In responses to Norheim’s editorial, this commentary offers reflections from Thailand, how the five unacceptable trade-offs were applied to the universal health coverage (UHC) reforms between 1975 and 2002 when the whole 64 million people were covered by one of the three public health insurance systems. This commentary aims to generate global discussions on how best UHC can be gradually achieved. Not only the proposed five discrete trade-offs within each dimension, there are also trade-offs between the three dimensions of UHC such as population coverage, service coverage and cost coverage. Findings from Thai UHC show that equity is applied for the population coverage extension, when the low income households and the informal sector were the priority population groups for coverage extension by different prepayment schemes in 1975 and 1984, respectively. With an exception of public sector employees who were historically covered as part of fringe benefits were covered well before the poor. The private sector employees were covered last in 1990. Historically, Thailand applied a comprehensive benefit package where a few items are excluded using the negative list; until there was improved capacities on technology assessment that cost-effectiveness are used for the inclusion of new interventions into the benefit package. Not only cost-effectiveness, but long term budget impact, equity and ethical considerations are taken into account. Cost coverage is mostly determined by the fiscal capacities. Close ended budget with mix of provider payment methods are used as a tool for trade-off service coverage and financial risk protection. Introducing copayment in the context of fee-for-service can be harmful to beneficiaries due to supplier induced demands, inefficiency and unpredictable out of pocket payment by households. UHC achieves favorable outcomes as it was implemented when there was a full geographical coverage of primary healthcare coverage in all districts and sub-districts after three decade of health infrastructure investment and health workforce development since 1980s. The legacy of targeting population group by different prepayment mechanisms, leading to fragmentation, discrepancies and inequity across schemes, can be rectified by harmonization at the early phase when these schemes were introduced. Robust public accountability and participation mechanisms are recommended when deciding the UHC strategy. Our analysis found that three out of these five are related to equity goal; trade-off II , trade-off IV (prioritize the well-off than the worse-off) and trade-off V (move from out of pocket payment by households to a less progressive mandatory prepayment source of finance).In Norheim’s editorial,The remaining two are related to efficiency goal: trade-off I (extend coverage to the low or medium priority services than the high priority services) and trade-off III . Note that the unacceptable trade-off II and IV are very close. In this editorial, efficiency is regarded as part of fairness.Since health resources are finite, using it for one purpose, policy-makers have to sacrifice other alternates; hence trade-off and priority setting is unavoidable. These unacceptable trade-offs are theoretically sound and convincing; they are useful caveats for which policy-makers in low- and middle-income countries (LMICs) may use to make informed, fair and ethical choices in their paths towards UHC.2 and achieved favourable outcomes.3 This commentary aims to generate global discussions on how best UHC can be gradually achieved.This commentary offers reflections from a country experiences, Thailand, how these trade-offs were (or were not) applied in implementing UHC reforms since 1975 until UHC was reached in 2002,Trade-off is a situation where one must decide to choose between or balance the two alternatives that are opposite or cannot be taken at the same time. There are three dimensions of the UHC cube see . The X aWithin each of the three dimensions, there are trade-offs. On the X axis, we concur with Norheim’s assertion, on an ethical ground, that the poor, the worse off and certain disadvantaged groups who are unable to pay their medical bills should be covered first. This ethical choice will gain high political support, if these population sub-groups are vocal constituencies who cast their votes or influence others in an election every four to five years in developing countries. Unfortunately, very often they are voiceless and powerless.Increasingly, private sector employment are growing especially in middle-income context, that payroll tax financed social health insurance (SHI) systems should be introduced as soon as possible, in order to minimize the regressive out of pocket payment, with a caveat that payroll tax finance must be designed as a progressive source, where the higher income employees pay higher contribution than the lower income counterparts. When window of opportunities open, SHI can be introduced immediately, and no need to wait for full coverage of the poor.In developing countries, implementing tax financed scheme dedicated for the poor, or exempting them from paying user fees is challenging. Thailand medical welfare scheme for the low income households introduced in 1975; premium funded voluntary health card scheme for the informal sector in 1984; and payroll tax financed SHI scheme for private sector employees in 1990 demonstrated the explicit political decision on population extension on the X axis based on ethical principles, the more vulnerable they are, the higher priority they receive.4 and Cost-effectiveness and strategic planning (WHO-CHOICE),5 countries need capacities to translate them into policies and implementation. As comprehensive benefit package was fully applied in all financial risk protection schemes, it is not possible to apply a new positive list covering basic essential package; hence pragmatism is applied by Thai reform, with the application of negative list, where all services are covered except a few in the list.On the Z axis, which service package is offered to different population group is a political choice, often governed by the government fiscal spaces and how priority is made; either informed by cost-effectiveness evidence, financial risk protection, equity or pragmatism. Often there is limited technology assessment capacity in developing countries. Though global evidence is available such as Disease Control Priority8Despite the cost-ineffectiveness and large budget impact, renal replacement therapy for kidney failure patients, a life threatening condition, was approved by the government in 2008 on equity ground and financial protection. Two other schemes, the government employee and the private sector employee schemes have full coverage of renal replacement treatment; should not the universal coverage scheme (UCS) get this similar service? Cost of treatment is catastrophic to UCS members, certain patients died from inadequate out of pocket financed dialysis, leaving behind a large debt to repay by family.9 and Philippines.10 While extensive geographical coverage of functioning primary healthcare determines the pro-poor UHC outcomes.12Inequity arises when certain services are not available in remote rural areas where the poor live, but enjoyed by urban rich population. Introducing UHC without adequate and equitable distribution of supply side capacity is prone to pro-rich outcomes, as demonstrated in China13 Skilled birth attendance had reached 99.3% of total births; and contraceptive prevalence 79.2% of women age 15-49 in 2000, well before UHC achievement in 2002.14 An extensive geographical coverage of functioning health services is the foundation for effective UHC implementation.The Thai UHC was introduced after three decades of government investment in health service infrastructure in particular district health systems, and ensuring functioning of health service through mandatory rural services by health professional graduates.On Y axis, cost sharing is interlinked with X axis, which population group should or should not co-pay, and interlink with Z axis, which services should be fully subsidized. Clearly, Thailand applied equity principle where the poor are exempted from payment or copayment; and efficiency principle where services such as maternal and child health, immunization, cost effective interventions and community-based public health interventions are fully subsidized to the whole population, not only the poor due to external benefits. Until recently when UHC was achieved in 2002 that all services in the benefit package are fully covered, free at point of services; this is not because improved fiscal capacity but the application of close end payment which has the merits of cost containment and system efficiency. Efficiency frees up more resources for zero co-pays.15 Copayment can be applied to discourage bypassing primary healthcare. However, ensuring quality at primary healthcare to gain citizens’ trust and confidence are important prerequisites.A caveat on copayment, introducing copayment as percentage of the medical bills in particular when insurance agency applies fee-for-service is harmful to the patients in particular the low income; ample evidence shows that fee-for-service stimulates supplier induced demand (demand in excess of what patient would choose) because of information asymmetry in healthcare market, hence professional acts as patients’ agent and making decision on their behalf. Fee-for-service provides opportunities for professionals to maximize services.3 Cost coverage in Y and service coverage in Z axes are interlinked under the strategic purchasing design and implementation.13Strategic purchasing comes into play to contain cost and protect members from catastrophic spending and medical impoverishment. Institutional capacities to manage purchasing by insurance agencies are contributing factors to efficiency and equity.UHC achieves favorable outcomes as it was implemented when there was a full geographical coverage of primary healthcare coverage in all districts and sub-districts after three decade of health infrastructure investment and health workforce development since 1980s.At the bottom layer, people living below national poverty line was covered by publicly financed medical welfare schemes, launched in 1975, which gradually extended to cover all elderly, children under 12 years old, persons with disability and village health volunteers.At the top layer, government employees and their dependents are historically covered by non-contributed tax financed scheme, as part of the comprehensive welfare. Civil servants’ salary is claimed to be lower than labour market. The private sector employees are covered by payroll tax financed SHI, launched in 1990, as part of the comprehensive social security including pensions and unemployment benefits.The informal sector, at the middle layer, was covered by voluntary premium financed public insurance launched in 1984 by the Ministry of Public Health, and later 50% of premium was subsidized by the government in 1992. Despite Ministry of Public Health’s efforts, coverage remained low; by 2001, 30% of total population was uninsured. Clearly, voluntary nature of prepayment scheme cannot achieve UHC.3 Squeezing from the bottom described in In 2002, in keeping UHC political manifesto in the 2001 general election, decisive political decision was made to cover the whole bottom and middle layers by UCS, financed by general tax. Tax is one of the most progressive sources of financing.16 Advocates to abolish contribution in SHI is not a political correct and economic sound proposal given the increased size of formal sector.Arguments arise on inequity in financial contribution that private sector employees have double contributions: payroll-tax for SHI coverage and general tax (direct and indirect) while UCS members only contribute to general tax. This may incentivize an increased informality in the economy. Tax financed UHC is the political choice and societal preference to maintain payroll tax financed SHI. It is a political correct and technical sound decisions to apply tax-finance UHC in 2002 when Thailand decided to achieve UHC fairly quickly.Finally, the reform in 2002 resulted in UCS for the bottom and middle layers, 75% of total people; while keeping intact the Civil Servant Medical Benefit Scheme for government employees and SHI for private employees. The application of close ended budget facilitates more comprehensive service coverage and high financial risk protection for UCS and SHI members.3; contracting with district health systems contributes to pro-poor use of services and public resources as measured by benefit incidence.11The key designs contributing to favorable outcomes are; a comprehensive benefit package and free at point of service contributes to high level of financial risk protection as measured by the incidence of catastrophic health expenditure and medical impoverishmentFragmented schemes are essential feature of UHC transition when most countries apply targeting population groups. Norheim assumes a single entity in making decisions about how to expand coverage; there are many actors having stakes on UHC, such as Ministry of Health, Ministry of Labour and also private insurance agencies. Expansions of services that are privately financed are hard to influence in a laissez fair economy.2 China,17 Lao PDR.18 These trajectories result in discrepancies across different insurance schemes in term benefit package and provider payment — causing inequity and inefficiency.In the stride towards UHC, various countries extend coverage to different population sub-group when windows of opportunity opened, for example the poor subsidized by general tax, the private sector employees by SHI payroll tax financed scheme, and informal sector by premium financed voluntary community based health insurance, with or without government subsidies, such as the case of Thailand,In the paths towards UHC, LMICs should recognize these challenges facing the pathfinder countries; efforts should be given to minimize the gap of inequity through harmonization strategic purchasing  across different schemes, if unavoidably different schemes for different population groups are applied.We fully support Norheim’s recommendation that “Robust public accountability and participation mechanisms are, therefore, essential when deciding on the overall strategy and the appropriateness of central trade-offs on the path to UHC.” However, not all LMICs have such platform. Cross country learning and sharing lessons from UHC pathfinder countries convened by international development partners, as well as institutional capacity strengthening focusing on strategic purchasing function are further recommended.Expansions of financial risk protection are incremental processes where there is no “clean slate” furnished with all ethical options for making UHC choices; reformists should stand ready when the political windows open to re-orient toward more equitable and ethical choices.We pay tributes to the late professor Nikom Chantaravitura and Dr. Sanguan Nittayaramphong, who are the champions in marshalling Thailand adoption of Social Security Scheme in 1990 and Universal Coverage Scheme in 2002. We acknowledge policy-makers, managers, program implementers, front line health workers and researchers for their contributions in the design, implementation, monitoring, and evaluation of UHC.Not applicable.Authors declare that they have no competing interests.VT conceptualizes and starts the first draft. All authors contribute to strengthening the manuscript, reviewed and signed off the final version.


