The risk of Alzheimer’s disease (AD) increases with nonmodifiable conditions including age and lack of effective efficacious pharmacotherapy. During the past decades, the non-pharmacotherapy mode of treatment of dietary modification received extensive attention in AD research. In order to reduce the AD pathology and cognitive decline, various dietary patterns have been attempted including caloric restriction (CR), dietary approaches to stop hypertension (DASH), ketogenic diets (KD), Mediterranean diet (MedDi) and Mediterranean-DASH diet Intervention for Neurological Delay (MIND) diet. Higher adherence to the MedDi diet was associated with decreases in cardiovascular and neurological disorders including AD and related cognitive decline. However, another emerging healthy dietary pattern MIND diet has also been associated with slower rates of cognitive decline and significant reduction of AD rate. Olive serves as one of the building block components of MedDi and MIND diets and the exerted potential health beneficial might be suggested due to the presence of its bioactive constituents such as oleic acids and phenolic compounds (biophenols). A few trials using medical food showed an optimal result in presymptomatic or early stages of AD. The review supports the notion that MedDi and MIND diets display potential for maintaining the cognitive function as nonpharmacological agents against AD pathology and proposed preventative mechanism through the presence of olive biophenols and presents the gaps along with the future directions. Brain ageing may be considered as a progressive and inevitable physiological process related to the dramatic increase in the oxidative stress condition and inflammatory markers that potentially cause damage to cellular structures of the brain . The conFollowing continuous attempts of using synthetic origin molecules against AD and their unfortunate failure in pharmacotherapy, the research trend has been changed towards using plants in diet or medication form that have positive effects on cognitive disorders, as well as strong acetylcholinesterase (AChE) inhibitory, anti-inflammatory, and antioxidant activities, and are of potential clinical interest for the treatment of AD . EvidencSince 2000, it has become increasingly evident that biophenols, plant phenolic compounds containing foods received extreme attention due to their vast occurrence and versatile actions including several cellular functions modulation, processes that go well beyond their first-described natural antioxidant capacities and neuroprotection . VegetabThe present review summarizes the existing evidences of adherence to diets  and Mediterranean dietary approaches to stop hypertension (DASH) diet Intervention for Neurological Delay (MIND)) with the reduction in AD risk and cognitive function and exploring the involvement of olives as one of the backbone dietary components. In addition, the neurobiological mechanisms of the MedDi against AD or cognition decline are proposed.The published scientific literatures were searched for in vitro, in vivo, observational studies, prospective cohort studies and randomized controlled trials of adult human participants reporting diet/nutritional intake and AD biomarkers. The searches were conducted digitally by using the databases including MEDLINE, PubMed, ScienceDirect, Google Scholar, and SCOPUS to identify peer-reviewed articles in the last two decades . A Boolean search strategy was conducted with the following keywords and logic:  AND (“Alzheimer’s disease” OR “amyloid” OR “β amyloid” OR “β-amyloid” OR “Aβ42” OR “Aβ40”) AND (“olive biophenols” OR “olive polyphenols” OR “oleuropein” OR “hydroxytyrosol”). Alzheimer’s disease can be influenced by several factors and the potential effect of diet has become a topic of increasing scientific and public interest. It was suggested that AD patients were associated with a high intake of meat, butter, high-fat dairy products, eggs, and refined sugar . Animal 40, Aβ42 and phospho-tau in the hippocampus compared to the control diet group. Further, in a conditional double knockout of presenilin-1 and presenilin-2 (cDKO) mice study [Caloric restriction (CR) dietary interventions influence the ageing and age-related changes in the brain and ultimately extend the lifespan and health span. In a transgenic mouse model of AD study , two difce study , adminisce study , the CR p < 0.001).A few earlier pieces of epidemiological evidence indicated that individuals who habitually consume fewer calories have a reduced incidence of AD ,22. In aApart from the possible benefits of CR diets in the lowering of blood pressure and improved heart health to longevity, it was also suggested that a reduction up to 30 % of daily calorie intake can cause gaunt look or emaciated and experience dizziness, cognitive decline, loss of muscle mass, a faulty menstrual cycle and a lowered libido. Moreover, animal studies showed that eliminating more calories up to 60 % of daily intake resulted into signs of starvation in mice. The researchers asserted that long-term calorie restricted diet can increase stem cell functionality but may also reduce the performance of the immune system especially regarding bacterial infections. Although, there is experimental evidence for a calorie restricted diet as an ageing intervention, the potentially harmful effects and risks in elderly humans need to be studied further.However, initiating