate drug delivery to the tumor sites with minimum side effects, and that are performed by two targeting strategies, including passive and active targeting Even though nanocarriers can be passively targeted to tumor via EPR effect, it suffers from some serious limitations such as inefficient drug diffusion into tumor cells, the random nature of targeting, and the lack of EPR effect in some tumors. Thus, there is potential to improve the tumor targetability of the nanocarriers through active targeting strategies, such as ligand-mediated tumor targeting In vitro models showed that the JQ1-loaded HANPs significantly inhibited osteosarcoma cells migration and invasion but exhibited less cytotoxicity to primary fibroblasts In vivo. These functionalized, DOX-loaded nanoparticles demonstrated enhanced, prolonged tumor accumulation and significantly improved anticancer activities compared to nontargeted DOX-loaded nanocarriers or free DOX Bisphosphonates display a pyrophosphate-like structure and exhibit affinity toward bone by chelating with divalent calcium ions (Ca2+) present in the HAP matrix of the bone In vivo administration. Finally, LC09 were chose as osteosarcoma cell-targeted aptamers, and LC09-modified lipopolymers loading CRISPR/Cas9 plasmids encoding VEGFA gRNA and Cas9 were developed. The LC09-fuctionalized nanocomposites achieved selective distribution of CRISPR/Cas9 in both orthotopic osteosarcoma and lung metastasis, and reduced VEGFA expression and secretion, thus inhibiting osteosarcoma malignancy and lung metastasis. CD133 is considered to be a cancer stem cells (CSCs) marker in osteosarcoma or other tumors. Accordingly, CD133 aptamers have been used as targeting ligands for tracking osteosarcoma CSCs Aptamers are short, synthetic and single-stranded DNA or RNA molecules that can specifically bind to their targets with a high affinity. And aptamers have attracted broad interest in targeted drug delivery because of their high selectivity and affinity, low immunogenicity, easy synthesis with high reproducibility, facile modification, and relatively rapid tissue penetration with no toxicity HA, an endogenous polysaccharide, has hydroxyl and carboxylic groups, as well as an N-acetyl group, which can be used for further chemical modifications. HA exhibits some superior physiochemical natures, such as biodegradability, biocompatibility, and non-immunogenicity in vitro and in vivo than the nontargeted complexes.Folate is an ideal candidate for ligand-based targeted therapy, as numerous examples of folate receptor-targeted drug delivery carriers have been reported to transport anticancer drugs into cells through receptor mediated endocytosis In vitro over healthy osteoblast cells. YSA, a 12-amino acid peptide, is a ligand for ephrin type-A receptor 2 (EphA2), a surface molecule which is overexpressed in osteosarcoma cells and tissues In addition, peptide ligands, such as RGD and YSA, have shown active osteosarcoma cell targeting ability Osteosarcoma is characterized as high-grade malignant tumor due to the high local aggressiveness and rapid systemic metastasis, especially lung metastasis, and the 5-year survival rate is only about 15-30% for patients with lung metastasis. Precise and selective drug release in the tumor sites will further enhance the therapeutic efficacy and minimize the undesired side effects to normal tissues The pH of blood is approximately 7.4, whereas extracellular pH in tumors decreases to 6.0-7.2. The pH in the subcellular compartments decreases to 5.0-6.0 in endosomes and 4.0-5.0 in lysosomes In vitro release of DOX was well controlled at physiological conditions, but a burst release of more than 60% was observed in the presence of 10 mM GSH, compared to non-redox sensitive nanocarriers.Glutathione (GSH), a highly effective antioxidant, can reduce the disulfide bonds of nanocarriers. GSH present in intracellular environments (2-10 mM) is 100-fold higher than that in extracellular environments (2-10 μM). The concentration of GSH in the cancer cells was found to be much higher than that in normal cells. The difference in the redox potential between intracellular and extracellular concentration of GSH can be used for intracellularly controlled drug release once the nanocarrier is internalized In vitro and In vivo experiments. Near infrared (NIR) light has received considerable interest in photoactivated drug delivery because of its unique advantages such as deep tissue penetration, and limited photo damage Due to its