Ovarian cancer usually has a poor prognosis because it predominantly presents as high stage disease. New approaches are required to develop more effective early detection strategies and real-time treatment response monitoring. Nano-sized extracellular vesicles  may provide an approach to enrich tumor biomarker detection and address this clinical need. Exosomes are membranous extracellular vesicles of approximately 100 nm in diameter that have potential to be used as biomarkers and therapeutic delivery tools for ovarian cancer. Exosomal content (proteins and miRNA) is often parent cell specific thus providing an insight or “fingerprint” of the intracellular environment. Furthermore, exosomes can aid cell-cell communication and have the ability to modify target cells by transferring their content. Additionally, via the capacity to evade the immune system and remain stable over long periods in circulation, exosomes have potential as natural drug agents. This review examines the potential role of exosomes in diagnosis, drug delivery and real-time monitoring in ovarian cancer. Ovarian cancer is the most lethal gynaecological cancer surpassing other malignancies such as uterine and endometrial cancer . The 5-yExosomes are small ∼100nm), membranous vesicles of endocytic origin and can be found circulating in biological fluids such as blood, urine and milk , 10. Per0nm, membTherefore, this review will summarise the recent advances that have been made in understanding the relationship between ovarian cancer and exosomes. Specifically, the role of exosomes in screening and early detection, as biomarkers for prognosis and drug resistance and as drug delivery vehicles will be discussed.Despite current advances in research, ovarian cancer retains its position as the leading cause of gynaecological related deaths in females worldwide , 14. SubThe International Federation of Gynaecology and Obstetrics (FIGO)  has diviNonetheless, the most well-known factors involved in ovarian cancer susceptibility are mutations in the BRCA1 and BRAC2 genes  which arDue to the limitations presented by genetic testing, literature has shifted towards different techniques for susceptibility estimation such as, studying smaller molecules, example, RNA and their contribution to disease. In 2016, Permuth and colleagues suggested that RNA editing, which is a post-transcriptional modification where adenosine is converted to inosine by a family of enzymes known as adenosine deaminases acting on RNA (ADAR), may have a role in epithelial ovarian cancers. ADAR expression levels have been found to be significantly greater in peritoneal fluid obtained from patients with epithelial ovarian cancer compared to patients with benign disease . They coApproximately 20% of patients with high-grade serous subtype disease carry mutations (both somatic and germ-line) in the BRCA1 and BRCA2 genes . However“one size fits all” approach cannot always be applied to treatments. The different subtypes of ovarian cancer have diverse aetiologies and different sensitivity to treatments [Another factor that is negatively correlated with survival is the fact that ovarian cancer is a heterogeneous disease and therefore the eatments . Currenteatments . The cheeatments . The proeatments , 32. Of eatments .Invasion of the tumor cells into the pelvic and abdominal peritoneum is often representative of tumor aggressiveness . This inCancer Antigen-125 (CA-125) and Trans-vaginal imaging are currently routinely used as screening tests for ovarian cancer . CA-125 Zhao et al (2016) reported the development of a microfluidic chip approach to isolate exosomes that expressed certain markers , CA-125 and CD24) . It was It is thus clear that improved screening tests are required for early detection of disease. These screening tests need to fit a specific criteria including: the test can be performed easily, is inexpensive, clinically validated and highly sensitive and specific , 43. SevSmaller molecules such as circulating miRNA and cell-free DNA (cf-DNA) have also received attention in recent literature although progress is limited. Kamat and colleagues quantified cf-DNA and reported that patients with epithelial ovarian cancer had higher levels compared to healthy individuals . FurtherThis is also a limitation in the use of miRNA expression as an ovarian cancer early detection tool. Previous studies have identified changes in the expression of multiple miRNA between patients with epithelial ovarian cancer and healthy controls such as Let-7 and miR-205. Let-7 levels decreased in patients with ovarian cancer whereas miR-205 levels were higher . HoweverCell-cell signaling as well as particles released by tumor cells to aid cellular communication remains a major focus. However, cells release a diverse population of factors including signalling molecules, transmitters, apoptotic bodies and microvesicles. These factors combined with the multitude of other abundant proteins  in the blood render it difficult to identify molecules of interest from tumor cells as there is a high signal-to-noise ratio , 52. To The biogenesis of exosomes begins with an inward budding of the plasma membrane leading to the formation of an early endosome. The early endosome then matures to a late endosome. Invagination of the late endosomal membrane leads to the formation, within the endosome, of intraluminal vesicles known as multivesicular bodies. The multivesicular body can then either fuse with a lysosome leading to degradation or fuse with the plasma membrane of the cell leading to the release of exosomes -62. SecrExosomes express a range of proteins including those related to multivesicular body biogenesis, such as Alix, TSG101 and members of the tetraspanin family . The divExosomal cargo is composed of a range of molecules including but not limited to proteins ), cell surface receptors and miRNAs . InteresThe implications of exosomes in various biological processes as well as in pathological conditions have been examined although further research is required. For example, it has been reported that tumor-derived exosomes can promote oncogenic activity in recipient tumor cells. Moreover, exosomes can also aid in the development of suitable microenvironments for tumor growth and progression through processes such as angiogenesis. It has been shown that tumor-derived exosome can promote angiogenesis by activating myofibroblasts and endothelial cells . ExosomeTherefore, there is increasing research involving extracellular vesicles or exosomes, however, a major challenge is to understand the exact mechanisms underlying the role of exosomes in healthy cell transformation. Thus, the current focus is to decipher these mechanisms to facilitate early detection and prevention of ovarian cancer. Additionally, a challenge that remains in the field of extracellular vesicles is an ability to isolate and characterise exosomes as there is no consensus in current literature regarding an exosome isolation protocol , 77.Table The proposal of personalised medicine by Kewal Jain in 1998 has drastically changed the approach towards cancer research and therapeutics . HoweverIn 2009, Li et al showed that exosomes found in the circulation of approximately 32 (out of 63) patients with ovarian cancer contained claudins whereas only one out of 50 healthy individuals had claudin expressing exosomes present in their circulation . ClaudinKobayashi et al (2014) showed that highly invasive ovarian cancer cell line, SKOV-3 derived exosomes had greater expression of the miRNA, Let-7, when compared to the poorly invasive cell line, OVCAR-3. Additionally, a correlation between the miRNA profiles of OVCAR-3 cell-derived exosomes and the OVCAR-3 parent cells was noted . Vaksmanin vitro tumor cell-derived exosomes contained CD24 and that it was also present in exosomes obtained from malignant ascites [Therefore, the potential of exosomes as diagnostic and prognostic biomarkers was hypothesised and it was found that several molecules native to the tumor cells/tissue were expressed in exosomes. This is promising as the expression of low abundance molecules may be masked in circulation but detected in exosomes. In 2004, Graves et al showed that matrix-metalloproteinases MMP-2 and MMP-9 which are related to ovarian cancer metastasis are concentrated in membrane vesicles found within ascites. Furthermore, there are a greater number of these vesicles with increasing disease stage . The uti ascites . Moreove ascites .Exosomal research also provides a warrantable avenue in patients that do not respond to standard therapies or patients that present with tumor resistance and/or relapse. Recurrence often occurs between 12-24 months of treatment in advanced disease patients and is accompanied by chemotherapy resistant disease . ResearcSafaei et al showed that a cisplatin resistant ovarian cancer cell line released exosomes which had an increased expression of LAMP1 compared to a cisplatin sensitive cell line . LAMP1 iIn addition to their applications in diagnosis and treatment, exosomes provide an invaluable tool for drug delivery and monitoring response. This is attributed to the fact that exosomes can encapsulate and transport molecules through compartmentalisation . SpecifiResearch by Yang et al (2015) incorporated anti-cancer drugs, doxorubicin, and paclitaxel, into exosomes and then introduced the exosomes into zebrafish embryos . As a coAnother study by Tian and colleagues (2014) delivered exosome encapsulated doxorubicin to solid tumors in a mice model . They taIt has been suggested that circulating exosome levels before and after chemotherapy can be considered to determine a patient’s response to the treatment. Szajnik et al (2013) showed that patients that did not respond to treatment with carboplatin and paclitaxel showed no significant change in exosomal protein levels after treatment compared to prior to treatment . ConversWhiteside (2015) has suggested that the immuno-suppressive molecular profile of tumor exosomes could provide an understanding of the immune dysfunction resulting from the tumor and ultimately tumor progression . This waMost significantly, it has been proposed in literature that tumor-derived exosomes may provide a way forward in aiding early diagnosis, drug delivery and monitoring response to treatment in the field of ovarian cancer. Furthermore, exosomal cargo including proteins and miRNA may also provide an insight into the cellular environment including changes in the cell at a molecular level. This ability to gain a snapshot of the internal environment in a timely manner may eradicate the need for invasive biopsies and allow detection of disease prior to metastatic spread. Therefore, exosomes provide a minimally invasive “liquid biopsy” method to gain insight into the tumor microenvironment without the need for a highly invasive tumor biopsy.However, this achievement is dependent on a complete understanding of exosome biogenesis, secretion, interaction with target cells and the pathways underlying exosomal cell signalling. Additionally, it is important to note that exosomal cargo varies, depending on the releasing cell, and therefore, the process of cargo packaging into exosomes also needs to be fully elucidated. In terms of ovarian cancer, the cross-talk between cells and the microenvironment as well as the involvement of exosomes in facilitating this cross-talk also needs to be studied. This review provided a glimpse at the multi-faceted nature of exosomes by discussing recent publications which have aimed to decode the role of exosomes by examining the exosomal cargo, specifically miRNA and proteins. It is anticipated that this review will highlight exosomes as a precious biomedical tool under various contexts as shown in Figure