a CR intervention prior to adulthood imposes many practical, physiological, and ethical challenges, including consideration of the impact on growth and development in humans. Therefore, introducing a CR intervention prior to adulthood is not an option in human studies.The dietary approaches to stop hypertension (DASH) diet mainly contains a high intake of plant foods, fruits, vegetables, fish, poultry, whole grains, low-fat dairy products, and nuts, alongside low intake of red meat, sodium, sweets, and sugar-sweetened beverages . The DASp = 0.04) different systolic and diastolic blood pressures [Coronary heart disease and hypertension are independent risk factors for AD , while fressures . Moreoveressures , and theressures . Studiesressures , which mressures . Despite the known benefits of the DASH diet, adherence has been limited to the general population. The approach taken for DASH diet constructing an adherence score is a major limitation to the study. Incomplete data on the DASH diet made it difficult to take into account specific intake levels for each food group according to energy requirements for individuals because these data were not available. Additional research is needed to determine whether this finding also holds true for men and other race/ethnicity, as well as in the educated, socioeconomic status subgroups.40 and Aβ42. In an animal study [A ketogenic diet primarily consists of high-fats (55%–60%), moderate-proteins (30%–35%), and very-low-carbohydrates (5%–10%) and provides 2000 kcal per day diet . Adminisal study , showed al study . In a double-blind placebo-controlled study on AD patients , the oraIn summary, KD showed an improvement in cognitive outcomes, which was associated with the level and duration of ketosis . In addiA number of studies have shown the slow rate of cognitive decline and reduced risk of cognitive impairment and suggested that the decreased rate of dementia have been associated with a higher consumption of single nutrients  or of single foods  diet (fifteen dietary components make up the MIND diet ), observAcross four studies, the MIND diet has been associated with reduced AD risk  and bettThe exact pharmacological mechanism by virtue MedDi exerts a protective effect in brains are not fully understood. Despite the lack of studies investigating the underlying mechanisms, MedDi and MIND diets showed multiple pharmacological action including anti-oxidant, anti-inflammation, anti-atherogenic, and cognitive enhancement mediated through its food components. However, it may suggest that the health beneficial effects of the MedDi and MIND diet have been attributed to the use of olive as a major source of dietary fats and phenolic compounds .Triglycerides are the major constituents of olive oil (98–99% by weight) categorized into three main fatty acids fraction including monounsaturated fatty acid (oleic acid), saturated fatty acid  and polyunsaturated fatty acid (linoleic acid) respectively . There iPhenolic compounds called biophenols isolated from plant tissues or products are derived from shikimate-phenylpropanoid and/or polyketide pathway(s) including their derivatives, conjugates, degradation products and metabolites . Olive cp-HPEA) or its hydroxyl derivative hydroxytyrosol  are the most abundant class of phenolics found in all olive products. However, olive oil, specially the extra virgin olive oil (EVOO), is mostly represented by the presence of abundant secoiridoids as aldehydic forms of oleuropein and ligstroside aglucones  [−1 of biophenols  with the health claim “Olive oil polyphenols contribute to the protection of blood lipids from oxidative stress” [In general, secoiridoids compounds, and tyrosol (ctively) . In addi stress” . Olive f stress” . Olive lBiophenols serve as potent scavengers through capturing free radicals , combiniDysregulation of unsaturated fatty acids including oleic acid metabolism plays a major role in driving AD pathology . Oleic aInflammation, a well-known causative factor of several diseases showed a significant association with compromised vascular health, as well as neuronal damage in the brain, through amyloid peptides accumulation and subsequent activation of microglia and reactive astrocytes . PreventAs MedDi is a plant-based dietary pattern particularly rich in biophenols, usually olives and olive oil together provided approximately 11% of total biophenols intake, followed by red wine, which contributed approximately 6%. Multiple pharmacological activities  have beeDiabetes is an established link with cognitive decline, and olive biophenols showed antidiabetic effect through significantly improved insulin sensitivity and pancreatic β-cell secretory capacity . HoweverOverall, the findings provide insights into the proposed mechanisms of action of the MedDi containing olive biophenols in the context of prevention of neuroprotection and cognitive decline. Studies highlight significant cardioprotective and protection from age dependent cognitive decline which was suggested due the presence of oleic acid and biophenols in MedDi. Further experiments will involve the investigation into the effects of MedDi in aged and Alzheimer’s patients to provide insights into the potential anti-Alzheimer’s properties of the olive biophenols.Growing evidence from the recent experimental and clinical data show the key role of gut dysbiosis and gut microbiota-host interactions