noninvasiveness and spatiotemporal precision, light with a specific wavelength has been extensively used as an external stimulus for triggering on-demand drug delivery in vivo biosafety, biodistribution, drug release kinetics, and antitumor effects of this drug-loaded nanogel were not evaluated in this study.Magnetic nanoparticles (e.g. iron oxide nanoparticles) can convert magnetic energy into heat when exposed to an alternating magnetic field. The heat generated by these particles may cause structural alteration of the drug loaded nanocarriers, thus achieving an “on demand” drug release In vivo, and furthermore, animal models are different from human tissue, after all.The unknown etiology, high genetic instability, large histological heterogeneity, lack of specific biomarkers, high local aggressiveness, and a rapid metastasizing potential create challenges for osteosarcoma treatment


Although imaging techniques exist that can probe different facets of the TME, they face several limitations, including limited spatial resolution, extended scan times and poor specificity from confounding signals. Photoacoustic imaging (PAI) is an emerging modality, currently in clinical trials, that has the potential to overcome these limitations. Here, we review the biological properties of the TME and potential of existing imaging methods that have been developed to analyse these properties non-invasively. We then introduce PAI and explore the preclinical and clinical evidence that support its use in probing multiple features of the TME simultaneously, including blood vessel architecture, blood oxygenation, acidity, extracellular matrix deposition, lipid concentration and immune cell infiltration. Finally, we highlight the future prospects and outstanding challenges in the application of PAI as a tool in cancer research and as part of a clinical oncologist's arsenal.The tumour microenvironment (TME) is a complex cellular ecosystem subjected to chemical and physical signals that play a role in shaping tumour heterogeneity, invasion and metastasis. Studying the roles of the TME in cancer progression would strongly benefit from non-invasive visualisation of the tumour as a whole organ  Summary: This Review details the potential of photoacoustic imaging to visualise features of the tumour microenvironment such as blood vessels, hypoxia, fibrosis and immune infiltrate to provide unprecedented insight into tumour biology. Absorption coefficient: the fraction of incident radiant energy absorbed per unit mass or thickness of an absorber.Angiogenesis: the development of new blood vessels from pre-existing vessels. Driven by pro-angiogenic factors such as VEGF and inhibited by anti-angiogenic factors.Contrast agent: a substance or molecule that is administered into a living subject to enhance the visualisation of a particular structure or biological process within the body in medical imaging.Extracellular matrix (ECM): a three-dimensional, non-cellular structure produced and secreted by cells. It is composed primarily of fibrous elements such as collagen and forms the interstitial connective tissue and the basement membrane.Fibrosis: the thickening and scarring of connective tissue, usually as a result of injury.Hypoxia: a deficiency in the amount of oxygen reaching tissues.Isosbestic point: a wavelength at which the absorption coefficient of two molecules is equal.Light fluence: optical energy per unit area.Optical diffusion limit: the depth in biological tissue beyond which light propagating along the predefined linear trajectory becomes too weak to be detected in practice (practice .Perfusion: the flow rate of blood per mass of tissue.Photoacoustic (optoacoustic) imaging (PAI): a biomedical imaging modality based on the photoacoustic effect.Photoacoustic effect: the generation of sound waves following light absorption in a material such as tissue.Tumour microenvironment (TME): the cellular environment in which a tumour exists, including blood vessels, fibroblasts, immune cells and the ECM.The focus of cancer research has moved from treating the tumour as a homogenous mass of cancer cells to considering the wider tumour microenvironment TME; see . The infin vivo, both preclinically in mouse models of the disease, as well as in patients. Unfortunately, features of the TME remain challenging to resolve with non-invasive imaging. As a result, many studies still rely on excised tissues ex vivo, which only interrogate a small portion of the tumour at a fixed time point, and are