Canis lupus, are difficult to protect in mixed-use landscapes because some people perceive them as dangerous and because they sometimes threaten human property and safety. Governments may respond by killing carnivores in an effort to prevent repeated conflicts or threats, although the functional effectiveness of lethal methods has long been questioned. We evaluated two methods of government intervention following independent events of verified wolf predation on domestic animals (depredation) in the Upper Peninsula of Michigan, USA between 1998–2014, at three spatial scales. We evaluated two intervention methods using log-rank tests and conditional Cox recurrent event, gap time models based on retrospective analyses of the following quasi-experimental treatments: (1) selective killing of wolves by trapping near sites of verified depredation, and (2) advice to owners and haphazard use of non-lethal methods without wolf-killing. The government did not randomly assign treatments and used a pseudo-control , but the federal permission to intervene lethally was granted and rescinded independent of events on the ground. Hazard ratios suggest lethal intervention was associated with an insignificant 27% lower risk of recurrence of events at trapping sites, but offset by an insignificant 22% increase in risk of recurrence at sites up to 5.42 km distant in the same year, compared to the non-lethal treatment. Our results do not support the hypothesis that Michigan’s use of lethal intervention after wolf depredations was effective for reducing the future risk of recurrence in the vicinities of trapping sites. Examining only the sites of intervention is incomplete because neighbors near trapping sites may suffer the recurrence of depredations. We propose two new hypotheses for perceived effectiveness of lethal methods: (a) killing predators may be perceived as effective because of the benefits to a small minority of farmers, and (b) if neighbors experience side-effects of lethal intervention such as displaced depredations, they may perceive the problem growing and then demand more lethal intervention rather than detecting problems spreading from the first trapping site. Ethical wildlife management guided by the “best scientific and commercial data available” would suggest suspending the standard method of trapping wolves in favor of non-lethal methods (livestock guarding dogs or fladry) that have been proven effective in preventing livestock losses in Michigan and elsewhere.Large carnivores, such as gray wolves,  Canis lupus, are difficult to protect in mixed-use landscapes because some people perceive them as dangerous and because they sometimes threaten human property and safety. Traditionally, governments kill wild animals in an effort to prevent threats to property and safety  test: χ2 = 0.48, P = 0.488; Log rank tests could not distinguish the survival functions between treatments  and 99 followed by non-lethal intervention (79%). Log rank tests could not distinguish the survival functions between treatments . Similarly, all Cox models revealed no differences between treatments  and 99 followed by non-lethal intervention (79%). Again, log rank tests could not distinguish survival functions between treatments  between 1998–2014 resulted in lower risk of recurrence of depredations than if no wolves were killed. We found the delay to recurrence of depredations was unrelated to the number of wolves killed at all spatial scales. We found lethal management did not significantly shorten or lengthen the interval to the next depredation relative to non-lethal interventions. A small, statistically insignificant reduction in the risk of depredation at the section level was offset by a similar and also statistically insignificant increase in the risk of depredation at the township scale, which is about half the size of a wolf pack territory, and then a similar decrease in risk at the scale of neighborhoods of townships, which are four times larger than the average wolf pack territory . None ofOur methods or alternative models accounted for potential violations of the proportional hazards assumption, unlike a prior study of wolves in the Northern Rocky Mountains (see below); accounted for within-subject correlation; were unaffected when we restricted analysis to the period after 2003 when lethal interventions first became legal; and accounted for a change in definition of lethal methods to include the installation of lethal methods that did not kill any wolves . There iOverall, our analysis suggests that any potential beneficial effects of lethal interventions locally would be offset by detrimental effects for neighboring farms in the same township. If the small, local improvements were considered biologically, ethically, or economically important to one farm, then one would also have to admit the associated costs to neighboring farms and the biological, ethical and economic importance to that farm. Therefore, given the evidence available, we cannot conclude that lethal management had the desired effect of preventing future livestock losses.Over the 17 years of our study, the risk of depredation increased by 9 and 14% per year at the section and neighborhood  scales, respectively, in our main dataset. However, this effect of year is insignificant in our post-2003 dataset . In addiWe present our results guardedly rather than as a definitive conclusion about effectiveness because of insurmountable uncertainties about the government data. Retrospective analyses to evaluate the effectiveness of interventions to prevent predation on livestock are fraught with uncertainty because of various biases or challenges presented by field conditions . For exaSelection (or enrollment) bias would arise if subjects entered the study under varying conditions that affected outcomes. All sections containing farms (subjects) entered our study because of a verified depredation, but subjects entered at different times and some farm owners might have responded to depredations in undocumented ways including poaching wolves. Likewise, attrition bias would arise if subjects left the study for reasons that were not random with respect to their outcomes. This would occur systematically if a subset of the interventions led farmers not to complain in the future despite facing depredations, or to take matters into their own hands, as above. Compensation was offered throughout the study as well as state-financed non-lethal deterrence when lethal intervention was unavailable, so attrition by withholding complaints seems unlikely to have been frequent or widespread. However, we would guess that non-intervention might be construed as unhelpful by complainants, leading some of them to intervene independently. We consider unreported wolf-killings to be a more pronounced confounding variable after 2003, when state lethal management was allowed , substanS1 Data File). Given that authority for the state to kill wolves after verified depredations was granted or withheld by federal decisions unrelated to area attributes or recent depredation complaints and in several years of the study even high-risk areas received no interventions [Furthermore, treatment bias would arise if methods of intervention were not standardized. Treatment bias certainly arose among non-lethal deterrents because different complainants received different types of non-lethal methods and we do not know if they maintained or installed the methods appropriately or identically. Non-lethal deterrents were presumably negotiated with complainants and therefore most prone to treatment bias that would confound our results. However, only 8% of our eventual sample received non-lethal deterrents. Moreover, we have no data on other deterrents or precautions unilaterally implemented by complainants. Lethal interventions were more uniform in method  but we dventions , it seemFinally, wolf abundance was unlikely to confound our tests because the number of wolves within our spatial units was unlikely to change substantially from one incident to the next within a small area within one year.In sum, we find ample reason to expect confounding variables would weaken inference from a retrospective, quasi-experimental test of interventions to prevent livestock loss. Our attempts to detect and screen for biases were necessarily imperfect because we could not assign treatments randomly nor could we retrospectively assess if interventions were assigned haphazardly or subjectively. Our analyses controlled for variation in risk due to time and inter-farm differences using tvc and frailty models , but couNevertheless, there is value in the scientific examination of on-the-ground programs of predator management as they are actually carried out by the organizations that discharge them. Avoidance of selection, treatment or measurement biases would require enforcement of strict protocols that are rare worldwide , 39–41. 2) with uniform treatments, in which measurement is unbiased by blinding or independent, third party monitors, and data analysis is conducted by independent, third-party analysts without financial conflicts of interest involving the government or livestock industry. However, such an experiment would have to address the ethical implications for both animals and people of removing wild animals, possibly exposing more livestock to spill-over effects, and the broad public interest in preserving both wildlife and livelihoods. A step in that direction, albeit imperfect, may be to temporarily relocate predators to captivity until the analysis period ended in each area.An example of a gold-standard design that might achieve strong inference would be random-assignment of treatment to different, large areas  so our results are consistent. However, other differences in results between their study and ours could be due to different sites and methods.Our results appear to contradict those of the  in the NThe analysis in  includedFirst,  did not Second, apparent shortcomings of the statistical modeling in  may haveUsing a biomedical analogy,  identifiThird, differences with  could alFinally, some might argue that by defining our subjects as area-years and including the same area over different years we pseudo-replicated non-independent samples. In our dataset, only 16 out of 106 sections had depredation incidents in multiple years. To address that concern, we built an alternative model in which areas were omitted in succeeding years . ResultsLethal interventions by the State of Michigan against wolves in the vicinities of verified livestock losses did not appear to reduce future losses. We view our findings as preliminary pending experiments with stronger inference. Our inferences could not overcome a lack of systematic information on government interventions and no effort to control for their treatments, despite a call for such shortly after the legalization of lethal removal of wolves in 2003 . We deteFollowing recommendations for ethical wildlife management , 49, letS1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S5 File(DOCX)Click here for additional data file.S1 Data File(XLSX)Click here for additional data file.