in neurodegeneration, and may contribute in the pathogenesis of AD in elderly through the convergence of gut-derived inflammatory response together with ageing and poor diet . Eubacterium rectale and Bacteroidesfragilis counts having anti-inflammatory action and rise of pro-inflammatory bacteria Escherichia/Shigella counts were observed in the cognitively impaired elderly patients with brain amyloidosis [Bifidobacteria counts and anti-inflammatory activity with reduction in C-reactive protein and plasma cholesterol concentrations [Bifidobacteria population [Significant reduction of gut bacteria loidosis . Studiestrations ,117. Furpulation .There is a need of research regarding MedDi diet-microbiota and specific inflammatory biomarkers in AD, as there are not many studies that explore this connection. Moreover, probiotic therapy by using measured quantity of specific olive biophenols are also warranted which may offer an intriguing approach to promote host health via delivering anti-inflammatory mediators.Based on the recognized scientific principles and medical evaluation, a medical food is formulated to be consumed or administered under the direct supervision of physician and intended to meet specific dietary or nutritional requirement for management of a disease or condition. The products are considered medical foods in the European Union, however in the United States the products are regulated as dietary foods for special medical purposes. It was found that the symptomatic benefits in AD have been claimed for three medical foods  includinThe dietary intake of coconut or palm kernel oils was suggested to be unable to provide caprylic triglyceride in sufficient quantities to meet the needs of people with AD . Axona cSouvenaid is a multinutrient beverage enriched with 11 vitamins and supplements, approved for early AD in some European countries and Australia. Souvenaid formulation consists of phosphatide precursors along with the supporting nutrients, which may intend to act synergistically to enhance the membrane formation and synaptic function improvement in individuals with AD . In addiCerefolinNAC is a mixture of L-methylfolate, methylcobalamin, and N-acetyl-cysteine, mainly to address the metabolic imbalances of hyperhomocysteinemia and neurovascular oxidative stress involved in the cognitive impairment. Administration of CerefolinNAC in AD or cognitive impaired patients having hyperhomocysteinemia due to cerebrovascular disease resulted in an adjusted hippocampal atrophy rate 4.25 times slower than those with AD and related disorders but not those with hyperhomocysteinemia who did not receive CerefolinNAC .However, the study has not been further verified and needs to be investigated in the future in a large sample size with a longer follow-up period and a randomized assignment to the CerefolinNAC treatment and placebo. Moreover, there is a need to emphasize that the findings in individuals with hyperhomocysteinemia cannot be extrapolated to those with normal homocysteine levels.The neuroprotective activity of olive phenolic constituents including oleuropein, hydroxytyrosol, verbascosides, and oleocanthal are well documented ,126,127.In summary, accumulated evidence from animal studies, epidemiological studies, and clinical trials linked higher adherence to a MedDi to a reduced risk of cognitive decline and dementia. However, the prescription of the MedDi as a preventive or therapeutic measure in AD is hampered by the lack of established levels of individual dietary components, especially of bioactive phytochemicals whose identification, content, and efficacy are well characterized.The health benefits of olive’s major and minor constituents i.e., oleic acid and biophenols are well established in context to cardioprotective and neuroprotective activity. Furthermore, it is also well documented that the most important health-promoting mechanisms induced by a MedDi are: a lipid-lowering effect, protection against oxidative stress, inflammation and platelet aggregation, modification of hormones and growth factors involved in cancer, the inhibition of nutrient sensing pathways by restricting specific amino acids, and gut microbiota-mediated production of metabolites .The need to stick with a uniform source of olive intake (virgin or extra virgin olive oil or fruit) in the MedDi and MIND diet.The measured portion or percentage of olive or olive biophenols are missing in the MedDi and MIND diet. There is a need for a specified and uniform amount of olive inclusion in both diets.However, the cognitive benefits of MedDi and MIND diet are not very well investigated in context to the use of olive or olive biophenols. Following considerations are warranted:Indeed, there is a need to investigate the randomized trial in placebo versus MedDi or MIND diet group which may include or exclude olive in cognitive decline patients.