not able to capture its full spatial and temporal heterogeneity. Visualising the dynamic TME in vivo would allow researchers to investigate key questions about the interplay between different features of the microenvironment, for example, the functional relationship between hypoxia and fibrosis  imaging  to accumulate in the TME, giving rise to acidosis  are the predominant stromal cell type secreting ECM in the TME and have a complex role that has been extensively reviewed elsewhere . It is wAnti-tumour immunityImmune cells can recognise cancer cells as foreign and initiate an immune response against them  can skew CD4+ T-helper cells to become immunosuppressive and begin to inhibit the effect of cytotoxic cell types. Innate immunosuppressive cells such as tumour-associated macrophages (TAMs) are also anti-inflammatory and pro-angiogenic, driving tumour progression . TAMs arHowever, a major cellular component that merits discussion is the immune cell infiltrate. It is now well known that immune cells play a vital role in tumour development , which hMonitoring tumour response to immunotherapy is currently a major clinical challenge due to the dynamic nature of the immune TME. To optimise immunotherapy use whilst minimising toxicity and cost in non-responders, appropriately validated non-invasive imaging techniques could be applied to monitor the evolution of the TME in response to immunotherapy in real time and avoid repeated tissue sampling . There iIt is increasingly evident that features of the TME regulate each other. Hypoxia plays a central role in these interactions. For example, increased deposition of ECM proteins and recruitment of stromal cells leads to fibrotic contraction of the interstitial tissue space, which is associated with: increased interstitial fluid pressure ; the col+ and cluster around existing blood vessels, promoting angiogenesis  as surrogate markers of hypoxia  for illumination, several vital endogenous molecules for the TME strongly absorb light B. Using by cells  or expreby cells , work byby cells B.Compared to existing optical techniques such as DOSI and intravital microscopy, PAI maintains high spatial resolution to a greater imaging depth due to the detection of ultrasound waves, which scatter less than light in tissue. By utilising optical absorption and contrast, PAI maintains high molecular specificity  compared2 (e.g. 532 nm). This method has been used to monitor the developing vasculature in early tumours, showing changes in structure such as increased tortuosity  and blood oxygen saturation (sO2=HbO2/THb). These functional parameters can provide further insight into the TME, as detailed below.Alternatively, utilising the differential absorption spectra of Hb and HbO2 B, PAI daPAI THb tends to be higher in tumours compared to normal tissue  because 2 values in tumours compared to normal tissue  and poorly perfused (with low ΔsO2) prostate cancer models (2 and ΔsO2 spatially correlate with regions of tissue hypoxia and necrosis (in vivo using Indocyanine Green, a clinically approved near-infrared dye. In general, functional PAI studies have focussed on blood flow (PAI generally measures lower sOl tissue , consist ex vivo . Alterinr models B (Bendinr models . Importanecrosis . In the ood flow  rather tood flow  due to a2 has been seen in ovarian mouse tumour models following anti-angiogenic therapies, indicating vessel normalisation, which was shown ex vivo by an increase in pericyte coverage of vessels and decreased vessel density (2 was demonstrated to be an early biomarker of radiotherapy response (PAI biomarkers relating to vascular function have also been used to predict and monitor treatment response in preclinical cancer models. A decrease in THb and a corresponding increase in HbO density . High turesponse  and coulresponse . These sExogenous contrast agents can also be used to assess metabolic changes in the hypoxic TME. Amine-oxide probes can give a direct measure of tissue hypoxia , complemIn a clinical context, application of PAI to monitor vascular features of the TME have focussed on breast cancer, where PAI can be combined with existing ultrasound imaging approaches. Hand-held PAI probes, similar to existing ultrasound probes, have been used in clinical trials to demonstrate higher THb in tumours compared to normal tissue  and an iin vivo has been focused mainly on diseases beyond cancer, such as Crohn's disease (To date, the application of PAI to detect fibrosis and lipids  disease  and athe disease ; howeverex vivo and in vivo rat intestine (in vivo remains challenging due to limited knowledge of the spatial distribution of optical parameters in living tissue (In preclinical studies of chronic fibrotic diseases such as Crohn's disease, collagen content has been probed at wavelengths above 1000 nm to detect fibrotic disease in ntestine , with rentestine . In a prntestine . Visualintestine , which mntestine  correctig tissue . Alternain vivo is of interest.Monitoring the production or activity of MMPs in the TME could provide a diagnostic or predictive biomarker of metastasis and has also been achieved with PAI using exogenous contrast agents . The proex vivo human coronary arteries (PAI of lipids has been mainly applied to monitor atherosclerotic plaques using an intravascular-imaging catheter in either rabbit aorta or arteries , althougarteries , whereasarteries . This mearteries .in vivo multi-wavelength PAI has demonstrated a decrease in lipid content with increasing tumour stage (As mentioned earlier, lipids play a vital role in the breast TME. In transgenic mice, ur stage A, presumur stage . The cliur stage . Additiour stage .Fig. 4.ex vivo (in vivo was monitored with PAI after labelling with a near-infrared fluorescent dye (in vivo with PAI (in vivo (Exogenous contrast agents can theoretically label any cell type of interest and track these cells in the TME. Strong absorbers with little or no fluorescence provide the best contrast because the absorbed energy can be converted efficiently into ultrasound signals rather than being re-emitted as fluorescence. Fluorophores with a low-quantum yield have also proved successful in providing photoacoustic contrast. Fluorescently labelled T cells were first imaged with PAI ex vivo . Since tcent dye B (Zheng cent dye . This iscent dye . Organicwith PAI , a methowith PAI . Such prwith PAI . In addiwith PAI . For exa(in vivo . The ext(in vivo . Reporte(in vivo .We have moved into an age of cancer biology research where tumours are considered not as isolated masses of cancer cells but as ‘organs’ with multiple cell types supporting growth and development. The complex contribution of the TME to tumour evolution is now well recognised as influencing cancer cell gene expression, growth, survival, motility, local invasion and metastasis. There are also dynamic interactions between different TME features that regulate processes such as blood vessel outgrowth that we are just beginning to understand.Given the importance of the TME in tumour development and progression, it is surprising that our ability to image TME features is lacking both in preclinical models and in clinical applications. Many existing imaging modalities have been used to visualise vasculature, hypoxia, fibrosis, lipid synthesis and immune cells ; howeverin vivo and hence is an active area of research in the field (The studies reviewed here indicate that PAI can inform on TME features in preclinical models and holds promise for clinical translation. Currently, further technical and biological validation of PAI biomarkers is needed to increase uptake of the modality in studies of TME biology or clinical evaluation of TME features . In termhe field .in vivo PAI data with other well-validated in vivo imaging methods as well as ex vivo analyses such as immunohistochemistry and biochemical assays. In the few studies where this has been achieved preclinically, PAI provided in vivo biomarkers of vascular maturity and function that correlate with hypoxia (in vivo distribution of haemoglobin signal that had good colocalisation with DCE-MRI and correspondence with vascular patterns measured ex vivo (In terms of biological validation, the extensive use of cell-line-derived cancer models in PAI studies limits the clinical applicability of the work due to the stark genomic and phenotypic differences between clonal cell lines and patient samples . More cl hypoxia . Additio ex vivo .Most clinical studies discussed in this Review are observational and/or conducted in a limited number of patients . Their pTo conclude, PAI can visualise multiple TME features with a single modality; high spatiotemporal resolution, low cost, use of non-ionising radiation and non-invasive properties with potentially easy integration into existing ultrasound systems make PAI an attractive option for monitoring dynamic TME features not only in a preclinical setting but also throughout a patient's treatment regime, from diagnosis and staging to monitoring treatment response.