Despite considerable effort and significant therapeutic advances, age-related macular degeneration (AMD) remains the commonest cause of blindness in the developed world. Progressive late-stage AMD with outer retinal degeneration currently has no proven treatment. There has been significant interest in the possibility that cellular treatments may slow or reverse visual loss in AMD. A number of modes of action have been suggested, including cell replacement and rescue, as well as immune modulation to delay the neurodegenerative process. Their appeal in this enigmatic disease relate to their generic, non-pathway-specific effects. The outer retina in particular has been at the forefront of developments in cellular regenerative therapies being surgically accessible, easily observable, as well as having a relatively simple architecture. Both the retinal pigment epithelium (RPE) and photoreceptors have been considered for replacement therapies as both sheets and cell suspensions. Studies using autologous RPE, and to a lesser extent, foetal retina, have shown proof of principle. A wide variety of cell sources have been proposed with pluripotent stem cell-derived cells currently holding the centre stage. Recent early-phase trials using these cells for RPE replacement have met safety endpoints and hinted at possible efficacy. Animal studies have confirmed the promise that photoreceptor replacement, even in a completely degenerated outer retina may restore some vision. Many challenges, however, remain, not least of which include avoiding immune rejection, ensuring long-term cellular survival and maximising effect. This review provides an overview of progress made, ongoing studies and challenges ahead. Age-related macular degeneration (AMD) is the commonest cause of blindness in the developed world. The number of patients with currently non-treatable AMD is staggering, being responsible for approximately half of the 370,000 people registered as blind or partially sighted in the UK alone . Late-stAMD is manifested fundoscopically in the early and intermediate stages by the appearance of yellowish subretinal deposits, called drusen deep to the retinal pigment epithelium (RPE) in the macular retina. At this stage, the effect on vision is relatively mild, although acuity in low-contrast conditions is frequently affected. At least 15% of patients progress however to the more advanced ‘wet’ and ‘dry’ forms of the disease. Dry AMD is characterised by degeneration of the RPE and subsequently the overlying photoreceptors. ‘Wet’ AMD is characterised by aberrant choroidal blood vessel growth beneath or through the RPE, affecting the function of the overlying neurosensory retina by vascular leak, haemorrhage and fibrosis with subsequent outer retinal degeneration. Treatments are available and evolving for wet AMD, most notably, anti-vascular endothelial growth factor (VEGF) treatment . HoweverGene therapy and a variety of other therapies are being investigated as possible treatments for these diseases, but they are unlikely to restore vision once photoreceptors loss has occurred nor do they aim to restore the RPE . ElectroA variety of cellular regenerative therapies with a range of cell types are being studied for the treatment of AMD and other outer retinal conditions. Transplantation is perhaps the most obvious mode of action and the idea of ‘young’ cells restoring and replacing the old is a particularly alluring concept. In advanced AMD, despite extensive outer retinal degeneration, the inner retina with its intricate neural connectivity and output via the ganglion cells to the brain, is anatomically intact, allowing the realistic possibility that cell replacement in the outer retina may restore vision . AMD is However, the appeal of cellular therapies also rests in their potential multiple mechanisms of action and their generic, non-disease-specific effects that are particularly attractive in a disease like AMD. Cell therapies can rescue host cells by the production of cytokines and neurotrophic factors as well as altering the neurodegenerative process by immunomodulatory effects , 11. TreProgress has been rapid, and transplanted cells from a range of types and sources have been shown to restore visual function in animal models of human disease, and some are in early-phase human trials. Indeed the eye is at the forefront of cell-based therapies, being an ideal site to evaluate their effects. This is particularly true for outer retinal diseases, where the confined, relatively immune privileged, easily observed and surgically accessible space between the photoreceptors and RPE is a particularly tractable place to inject and evaluate treatments. The anatomical connections required for proposed cellular transplantation are relatively simple for both RPE cells and photoreceptors relative to other central nervous system locations, and retinal imaging and functional assessment is at an advanced level with cellular level imaging possible. The number of cells required to provide measurable visual function is potentially low, with as few as 25,000 cells restoring some vision in murine disease, reducing the antigenic load and cell production logistics . IntraviThe history and ongoing efforts to produce an effective method of regenerating the degenerate outer retina, and in particular AMD is a compelling story covered by several excellent reviews, which we draw upon –21. Our The RPE is the name given to the monolayer of hexagonal-shaped pigmented epithelial cells that underlie the neurosensory retina. They grow on a specialised basement membrane, BrM and tight junctions between the RPE cells form the outer retinal blood barrier. The RPE is vital to the survival and function of the photoreceptors, and in addition to phagocytosing photoreceptor outer segments and recycling retinol as part of the visual cycle, it has several additional key functions, including polarised growth factor secretion, nutrient transport, stray light absorption and antioxidant functions. With age, the ability of the RPE to phagocytose and remove toxic by-products of phototransduction reduces, resulting in the accumulation of toxic material that in turn reduces the ability of the cell to function optimally. This process, coupled with other age-associated changes in the RPE and BrM are key stages in the development of AMD . ClinicaThere are two potential roles of RPE cell transplantation in AMD, namely replacement of RPE function and trophic support to dying cells . RPE trThe concept of RPE transplantation was quickly transferred to human studies with autologous RPE sheet transplantation in advanced wet AMD. Both macular relocation surgery, whereby the neurosensory retina is relocated from a fresh non-diseased area of RPE, and autologous RPE patches from the peripheral retina have demonstrated that, in selected cases, it is possible to restore vision long term in patients with recent onset wet AMD, where irreversible photoreceptor degeneration has not yet occurred –32 Figs and 5.FiWhen irreversible photoreceptor loss has occurred in AMD, combined RPE and photoreceptor transplantation will be required. As mentioned, despite profound outer retinal degeneration, the neurons of the inner nuclear layer survive for extended time periods after photoreceptor degeneration despite significant dendritic retraction and circuit remodelling. Studies with retinal prosthetic devices have shown that patients appear to retain a retinotopic map even after extensive outer retinal degeneration if the retina can be stimulated appropriately. Replacing degenerated photoreceptors would be one way to do this. Transplanted cells would only need to make one synaptic connection to the bipolar cells. The synaptic remodelling that developed during regeneration would need to reverse; however, it is known that plasticity exists in this regard .There have been two approaches to photoreceptor replacement considered, namely disassociated photoreceptor cells delivered as a suspension and retinal sheet or micro-aggregate transplantation (reviewed by Seiler and Aramant ).The first transplants with disassociated neural retinal cells into the subretinal space were carried out in light damaged and RCS rats , 36. TraUntil recently, it was thought that donor photoreceptors were able to physically migrate and integrate into the recipient outer nuclear layer based on studies using immunofluorescent (IF) labelling of proteins that could only originate from the donor. However, recent studies have suggested that this is a relatively rare event in animals with some degree of photoreceptor preservation, and instead there appears to be some form of cytoplasmic exchange between the donor and host cells to account for the IF findings , 42–44. Another way to transplant photoreceptors is as part of full thickness retinal sheet, ideally when they are about to start forming their synaptic connections, and when considerable plasticity exists (reviewed by Seiler and Aramant ). IndeedSeveral patients have been treated with foetal retinal transplants delivered as microaggregates or sheets –60. A PhIn human trials, sheet transplants seem to have longer survival that microaggregates and probably single cells; a foetal retinal graft sheet in a clinical trial was observed to survive 3 years after the transplantation, while transplants in the form of microaggregates were no longer able to be detected . HoweverThe optimum age of foetal donor tissue for both sheet and single-cell photoreceptor transplants for these approaches is in the second trimester with obvious ethical and supply problems. Other sources of cells have therefore been sought and these are discussed later.A variety of cell types have been shown to rescue photoreceptors in preclinical models of human retinal degenerative disease, including bone-marrow-derived hematopoietic and MSCs, adipocyte-derived cells, umbilical tissue cells and neural progenitor cells (NPCs). Although bone-marrow-derived MSCs can differentiate into cells expressing photoreceptor proteins when injected into the subretinal space, their ability to differentiate into functionally useful retinal cells is under debate. Their action is thought to be largely related to paracrine effects from neurotrophic factors (NTF) production. NTF are a family of proteins that participate in the regulation of the development, function, and survival of neurons and other cells in the nervous system , 64. EarAs mentioned previously, RPE cells may also exert a significant effect on photoreceptor regeneration and survival by trophic effects alone, thought largely to be due to the production of pigment epithelium-derived factor (PEDF).Delivery of cells to a subfoveal location requires foveal detachment, which could compromise the vision of the patient, particularly in the setting of retinal degenerative disease. Transplantation of NPCs subretinally has, however, demonstrated that they can migrate from the subretinal injection site quite extensively , 73, allMany of these classes of cells also display variable immunomodulatory activities. The most well established is the suppression of immune responses and inflammation by bone-marrow-derived MSCs. Under various conditions, MSCs induce the expression of immune-modulatory proteins, including Ym1, insulin-like growth factor-1 (IGF-1), Th2-related cytokines, galectin-3, and class