Parkinson’s disease (PD) is the second most common progressive neurodegenerative disease, affecting 1–2% of people over 65. The classic motor symptoms of PD result from selective degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), resulting in a loss of their long axonal projections to the striatum. Current treatment strategies such as dopamine replacement and deep brain stimulation (DBS) can only minimize the symptoms of nigrostriatal degeneration, not directly replace the lost pathway. Regenerative medicine-based solutions are being aggressively pursued with the goal of restoring dopamine levels in the striatum, with several emerging techniques attempting to reconstruct the entire nigrostriatal pathway—a key goal to recreate feedback pathways to ensure proper dopamine regulation. Although many pharmacological, genetic, and optogenetic treatments are being developed, this article focuses on the evolution of transplant therapies for the treatment of PD, including fetal grafts, cell-based implants, and more recent tissue-engineered constructs. Attention is given to cell/tissue sources, efficacy to date, and future challenges that must be overcome to enable robust translation into clinical use. Emerging regenerative medicine therapies are being developed using neurons derived from autologous stem cells, enabling the construction of patient-specific constructs tailored to their particular extent of degeneration. In the upcoming era of restorative neurosurgery, such constructs may directly replace SNpc neurons, restore axon-based dopaminergic inputs to the striatum, and ameliorate motor deficits. These solutions may provide a transformative and scalable solution to permanently replace lost neuroanatomy and improve the lives of millions of people afflicted by PD. Tissue engineering techniques generally combine aspects of biomaterial scaffolds and cell replacement techniques to create a three-dimensional (3-D) environment to influence cell (native and/or implant) behavior such as phenotype, architecture, migration, and survival. Indeed, biomaterials can provide 3-D structure for host cell infiltration, differentiation, and organization, and may also serve as a means for drug administration . In the central nervous system (CNS), both cell replacement and tissue engineering strategies are being vigorously pursued to facilitate regeneration of native tissue and/or to directly restore lost function based on permanent structural integration.5 Although the primary long-term goal for the treatment of Parkinson’s disease (PD) is to develop a means to arrest the progressive neurodegenerative pathology, the objective of the field of regenerative medicine is to advance restorative treatments to functionally replace and/or reconstruct neuronal circuitry that has succumbed to the disease. This article reviews the historic, current, and emerging regenerative medicine strategies for PD, emphasizing the importance of reconstructing the entire nigrostriatal pathway for consistent and durable recovery of function. Further, we will discuss the challenges associated with translating these solutions into clinical practice.The field of regenerative medicine encompasses the use of cell replacement strategies and tissue engineering to promote regeneration and functional restoration following injury or disease.7 In the United States, 50,000–60,000 cases are diagnosed each year with a prevalence of over one million people.7 As the disease affects 1–2% of people over 65, its prevalence is expected to double in the next 20 years.8 The cost of PD is estimated to be ~$35 billion per year in the United States alone.9 Recent studies have estimated that arresting PD progression would result in net monetary benefits of almost $450,000 per patient, and if PD progression was slowed by 20%, a benefit of over $75,000 per patient would be realized.9PD is a progressive neurodegenerative disease that causes significant morbidity across a prolonged and progressive disease course. PD is characterized by resting tremor, bradykinesia (slowness of movement), rigidity, and other symptoms that decrease quality of life, ultimately leading to significant disability via the inability to control motor function.10 The primary components of Lewy pathology are filaments of α-synuclein, a neuronal protein normally found in synaptic terminals.11 Lewy pathology also occurs outside of the SNpc and striatum, including in the olfactory bulb, medulla, pons, spinal cord, and peripheral autonomic system, and in later stages of PD, the midbrain, amygdala, hippocampus, and several other cortical regions.13 At the time of motor symptom onset, at least 60% of SNpc cells have degenerated.10 There are several theories as to why dopaminergic neurons are preferentially impacted, but many believe that a combination of factors including structure, function, and metabolic needs make dopaminergic neurons particularly vulnerable to the pathophysiology of PD.14Motor symptoms of PD are caused by the selective loss of dopaminergic neurons in the substania nigra pars compacta (SNpc) projecting to the dorsal striatum, which is composed of the caudate and putamen  in multiple interconnected CNS pathways, specifically between the striatum, prefrontal cortex, limbic system, and spinal cord, in the manifestation of many non-motor symptoms of PD.19Non-motor symptoms associated with PD affect nearly the entire body, encompassing a wide range of manifestations including sensory deficits and pain, irregular sleep cycle patterns and dream re-enactment behavior, cognitive decline, depression, anxiety, apathy, psychosis, urinary dysfunction, constipation, and other autonomic dysfunctions.20 The direct pathway serves to increase movement with inhibitory projections from the striatum to the SNpr and GPi. The indirect pathway serves to decrease movement with inhibitory projections to the external segment of the globus pallidus (GPe), which in turn sends inhibitory projections to the STN that sends excitatory projections to the GPi and SNpr.21 These pathways are antagonistic: the direct pathway leads to motor facilitation, and the indirect pathway suppresses unwanted motor movement (motor cessation).As SNpc neurons send long axonal projections to the striatum, the stereotypical neurodegeneration that occurs in PD deprives the striatum of crucial dopaminergic inputs and thereby interrupts important motor feedback pathways. The nigrostriatal pathway is a major component of the basal ganglia, the interconnected structures within the brain involved in motor control Fig. . The maiAs such, a main component of the basal ganglia motor circuit is the nigrostriatal pathway with dopaminergic projections originating from neurons in the SNpc. The projections comprise the highly dense axonal arborization found mostly in the striatum with some collateral arborization in the GPe. Dopamine-releasing axons in the nigrostriatal pathway synapse onto GABAergic medium spiny neurons in the striatum. In the direct pathway, the terminals synapse to D1 receptors, and in the indirect pathway, the terminals synapse to D2 receptors. Dopamine release activates the direct D1-receptor-mediated pathway that ultimately facilitates motor movement. In non-pathologic conditions, the cortex activates the indirect pathway with a smaller contribution of inhibition through SNpc dopamine release on D2-receptor cells in the striatum. In PD, the degeneration of SNpc dopamine neurons shifts the balance of direct and indirect pathways. The net result of both pathways is inhibitory cells in the GPi/SNpr are more active to create a stronger inhibition of the thalamus that culminates in less activation of the motor cortex.25 It is probable that these inputs to the SNpc are vital for highly functioning motor feedback circuits.27The diagram of the indirect and direct pathways Fig.  is meant28 The matrix compartment relays information from sensorimotor input and output neurons to the GPi and SNpr via the direct and indirect pathways. Striosomes, also known as patch compartments, are widely distributed regions within the striatum comprising ~15% of the volume in the striatum. The striosome compartments form an interconnected 3-D labyrinthine network within the striatum. Striosome neurons are thought to modulate the entire dorsal striatum via the inhibitory projections to the dopaminergic neurons in the SNpc .28 Moreover, because of the redundant overlapping arborization, it is estimated that a single striatal neuron is under the influence of 95–194 dopaminergic neurons from the SNpc. Consequently, this highly redundant organization might contribute to the clinical presentation of PD; patients only develop symptoms of Parkinsonism following an extensive loss of dopaminergic neurons (>60%).30 The complexity and nuances of the basal ganglia circuitry and the widespread innervation of a single nigrostriatal axon, let alone all nigrostriatal inputs, warrant thoughtful consideration regarding techniques for adequate circuit reconstruction.Long-projecting axons from SNpc neurons exhibit an exquisite arbor in the striatum to release dopamine at highly dense varicosities along the axon, which are organized nts Fig. . Dopamin31 Surgeons discovered that ablation or lesion therapy could modulate brain activity and ameliorate the symptoms of PD.32 However, the discovery of levodopa (or other dopamine agonists) led to a shift away from surgical therapy to chronic pharmaceutical treatment.33 These agents attempt to compensate for the underproduction of dopamine owing to cell loss. In the early 1990s, Laitinen et al. reintroduced the posteroventral pallidotomy and reported significant improvements for all motor complications of PD.34 During pre-thalamotomy mapping studies, high-frequency electrical stimulation ameliorated PD symptoms, leading to the development of DBS.35 By the mid-1990s, DBS of the GPi and STN was also shown to be effective. Indeed, in appropriately selected patients where levodopa-related complications are disabling, modulation of brain function via DBS of the GPi or STN has been shown to be superior to continued medical management.32 In many cases, pharmacological treatments are the first step in treatment, followed by DBS as a second step. Often, DBS patients continue levodopa or other pharmacological treatments, although often at a lower dose.As far back as the 1940s, the advent of the stereotactic head frame allowed the targeting of subcortical structures with great precision.36 Moreover, any current treatment regimen—pharmaceutical management, DBS, or a combination of the two—often fails to give the same clinical benefit over time. The lack of long-term efficacy is generally attributed to one of two issues: (1) the advent of adverse side effects ,37 or (2) the ongoing degeneration of nigrostriatal neurons decreasing clinical benefits. As such, even with state-of-the-art management, the average duration from minimal disability to confinement to bed or wheelchair is ~13 years.9Although these treatments have led to significant improvements in movement disabilities and improved the quality of life for patients with PD, they are treating symptoms resulting from the loss of dopaminergic input to the striatum rather than resolving the underlying neurodegeneration. In addition, there are often unwanted side effects. For instance, DBS stimulation may