II major histocompatibility complex (MHC) antigens . HoweverMany species of amphibians and fish have a remarkable ability to regenerate damaged retinas. The source of cells for this appears to come from three main sources of resident stem cells dependent on species and age; the anterior retina in the ciliary margin zone, Muller cells (MC), and RPE –80. HumaA wide variety of exogenous cell sources for transplantation have been considered and are listed in Table For photoreceptor cells, as described above, the developmental stage and the environment the cells are grown in are vital to their ability to fully develop after transplantation and to engraft into the retina. Pluripotent stem cells (PSCs) are currently considered to be the optimum cell source for these but the potential of foetal-derived RPC will also be discussed.PSCs could also provide a ready source of RPE cells. However, in contrast to photoreceptor transplants, transplanted RPE cells, virtually regardless of age or origin, readily extend apical villous process around host photoreceptors and phagocytose shed outer segment discs . AutologIris pigmented epithelial (IPE) cells are derived from the same embryonic cell line as RPE and are similar in many respects to RPE cells with apical/basal polarisation, microvilli, and the same type of tight junctions. Their appeal lies in the fact that they can be easily collected by surgical iridectomy and so could be autologous –90. TherFoetal RPE overcomes the problems of senescence but has obvious problems of ethical acceptability and supply. However, the recent finding that the human RPE contains a small subpopulation of cells that can self-renew and act as adult RPE stem cells  capable In vivo RPC exit the cell cycle and start differentiating into photoreceptors between 14 and 20 weeks of gestation and aside from ethical concerns, they would appear to offer a feasible source of cells for photoreceptor replacement. They also have relatively low immunogenicity and are not always rejected. Several studies have evaluated the use of disassociated foetal RPC for cellular transplantation  and at lHuman PSCs can produce all the cell types needed for retinal regeneration . They aThe umbrella term PSC is most commonly used to describe two types of stem cells: embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Both are defined by two key properties, namely an indefinite self-renewal ability and the capacity to give rise to any adult somatic cell type  Fig. 6)6).Fig. 6Human ESCs (hESCs) are derived from the inner cell mass of a preimplantation blastocyst. Several lines have been created since their discovery in 1998 and used to understand the mechanisms behind human embryonic development and congenital disease . The useIn 2006, Yamanaka and Takahashi, building on the work of Gurdon, isolated four key transcription factors (gene expression ‘controllers’) that when expressed exogenously in somatic cells induced the formation of pluripotent cells in a process termed reprogramming , 110. ThBeing autologous, however, also creates a potential problem for transplantation in that the cells will carry the same disease-causing gene mutations of the patient. This might not always be a problem in some diseases, particularly AMD, where onset is in later life, but even then gene editing may be useful to enhance integration or reduce recurrence. Current gene therapy approaches in the context of a patient with a known gene mutation uses viral vectors to transfect a corrected copy of the gene and a suitable promoter either in the cytoplasm or a ‘safe’ site in the genome in either the RPE or retinal cell type affected. These cells, being mature and non-dividing, only need this transfection event to occur once for prolonged protein expression. Conversely, in a PSC-derived cell line being delivered for transplantation, the optimum way of carrying out gene therapy would be to edit the genome at the PSC stage, to precisely correct for the mutation, thus correcting all derived differentiated cells thereafter. New gene editing technologies use specifically designed site-specific restriction endonucleases, such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and clustered regularly interspaced short palindromic repeat/CAS9 RNA guided nucleases (CRISPR/Cas) −116 to eThe use of PSC also carries another concern namely tumour formation. Transplantation of undifferentiated ESC and iPSC, which could exist in a differentiating colony, can result in teratoma formation. In one study, transplantation of ESC-derived neural precursors into the subretinal space of a mouse model of RP resulted in teratoma formation in 50% of the mice within 8 weeks . SortingA third type of autologous PSCs can be produced by cloning. In this technique, termed somatic cell nuclear transfer, the nucleus of a donor adult somatic cell is transplanted into an enucleated oocyte to produce a near genetically identical cell to the original adult nuclear donor, akin to an ESC and as used subsequently to produce ‘Dolly the sheep’ . These EOn the basis of early publications describing differentiation of hESCs, all retinal cell types have now been differentiated from human iPSCs (hiPSC). Differentiation models development in vivo in which the initial cell mass, representing the epiblast divides into the three embryonic germ layers before the ectoderm differentiates further into neuroectoderm then retinal cell types −126. At In vitro retinal differentiation protocols for PSCs can be broadly divided into default differentiation, in which cells differentiate to retinal lineages in the absence of extrinsic growth factors  and direiPSC lines vary greatly in their ability to differentiate into RPE and photoreceptors. These differences can be attributed to multiple factors. Variation in endogenous gene expression controlling differentiation have been shown to be one of the factors , 139, inA major leap forward in photoreceptor differentiation was made in 2011 when Eiraku et al. showed that by suspending developing PSC in 3D culture conditions, the retina could be observed to arise from a neuroectodermal structure mimicking the anterior part of the primitive forebrain, with optic grooves protruding to form optic vesicles, and then invaginating spontaneously (rarely) to form a double walled optic cup structure, without the requirement for surface ectoderm . SeveralRPE cells can be easily recognised by their characteristic pigmented polygonal appearance from an early stage in culture and subsequently dissected out to purify and mature. Their maturity can be assessed by a variety of morphological, molecular and functional features. RPE-specific markers include those for phagocytosis such as MERTK and the basal marker BEST1. Apical membrane-associated markers include Na+/K+ATPase. Visual cycle markers include RPE65, LRAT and CRALBP . PhagocyBeing non-pigmented, photoreceptor cells suitable for transplantation are less easy to identify. The intracellular markers used in vitro to identify photoreceptors such as CRX and NRL cannot be used to sort cells for transplantation using fluorescent- or magnetic-activated cell sorting . StudiesSuccessful cell replacement does not equate solely to the ability to produce, and isolate the desired cell types, the cells also have to be delivered successfully to the subretinal space, survive and be able to replace the function of the degenerated cells. We now discuss the various approaches being evaluated to achieve this and the challenges faced.RPE replacement, in particular, using PSCs has progressed relatively rapidly and two approaches are currently being considered for their transplantation: RPE cell suspension injection into the subretinal space and RPE sheet transplantation.Suspension of RPE cells transplanted into the subretinal space has rescued photoreceptors in numerous preclinical models of retinal degeneration . Ocata TVarious possibilities have been suggested to counteract this, including re-surfacing BrM to enhance RPE attachment. Research by Priore and colleagues showed that coating aged BrM with a mixture of laminin, fibronectin and vitronectin significantly improved cell adhesion, survival and proliferation and phagocytosis −161. AnoFormation of a monolayer with apical-basal polarity and attachment to a surface are critical to RPE cell function and survival. Another approach to transplantation is, therefore, to deliver RPE cells as an intact monolayer on a carrier to replace BrM . Uhttps://clinicaltrials.gov/ Identifier: NCT01691261) uses hESC-derived RPE on a polyester sheet. The Riken Institute in Japan (JPRNUMIN000011929) used hiPSC-derived RPE transplanted on its own type 4 collagen layer without the use of any artificial scaffolds. The Riken trial has been halted partly due to concerns regarding the genomic integrity in the donor cells but has reported results in one patient to date. Safety and tolerability have been shown and long-term cell survival suggested by graft appearance at 6 months [Two RPE sheet transplantation trials in humans are in progress both in patients with wet AMD of recent onset and both inserting RPE sheets grown from PSCs subretinally. The London Project to Cure Blindness , induced partly by the retinotomy site to introduce the patch was a problem, but it is hoped that the avoidance of a donor retrieval site in the eye will reduce this. Sheet transplantation requires a significantly larger incision than suspension injection but multiple sheets rather than single large ones could be transplanted to cover larger areas. Autologous work has also suggested that peripheral retinotomies may be better tolerated than posterior ones as used in both of the current sheet trials and this is something that could be explored albeit with more extensive surgery required . AnotherTo date, there have been no clinical studies with PSC-derived photoreceptor cells, although this will likely occur soon based on a number of compelling animal studies. Human trials have commenced with foetal-derived RPC and previously carried out with foetal-derived retinal sheets.Studies in animal models of retinal degeneration have now demonstrated that transplanted photoreceptor cell suspensions can restore visual function, including using PSC-derived cells albeit with reduced effect. Experiments can be divided into those with remaining photoreceptors and a completely absent photoreceptor layer.Studies in animals with some surviving photoreceptors have had several key findings. ‘Integration’  is very dependent on the subretinal microenvironment and this can affect the survival, morphology and functionality of transplanted cells . Both tThe number of successfully ‘integrating’ cells can be increased by a variety of treatments to the host environment, including temporary disruption of the host external limiting membrane, digestion of disease-associated ECM, especially chondroitin sulphate proteoglycans using chondroitinase ABC and also potentially by modulation of glial cell hypertrophy/activation and matrix metalloprotease activity −182. DesTransplantation in animals with a completely degenerate outer retina and no remaining photoreceptors (as may be the case at the fovea in advanced AMD) results in subretinal cell clumps in close contact with the remaining inner nuclear layer with limited migration into the host. Synapse formation, correct orientation with