detrimentally impact cognition and speech.38 In addition, these treatments would not restore innervation to the striatum, as they would not replace SNpc dopaminergic neurons that had degenerated before the onset of motor symptoms.10 Therefore, a method to diagnose PD pathology before the onset of motor symptoms would be necessary for these pharmacological methods to maintain motor function.39 Although there is growing evidence that non-motor symptoms have a key impact on quality of life, only a few large randomized clinical trials have focused on management of non-motor symptoms.16 Dopaminergic replacement strategies are ineffective for treatment of most of the non-motor symptoms, and are commonly associated with non-motor side effects including somnolence, orthostatic hypotension, visual hallucinations, and nausea.40New pharmaceutical therapies that focus on neuroprotection are being developed, although to date, there are no proven therapies to slow progression of disease.42 (2) enzymes to increase endogenous striatal dopamine synthesis or conversion of l-dopa to dopamine,43 and (3) neurturin—a ligand similar to glial cell line-derived neurotrophic factor (GDNF) that has been shown to enhance survival and outgrowth of dopaminergic neurons.45 To date, out of these gene therapy clinical trials, only the vector with the gene encoding for increased production of glutamic acid decarboxylase has demonstrated beneficial motor function.46 Therefore, although gene therapy might be a promising strategy, development of an effective treatment requires more research.Gene therapies to relieve PD symptoms have recently reached clinical trials evaluating the efficacy and safety of delivering vectors with genes encoding for overexpression of (1) glutamic acid decarboxylase in the subthalamic nuclei to increase the GABA basal tone,48 Since then, many studies have examined the potential of cell transplant-based treatments to restore dopamine in the striatum and ameliorate motor deficits of PD.49 As of 2004, over 350 patients had received cell replacement therapies, and in many cases, patients were tapered off drugs and witnessed a decrease in motor symptoms.50 Successful fetal tissue grafts have survived over two decades in some patients despite ongoing PD pathology.51 Indeed, long-term survival of grafted dopaminergic neurons with extensive putamenal dopaminergic innervation was reported in PD patients at postmortem.52 In addition, 18F-fluorodopa imaging demonstrated robust dopaminergic uptake within the striatum. Clinical trials have indicated beneficial results from cellular grafts when methods sustain a novel population of at least 80,000 dopaminergic neurons, although, in a recent study, motor benefits were observed with only 40,000 neurons surviving after 24 years.53Prior to the 1970s, it was widely believed that restorative treatments for neurodegenerative diseases of the CNS were not possible. However, two articles published in the same year demonstrated that fetal mesencephalic grafts rich in dopaminergic neurons could ameliorate Parkinsonism symptoms in rodent models.50 Moreover, grafts were sourced from fetal tissue, and the variability of source tissue likely contributed to some trials showing limited to no efficacy. In addition, multiple fetal donors  must be pooled to source a sufficient number of cells for one patient, which may contribute to the heterogeneity of outcomes and likely indicates a lack of material for widespread clinical usage. This is exacerbated by ethical concerns associated with fetal tissue that make access difficult in some instances, thereby limiting procedures. Also, trials have had varied preparation, storage, and immunosuppression regimes that may have affected the health of the grafts, again creating a source of variability and likely affecting efficacy. Thus, fetal grafts have inherent issues with consistency and requirements for immunosuppression.54Although inconsistent, clinical improvements, such as decreased motor symptoms, were observed in some cases; however, widespread clinical adoption of tissues grafts has been stymied by several notable limitations. A subset of patients had adverse side effects or no significant improvements.27 The tradeoffs between different options for placement of cells/grafts is covered in more depth below, but complete function of the motor control system relies on well-controlled feedback loops that fine-tune dopamine levels. Although significant progress has been made, fetal tissue grafts do not appear to present a viable or sustainable strategy to repair the nigrostriatal pathway and replicate both the outputs and inputs for dopamine regulation to the striatum from the SNpc. However, success of fetal grafts in PD patients demonstrates the potential for dopaminergic neuron transplants to provide long-term benefits, provided there are improvements in cell/tissue supply, consistency, and ideally, actual recreation of the nigrostriatal pathway.Another concern is that the majority of these studies involved cellular graft implantations in the striatum, not the SNpc—the location of dopaminergic neurons naturally providing input to the striatum. Whereas cells implanted into the striatum may create new “factories” for dopamine, these cells do not receive their normal inputs to control their activity, potentially resulting in dysregulated dopamine release and related side effects. As discussed previously, the SNpc receives a wide range of inputs, and these inputs are thought to be important for restoring the full motor circuit.55 Degeneration and disconnection of these axonal pathways as well as localized neuronal degeneration frequently occur in many CNS disorders, including traumatic injury, stroke, PD, and others.56 Unfortunately, functional regeneration rarely occurs in the CNS and neurogenesis is restricted to a few distinct domains, such as the subventricular zone and the dentate gyrus of the hippocampal formation. In the majority of the CNS, natural regeneration of long axon pathways does not occur, mainly owing to endogenous inhibition of axon growth, absence of directed guidance to far distant targets, and loss of intrinsic capacity for long outgrowth in mature neurons. The lack of neurogenesis and correctly targeted axonal regrowth are key limitations in endogenous CNS regeneration and repair, thereby resulting in diminished recovery and continued functional deficits.The extraordinary computational capabilities of the human brain rely on vast axonal connections spanning long distances that form sophisticated neural circuits and enable profound parallel processing, often referred to as the connectome.The field of regenerative medicine is pursuing novel approaches to develop cellular and tissue constructs to facilitate regeneration and/or restore function following injury, aging, or disease. These techniques are being applied to address the limitations in repair and regeneration in the CNS, and in the particular case of PD, aim to build upon the benefits of cellular grafts seen in human patients. Using novel biomaterials, tissue culture techniques, and knowledge from previous experiments, the concerns associated with cellular grafts, such as consistency of cells, need for immunosuppression, placement of cells, and source of tissue (supply and ethics), can be addressed.57Long-term survival of dopaminergic neurons into the host striatum with robust fiber outgrowth in the adult rodent brain.59Afferent and efferent synaptic integration with the host rodent brain  following implant of dopaminergic neurons into a cortical cavity.60Adequate dopamine release in a controlled fashion following intrastriatal grafts in adult rats.61Amelioration of motor deficits in rodent models of PD following intracerebral grafting of dopaminergic neurons.The pioneering work from the 1970s and 1980s demonstrated the potential for allografted fetal tissue to improve striatal dopamine levels in some patients with PD. These studies also paved the way to design criteria for cell-based therapies prior to clinical trials including:m in the striatum for functional benefits to be seen.63 Based on human fetal graft studies, these numbers translate to 40,000–80,000 healthy dopaminergic neurons, unidirectional axonal architecture of ≥3–5 cm in length, and dopamine release of ≥7 ng/mg of tissue for nigrostriatal pathway restoration in humans , although the exact rates of attrition would require further research.To actually recapitulate the native anatomy of the nigrostriatal pathway would call for grafts, originating in the substantia nigra and terminating in the striatum. Initially, for proof-of-concept studies in rats, the literature suggests a requirement of ≥1200 morphologically healthy dopaminergic neurons with a unidirectional axonal architecture and ≥5–6 mm in length that result in dopamine release in the range of 50–100 n65 Cell replacement strategies use endogenous or exogenous cell sources, including stem cells, and are similar in concept to the tissue graft techniques but generally involve cell dissociation and/or expansion.4 The usage of stem cells allows for therapies that can include secretion of neuroprotective factors as well as self-renewing cells that give rise to additional neurons or other cell types.3 To restore the entire nigrostriatal pathway with appropriate inputs and outputs remains a considerable challenge, as implantation of only dissociated cells cannot restore the key anatomic features of damaged pathways—notably long axon tracts projecting to proper anatomical structures. Key considerations include (1) cell source, (2) location of the transplant, and (3) strategy to facilitate sufficient and appropriate axonal outgrowth to the striatum , sympathetic ganglia, carotid body cells, retinal pigmentary epithelial cells linked to microcarriers, and porcine ventral mesencephalon tissue.66 However, the most-effective transplantation strategy has been with human fetal mesencephalic tissue. Preclinical rodent studies have shown that transplanted dopaminergic neurons from human fetal mesencephalic grafts can innervate the dopamine-deprived striatum, receive inputs from host neurons, and diminish rotational behavioral symptoms.68 These developments led to clinical trials using human ventral mesencephalic tissue that demonstrated survival of grafted dopaminergic neurons up to 24 years after transplantation, integration with the brain circuitry, and improvement in motor symptoms.72 Despite these promising findings, clinical improvement has varied across different trials likely owing to inadequate standardization of patient enrollment, tissue harvest, graft location, surgical implantation, and immunosuppression.73 Although the early preclinical experience and clinical successes using fetal grafts has been crucial to establish the potential for a cell