respect to the RPE and visual responses have however been demonstrated and related to the number of surviving cells , 186. PhOne other interesting development, which although not directly related to macular degeneration may have implications for retinal regeneration, has been a case report of a free autologous retinal patch transplanted from the peripheral retina to the fovea in a patient with a prior retinal detachment and large macular hole. The patient regained a visual acuity of 0.6logMAR with gradually improving retinal sensitivity and the authors postulated that the free graft played some role in retinal regeneration . FurtherThe exact mechanism of action of human RPC transplantation is unclear at present as discussed previously. They have rescued photoreceptor death in a number of animal models and current data suggests it may be chiefly neuroprotective with growth factor release. The transplanted cells may also differentiate into, attract or retain neuroprotective glial cells.At least two human trials are in progress in the USA using foetal-derived RPC. ReNeuron  have a Phase I/II open-label, dose escalation clinical trial in progress on 20 patients with RP to evaluate the safety, tolerability and preliminary efficacy of a subretinal injection of RPC. jCyte, Inc   have a pAs mentioned previously foetal retinal sheet transplantation has been carried out in adults with advanced AMD and retinal sheet transplantation may be a promising strategy especially in focal disease such as AMD. PSC-derived retinal organoids are an exciting development in this regard as they offer the possibility of deriving retinal sheets for transplantation of any developmental stage and in practical quantities. Takahashi and colleagues have used the ~2 mm optic vesicles derived from hESCs, sectioned them into halves and transplanted them into murine and primate models of outer retinal degeneration , 191. ThIn advanced AMD, both RPE and photoreceptors would need to be transplanted. RPE often grows adjacent to neural retina in organoid models, but not as a continuous layer underlying the developing photoreceptors as occurs in vivo. It may be possible to develop culture conditions that encourage RPE growth and it has recently been shown that 0.5% alginate increases RPE growth alongside neural retina . AlternaThere has been relatively little investigation into choroidal replacement in AMD regenerative strategies. Replacement of healthy RPE may rescue a failing choroid by VEGF and other growth factor release; however, there is evidence that choroidal endothelial cells (ECs) are lost early in the pathogenesis of AMD and as mentioned previously with autologous RPE transplants, choroidal reperfusion is essential for graft survival. Developing a procedure to replace these cells would be an important consideration in advanced cases of AMD—both wet and dry. Human iPSC-derived ECs have been made and studies with human umbilical vein-derived ECs suggest that if grown with RPE cells, ECs may acquire choroidal characteristics . ChoroidThe subretinal space is considered to be a relatively immune-privileged site ; howeverBearing in mind that many cell types can have cell rescue effects, and conversely many have the potential for further differentiation into retinal cell types after transplantation, there are several cell lines being investigated, which are specifically being used for host cell rescue and undergoing clinical trials in patients with a variety of retinal diseases, including AMD Table . Cells hNeurotech  have used a cell capsule implanted into the vitreous cavity and anchored at the pars plana containing human RPE cells genetically modified to overexpress CNTF and encapsulated in a polyethylene terephthalate scaffold. Phase I and II trials in patients with AMD (and RP) have shown the device is well tolerated but evidence of efficacy was less clear and the technology is now being considered for other applications, including to deliver anti-angiogenic agents in AMD (NCT02228304).A wide variety of trials using MSC and other related cells are in progress. Early phase trials at the University of Sao Paulo, using autologous bone marrow-derived stem cells intravitreally in patients with advanced RP, showed some efficacy on macular oedema, but after 1 year, the effects were no longer evident . The UniSubretinal injection of human umbilical tissue-derived cells (hUTCs)  have been evaluated in a phase 1/2 trial in 35 patients with advanced dry AMD. These cells are derived from extraembryonic mesoderm and can be expanded significantly in vitro but are not classed as stem cells and do not differentiate into other cell types. hUTCs have been shown to secrete several key neurotrophic factors that rescue RPE cell and photoreceptor function, including phagocytic dysfunction and have also been shown to promote synaptogenesis via thrombospondin family proteins , 205. Vihttps://clinicaltrials.gov/, #NCT02467634). Trial results have not been reported and there appears to be no further studies planned.Preclinical studies using the RCS rat showed that subretinal transplantation of human NPCs derived from human prenatal cortex resulted in long-term rescue of visual function , 207. ItAdipose tissue-derived cells have been used in several trials and are also being used in non-trial situations by private clinics. Unfortunately, recently three patients were reported who developed severe visual loss with a variety of problems, including ocular hypertension, haemorrhagic retinopathy, vitreous haemorrhage, combined traction and rhegmatogenous retinal detachment, and lens dislocation after intravitreally delivered cells .The dream of replacing or rescuing old, dead or dying cells with new young ones is appealing and the developments in the field are very exciting. However, the path to an effective safe cellular therapy that can be used routinely in patients for late-stage AMD is likely to be a long one. IPSC technology and 3D retinal organoid production have been huge recent steps in this path, but several aspects of cellular therapies outlined in this review need further refinement. The optimum cell sources and age of cells used for transplant need to be identified, cells have to be produced in the numbers required in GMP and Xeno-free conditions, and they then need to be sorted so that the desired cell types are isolated before transplantation. Cell delivery needs further thought as well. Cell sheet transplants require large retinotomies with their associated risks of PVR and subretinal fibrosis, which could not only cause surgical complications but also affect integration of the graft. Fibrosis has been a major challenge in several transplant disciplines and lessons could be learned across disciplines. Cell suspensions have problems of cell death during delivery, reflux during injection and a less than perfect cellular distribution after injection aside from RPE adhesion problems, all potentially aided by tissue engineering approaches. Cellular integration and survival after delivery also need improving perhaps aided by pro-survival agents added to the transplanted cells including potentially the use of combined gene therapy.Preclinical testing would be easier to interpret if there were representative, near human size and accepted animal models of AMD. The commonly used model of the RCS rat is rescued by a wide variety of disparate cell sources and does not represent AMD very closely. Rabbit and pig models, although having visual streaks, lack foveas and in the case of rabbits are merangiotic, while primate models, as well as having ethical concerns and expense, do not have the problems of cell adherence to BrM seen in aged human subjects.Unmasked fellow eye control phase I studies have established the safety of many treatments, but evidence of unequivocal, reproducible and sustained efficacy needs to be demonstrated. Indeed, there are several limitations to using these studies to suggest efficacy in terms of asymmetrical disease and progression, protective cytokine release with surgery  and learning effects in terms of eccentric fixation in worse fellow eyes , 211 and


Value in health care represents the relationship between health outcomes achieved and resources used. In a highly prevalent condition it is important to ensure that the highest value interventions are employed effectively In COPD these are influenza vaccination, smoking cessation and pulmonary rehabilitation . Muscle fatigue is an important symptom limiting exercise. The main driver is physical inactivity and this occurs in early in the course of the disease in particular this may be before it has been diagnosed. Physical inactivity may itself drive lung disease progression.As well as a loss of muscle bulk there is a shift away from a Type I fibre, oxidative endurance muscle phenotype. The underlying biology is complex and in addition to inactivity, inflammation, corticosteroids, reduced anabolic hormones, corticosteroid treatment, hypoxia, poor nutrition and increased resting energy expenditure may all play a role. There is a recent ATS/ERS statement on limb muscle dysfunction in COPD.Exercise has a wide range of beneficial effects. It can improve exercise capacity, lipid profile, reduce falls, reduce cardiac risk, improve depression, insulin sensitivity, and systemic inflammation as well as protecting against cognitive decline and osteoporosis.Pulmonary rehabilitation has been defined as “an evidence-based, multidisciplinary, and comprehensive intervention for patients with chronic respiratory diseases who are symptomatic and often have decreased daily life activities. Integrated into the individualized treatment of the patient, pulmonary rehabilitation is designed to reduce symptoms, optimize functional status, increase participation, and reduce health care costs through stabilizing or reversing systemic manifestations of the disease.”A standard PR course might be ∼8 weeks of twice weekly sessions as well as home exercise being encouraged. A mixture of aerobic and strength training is used and it is supervised by health professionals. The venue can be a hospital gym or a community gym or other space. It should be integrated into clinical management and include education about self-management and other issues.There is a compelling evidence base for benefit of pulmonary rehabilitation in COPD, reversing the effects of deconditioning improving exercise capacity and quality of life. The cost per QALY of pulmonary rehabilitation is significantly better than for pharmacotherapy in COPD.Pulmonary rehabilitation in the early post-exacerbation phase reduces readmission and improves survival with a number needed to treat of 4 to prevent one readmission.This recent review on PR for non-COPD lung disease is useful.- Smoking cessation- Inhaler technique- Written self-management plan- Assessment for pulmonary rehabilitation- Follow up arrangement made before dischargeOne way to improve pulmonary rehabilitation uptake is the use of a COPD discharge care bundle has been developed with input from guidelines, clinicians and patients which includes the following items:Patients also complete a safe discharge checklist with the nurse responsible for their care. Evidence suggests that it may reduce readmission rates and improve quality of care. The documents are all available in the online supplement (open access).


Astronauts and vestibular patients face analogous challenges to orientation function due to adaptive exogenous (weightlessness-induced) or endogenous (pathology-induced) alterations in the processing of acceleration stimuli. Given some neurovestibular similarities between these challenges, both affected groups may benefit from shared research approaches and adaptation measurement/improvement strategies. This article reviews various past strategies and introduces two plausible ground-based approaches, the first of which is a method for eliciting and assessing vestibular adaptation-induced imbalance. Second, we review a strategy for mitigating imbalance associated with vestibular pathology and fostering readaptation. In discussing the first strategy , we review a pilot study wherein imbalance was elicited  via an adaptive challenge that caused a temporary/reversible disruption. The surrogate vestibular deficit was caused by a brief period of movement-induced adaptation to an altered (rotating) gravitoinertial frame of reference. This elicited adaptation and caused imbalance when head movements were made after reentry into the normal (non-rotating) frame of reference. We also review a strategy for fall mitigation, viz., a prototype tactile sway feedback device for aiding balance/recovery after disruptions caused by vestibular pathology. We introduce the device and review a preliminary exploration of its effectiveness in aiding clinical balance rehabilitation . Both strategies reviewed in this article represent cross-disciplinary research spin-offs: the ground-based vestibular challenge and tactile cueing display were derived from aeromedical research to benefit military aviators suffering from flight simulator-relevant aftereffects or inflight spatial disorientation, respectively. These strategies merit further evaluation using clinical and astronaut populations. Terrestrial spatial orientation and balance are maintained by overlapping multi-sensory information that is continually updated and cross-referenced. When processing of stimuli associated with body motion is altered, immediate changes in cognitive perceptions of self-orientation and motion occur, along with reflexive vestibulo-spinal, -ocular, and -autonomic responses . The sea or space voyager must then go through a second process of adaptation upon returning to Earth after a prolonged voyage. This is because the voyager must readapt to walking on a terrestrial substrate while being subjected to a steady and unidirectional acceleration due to Earth’s gravity. The same sequence of adaptation and readaptation accompanies many other sensory rearrangements, such as prolonged exposure to a military flight simulator .Regardless of which challenge causes altered processing of body motion, there is an initial period of disruption of functional abilities, leading to disorientation, imbalance, disruption of gaze control, or motion sickness symptoms. The process of disruption and subsequent adaptation is somewhat similar for patients recovering from many types of vestibular pathology as for healthy people adapting to the vestibular challenge posed by exposure to a flight simulator or a prolonged voyage at sea or into space. Once adaptation is achieved by sensorimotor recalibration to these new processing demands, coordination and well-being is restored. In the case of recovery from endogenous vestibular deficits, such adaptation represent an unqualified positive outcome. Unfortunately, in the case of adaptation to sea or space travel, the initial compensation confers resistance only to the sensorimotor effects of the voyage This review article compares the processes of disruption and adaptation associated with endogenous vestibular pathology to those associated with exogenous changes from the normal gravitational force level occurring during spaceflight. We briefly discuss how a preliminary ground-based gravitoinertial force alteration strategy might prove useful as a proxy for the induction and measurement of balance deficits caused by vestibular maladies or aftereffects of spaceflight. We also describe sensitive tests for balance deficits which have spun off from such work.In addition to discussing a possible balance deficit induction and assessment strategy, we discuss countermeasures. We review a few of the plausible strategies for mitigating imbalance due to space adaptation, including a sensory cueing strategy (and associated device) that we helped to initiate. The device is a tactile display that is known to reduce in-flight spatial disorientation. Explorations were made of its potential usefulness for avoiding imbalance and falls following vestibular pathology. We conclude that this strategy is worth exploring further, to determine if it will help with the space adaptation syndrome during/after spaceflight. To date, little is known concerning the potential space-related benefits of this strategy, particularly post-flight , and for perception of the body’s orientation and motion relative to the gravitational vertical. The many neurovestibular reactions and patterns of functioning mentioned in Table The essential neurovestibular aspects of the challenge represented by entry into microgravity can be most simply expressed as a mismatch between semicircular canal vs. otolith signals. After a lifelong tight coupling of canal-otolith information during head and body movement, astronauts in microgravity are subjected to a situation where the canal information is normal during head movement, but the otoliths are providing altered information from usual. This is analogous to many ground-based vestibular challenges that alter normal sensory integration. For example, on Earth, one is exposed to a canal-otolith mismatch when making head movements orthogonal to one’s body axis during prolonged (constant velocity) yaw rotation. In this case, the converse (of the spaceflight situation) occurs, in that the otoliths continue to provide normal signals, while the canal signals are altered. In either case, the mechanisms for central integration of canal and otolith information must adapt for normal coordination and well-being to be restoredFigure Welch and Mohler  and otheDuring orbital spaceflight, the unloading of the otoliths by microgravity and their shifts related to head movements represent relatively stable sensory rearrangements that provide sufficient opportunities for error-corrective feedback so that adaptation can occur. Unfortunately, many of the other factors that foster optimal adaptation are not present in microgravity. For example, astronauts presently cannot be conveniently exposed to gradual increases in their duration of exposure to constant microgravityThe most direct means of facilitating adaptation during G transitions associated with space exploration would be to employ artificial gravity. For example, upon entry into microgravity, living most of the time onboard a space vehicle rotating at a fairly large radius to produce Earth’s 1 G would allow adaptation to 0 G operations to proceed more gradually and via distributed exposures. This strategy could also be used to gradually adapt an astronaut to the 0.38 G field of Mars prior to landing. However, it is not known precisely how much artificial gravity  would be needed to prevent bone and muscle loss under microgravity in orbit or 0 G in deep space. If daily exposure to 1 G is needed, then a gradual change of artificial gravity from 1 G to 0 G to 0.38 G in preparation for a Mars mission may not be advisableAre there ways in which space adaptation can be fostered without (or in addition to) artificial gravity? While gradual introduction to longer periods of spaceflight is not feasible prior to an actual mission, the magnitude of the initial challenge can be partially blunted by: (1) Medication—i.e., the use of medications that alleviate motion sickness without slowing adaptation; (2) Adaptation—programs of ground-based pre-adaptation to spaceflight; (3) Behavioral strategies—such as limiting the speed and amplitude of head movements during initial exposure to space; or (4) Sensory cueing—providing additional orientation cues to decrease the magnitude of the initial rearrangement, e.g., by enhancing somatosensory inputs to make the astronaut feel more grounded relative to the perceived “floor.” All four of these methods have been attempted, but most of them are not yet mature or fully understood. Medications provide some relief from nausea related to spaceflight, but some can be sedating and their effects on the process of adaptation vary  their exposure to that part of the challenging stimulus which is under their control. In space, limiting one’s head movements means one is exposed only to the relatively static aspect of the vestibular challenge caused by the unloading of the otolith crystals, rather than also being exposed to the dynamic challenge associated with nonterrestrial movements of the otolith crystals during head movements under weightless conditions. Restricting head movement is a useful strategy for limiting the nauseogenic challenge. Unfortunately, since many astronaut and military duties must be performed quickly and require frequent head movement, this strategy can be applied less successfully in such occupations than it can in a less-demanding setting, such as during a pleasure cruise at sea. Furthermore, while astronauts and vestibular patients suffering from nausea or vertigo may prefer to lie quietly with their eyes closed, such behaviors could become counterproductive habits that limit beneficial adaptation. Vestibular patients often limit their activity when they should engage in movement in order to recover Herdman, . In factThe final strategy for helping astronauts with space adaptation syndrome (or readaptation to Earth) is to provide additional orientation cues to decrease the magnitude of the initial rearrangement, by enhancing somatosensory inputs to make the astronaut feel more grounded relative to the perceived “floor” or other significant frames of reference. It has long been known that situations where the locally-defined “up and down” are ambiguous or unexpected can elicit disorientation and motion sickness symptoms among astronauts  control after challenges such as labyrinthine concussion is largely a process of neurological adaptation based on sensory feedback. One of the key principles of vestibular rehabilitation is that movement, and the associated sensory feedback from movement, are critical for such adaptation. This fact is vividly illustrated by the large deleterious effect of restricted mobility on the rate of clinical recovery of baboons following unilateral transaction of the vestibular nerve Herdman, . UnstrucThe choice of an optimal sensory modality for sway-feedback is not trivial. A visual feedback device is simple to program and display and is readily available from various manufacturers. However, if visual cueing technology is eventually made wearable not only for convenient/portable adaptation training, but also for non-invasive sensory prosthetic applications , the display would produce light cues for orientation which interfere with one’s retinal sensitivity for natural, ambient visual environmental orientation cues in low light situations. Thus, balance cues would be enhanced by a visual prosthesis, but natural cues for avoiding collisions would be degraded even further than they are by existing head injuries or the natural aging process  of the balance test. After only 10 min of adaptation to the rotating room, the subjects had already adapted sufficiently to exhibit observable vestibulo-ocular reflexes and coordinated walking movements appropriate to the new rotating environment. This is a striking example of adaptive plasticity allowing rapid adjustment to a complex stimulus. The balance testing situation is shown in Figure Wilcoxon Signed-Rank Test: p < 0.05; Figure When subjects left the room to be tested on the balance platform, they showed significantly worse post-rotation balance performance during the modified SOT5 involving balancing while moving one’s headMeasures of sway are interesting for sensitively quantifying deficits in standing balance, but the critical functional indicator of instability among patients and astronauts is falling. Falling was common during the first post-test, occurring among 25 (of the 32) subjects on the first trial of the first post-testing session. This suggests that the adaptation protocol created a profound aftereffect. As desired, the falling aftereffect was temporary and readaptation to normal conditions was rapid. This statement is supported by the fact that only five subjects fell during all three trials of the second post-testing session. In summary, the ground-based adaptation task worked as planned, inducing a temporary and reversible vestibular insult with functional consequences for standing balance detectible during a challenging balance task. As with imbalance following spaceflight, the elicited balance deficit was attributable to the aftereffects of adaptation to an unusual gravitoinertial force environment.The addition of head movements to SOT5 was an interesting feature of this pilot study  showing a nonsignificant trend for earlier improvement of balance performance scores during computerized posturography compared to subjects who received standard-of-care physical therapy (n = 11). Calculations of percent-of-change in mean composite scores on the SOT (#1–6) determined that the Device group realized maximum improvement after eight sessions. Controls reached maximum improvement after 12 sessions (by which time both groups were performing similarly).Both groups reached the same sway improvement eventually, with the group in the enhanced tactile feedback condition  increased by five from pre-testing vs. testing after two sessions of enhanced tactile cueing device therapy, whereas the number of Control group (standard of care therapy) participants who did not fall did not change at all over the same period . The device is going to market soon, so improved, widespread evaluations should be forthcoming from multiple clinics.BDL: led the writing effort, assisted with the studies reviewed, assisted with development of apparatus. AHR: contributed to the writing effort, initiated studies 1 and 2, assisted with development of apparatus. BJM: contributed to analyses, led study 1, developed apparatus.Studies #1 and #2 described herein were primarily sponsored by the Naval Air Systems Command and the U.S. Army Medical Research and Material Command, respectively.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.


In recent years, there has been a deluge of papers linking altered microbiome compositions to a myriad of diseases. Mechanistic insight into microbial drivers of disease phenotypes is essential for translation to novel therapies. In recent years, there has been a deluge of papers linking altered microbiome compositions to a myriad of diseases. Mechanistic insight into microbial drivers of disease phenotypes is essential for translation to novel therapies. A key mechanism by which microbes influence health is immune modulation by components of their capsule and cell envelope and their metabolites. A major research focus of my laboratory is to gain mechanistic insight into which microbes modulate host immunity generally and in the context of disease. Using 16S rRNA-targeted sequencing, we have established associations between gut microbiome composition and immune-modulated disease phenotypes in diseases such as graft-versus-host disease in cancer patients undergoing stem cell transplantation. By integrating omics and computational approaches with laboratory experiments, we have expanded knowledge of mechanisms used by host-associated microbes to dampen inflammatory responses. This work has promise for development of novel microbiome-targeted therapeutics. Many diseases that plague our population share the common basis of a dysregulated immune system, including those characterized by a hyperactive immune response  or by an ineffective immune response  and those for which chronic low-grade inflammation is a potential contributing factor . Since our microbial inhabitants are in constant communication with the immune system on every surface of our body and are important for training the immune system in early life , understIt is well established that microbes locally influence immune status at the sites that they occupy . However+ T cell differentiation into proinflammatory or anti-inflammatory (Treg) phenotypes depending on the concentration and on which costimulatory factors are present  and immune phenotypes is hampered by a poor understanding of what many of these organisms, genes, and transcripts actually do . FurtherBacteroides fragilis, can suppress inflammation by stimulating CD4+ T cells to differentiate into an anti-inflammatory T regulatory (Treg) phenotype  in blood have been linked with chronic inflammation in diseases such as HIV infection, indicating that therapies that discourage proinflammatory gut microbe growth and/or enhance intestinal barrier function have therapeutic potential . Howeverhenotype . We firspulation .Streptococcus pneumoniae, and PSA2, a structural variant of PSA carried by a different strain of B. fragilis (in vitro assays with peripheral blood mononuclear cells and also by evaluating protection in a mouse model of inflammatory disease. We further confirmed ZPS to be a driving factor of Treg and IL-10 induction in in vitro assays performed with Bacteroides cellulosilyticus by genetically disrupting ZPS operons (in vitro systems to establish whether any of these novel ZPS molecules or the microbes that produce them have therapeutic potential for diseases of chronic inflammation, including HIV infection. Understanding how to identify ZPS activity based on gene sequences will also allow us to investigate how the presence and expression of this function differ in various disease contexts using shotgun metagenomics and metatranscriptomic data sets, both generated in our laboratory and from the public domain.When we began our work, ZPSs of related immune-modulatory properties that could be produced by a few phylogenetically divergent bacteria were known, including PSA, SP1 of fragilis , but ZPSfragilis . We veri operons . We are The microbiome field has come a long way in a short time. Although I am early in my scientific career, I can look back to how different things were just over a decade ago, when I was a Ph.D. student working with Rob Knight to develop a tool called UniFrac, which uses the information in phylogenetic trees to compute distances between microbial communities . When weAlthough advances in next-generation sequencing and bioinformatics have been major drivers of progress in human microbiome research, there have also been other key advances that have enabled mechanistic insights. These include advances in our ability to culture diverse intestinal microbes and to genetically manipulate bacteria so that the effects of gain or loss of particular functions can be evaluated, the development of animal models such as gnotobiotic mice for establishing causality, and integration of other cutting-edge technologies such as metabolomics. Despite these gains, there are still many challenges, such as the high number of genes and metabolites of unknown function and of bacteria with poorly understood properties that evade cultivation and the lack of availability of tools to genetically manipulate most of those that we can culture. The most exciting and translational work integrates complex multi-omic and bioinformatics methods with confirmatory experimental work to establish a mechanistic link between microbes and disease. As a scientist early in my career with a particular expertise in computation in comparison to bench science, I have worked to establish strong collaborations with individuals with complementary expertise to perform integrated multidisciplinary research with the goal of developing effective microbiome-targeted therapeutics. With microbiome research accelerating at an impressive rate, one cannot help but be excited by the vast amounts of knowledge to be gained from the tiny world inside each of us.


Recent epidemiological and clinical studies have shown that type 2 diabetic patients can develop diabetic vascular complications even after intensive glycaemic control. It has been suggested that this phenomenon could be explained by the hypothesis of ‘metabolic memory’. The underlying mechanisms between these enduring effects and the prior hyperglycaemic state are still not well understood. Preliminary studies demonstrate that hyperglycaemia can regulate gene expression by epigenetic modifications, such as DNA methylation, which can persistently exist even after glucose normalization. Increasing evidence shows that epigenetic mechanisms may play a substantial role in the pathophysiology of diabetes and its associated vascular complications, including atherosclerosis, diabetic cardiomyopathy (DCM), nephropathy and retinopathy. In this review, we will examine the growing role of DNA methylation in diabetes and its vascular complications, thus it can provide critical implications for the early prevention of diabetes and its vascular complications. The epidemic of diabetes is a serious and growing public health problem. In 2015, the International Diabetes Federation (IDF) declared that 415 million people have diabetes and the number will rise to 642 million by 2040 worldwide ,4. DiabeAlthough the prevalence of diabetes and its vascular complications are increasing, the pathogenesis of diabetes and its complications have not been clearly understood. There is growing evidence supporting the role of ‘metabolic memory’ in diabetic complications and metabolic memory plays a critical role in the development of vascular complications in diabetic patients ,10. MetaEpigenetics has been widely accepted as the heritable changes in gene function, which is independent of DNA sequence . It can DNA methylation is the earliest discovered and most important epigenetic modification with extensive investigations. DNA methylation is exerted by DNMTs at the 5′-position of cytosine residues in CpG dinucleotides (the p denotes the intervening phosphate group) by transferring methyl groups from S-adenosyl methionine (SAM), thus 5-methylcytosine is formed . Most CpIt is well known that atherosclerotic cardiovascular disease is the major cause of morbidity and mortality in diabetes. Atherosclerosis occurs earlier and with greater severity in diabetic patients, leading to a much higher risk of limb ischaemia, myocardial infarction and stroke . ArteriaCardiovascular complications are a primary cause of mortality and morbidity in diabetic patients. DCM was first proposed in 1972, which is a distinct primary disease process, independent of coronary artery disease . It can Diabetic nephropathy is a serious microvascular complication of diabetes mellitus and and has become the most common cause of end-stage renal disease (ESRD). There is increasing evidence to suggest that dysregulation of the epigenome is involved in diabetic nephropathy . One cliDiabetic retinopathy remains one of the major causes of blindness in adults. Clinical and experimental studies have demonstrated that hyperglycaemia has long-lasting effects on the retina and the damage continues even after termination of the hyperglycaemic abberration . Epigenein vitro experiments have shown that DNA methylation may play a substantial role in the pathophysiology of diabetes and its associated vascular complications, including atherosclerosis, DCM, nephropathy and retinopathy. The relevant evidence was summarized in Taken together, the aforementioned clinical studies, animal models and Traditionally, it was considered that epigenetic modifications were static in the regulation of gene expression. However, this idea is now being altered and epigenetic marks, including DNA methylation are dynamic induced by some factors. Some preliminary studies have showed that DNA methylation plays an important role in the reversibility and treatment of diabetic complications. One of the first study showed that epigenetic modifications was reversibly regulated in diabetic complications. It demonstrated that treatment of type 2 diabetic db/db mice with the angiotensin II type 1 receptor (AT1R) blocker losartan not only ameliorated diabetic nephropathy, but also reversed epigenetic changes. More specifically, it showed significantly increased expression of HMTs and HATs in db/db mice. However, these increases were abolished in losartan-treated mice, accompanied with decreased blood pressure, mesangial hypertrophy and proteinuria . Lou et In summary, DNA methylation plays a critical role in the pathogenesis of diabetic complications. A better understanding of the role and mechanism of DNA methylation and diabetic complications can inspire critical implications for the early prevention of type 2 diabetes and provide unique opportunities to develop novel therapeutic approaches of diabetic complications. Furthermore, increasing evidence shows that epigenetic modifications are not static, which are dynamic and even reversible. Therefore, in view of the reversibility of epigenetic mechanisms, intervention with pharmaceuticals or other interventions during early course of diabetes may ameliorate its complications in later life, which can generates long-lasting effects.