transplantation strategy in PD, further developments have been challenging owing to ethical concerns and limited tissue availability.For cell replacement strategies that aim to restore the entire nigrostriatal pathway, the first key consideration is the cell source of the graft material. The development of the 6-hydroxydopamine (6-OHDA)-lesioned rodent model of PD, which selectively and irreversibly degenerates dopaminergic neurons in the lesioned nigrostriatal pathway, has led to various approaches to determine whether restoration of dopaminergic tone could ameliorate Parkinsonian motor symptoms.49 However, these grafts did not demonstrate the same potential to restore the dopamine levels in the striatum and ameliorate behavioral deficits as the fetal grafts in preclinical studies; therefore, it was not surprising that clinical trials evaluating non-neuronal cell sources did not result in significant motor improvements compared with the relatively successful human fetal ventral mesencephalic grafts.66 These experiments illustrate the importance of validating potential grafting strategies by demonstrating robust cell survival, host integration, and dopamine release in animal models before clinical trials.Alternative, non-fetal cell source candidates have been developed to avoid these concerns with the goal of attaining the same outcomes as the early fetal transplantation studies. Non-neuronal, catecholamine-producing cell sources, such as AMCs and retinal pigmentary epithelial cells, have been considered as a potential alternative to fetal grafts by producing and secreting dopamine in the striatum, thereby restoring motor function.74 Indeed, various approaches have been developed to generate dopaminergic-like neurons from alternative cell sources, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), expanded neural precursor cells (NPCs), mesenchymal stem cells (MSC), and direct neuronal reprogramming.49Building on the knowledge gained from transplantation studies using fetal grafts or non-neuronal cell types, it has become clear that a successful grafting strategy likely requires use of a cell source capable of generating authentic dopaminergic neurons that resemble those found in the SNpc and can re-establish lost striatal innervation.75 Recent studies appear to have resolved these issues with improved differentiation protocols and cell culture techniques.77 However, despite these advancements, widespread development of ESC-derived dopaminergic cell therapies has remained limited for the same inherent reasons as fetal transplantations; lack of tissue availability and ethical concerns.Pluripotent stem cells are a promising alternative to fetal ventral mesencephalon grafts owing to their capability to provide a potentially unlimited source of any cell type. Advancements in the fields of stem cell neurobiology have led to the generation of two different types of pluripotent stem cells: (1) ESCs derived from the inner cell mass of early-stage embryos and (2) iPSCs derived from de-differentiated somatic cells. Early studies demonstrated that ESC-derived dopaminergic neurons were capable of surviving in an adult rodent brain, however, incomplete differentiation led to increased risk of tumor formation and neural overgrowth.65 Although early preclinical data did not demonstrate similar outgrowth or functional restoration in rodent and non-human primate (NHP) models of PD compared with fetal grafts, advancements in developmental neurobiology led to the discovery that dopaminergic neurons are derived from floor plate cells, not neuroepithelial progenitors like every other neuron in the brain.78 This new finding enabled the development of novel molecular differentiation techniques for generating more “authentic-like” dopaminergic neurons resembling those found in the SNpc.79 Furthermore, human ESC- and iPSC-derived dopaminergic neurons generated with these refined differentiation protocols seem to have solved the earlier issues with tumorgenicity, and enabled long-distance targeted striatal innervation.80 Although iPSC-derived neurons have been shown to survive transplantation, variable amounts of neurite outgrowth has been reported, which is likely responsible for inconsistent functional restoration.81The discovery of somatic cell de-differentiation has led to increased interest in iPSCs as a promising alternative cell source for future PD therapies owing to the potential of generating patient-specific iPSC-derived dopaminergic neurons.82 Unlike ESCs and iPSCs, NSCs have limited differentiation capability and are fated towards a brain cell phenotype, reducing the likelihood for tumor growth.83 NSCs have been shown to be an effective neural repair strategy in preclinical models of injury owing to their inherent ability to provide neuroprotection, decrease immunoreactivity, and secrete neurotrophic factors, such as glial-derived neurotrophic factor and brain-derived neurotrophic factor.84 Previous work has also shown that NSCs can differentiate into dopaminergic-like cells, survive transplantation, integrate with the host architecture, and improve functional recovery.85 Another promising direction is the development of an autologous cell source for transplantation using patient-specific iPSC-derived NSCs.82 Although intriguing, expansion of NSCs has remained challenging and likely requires further refinement for clinical trials. Moreover, further safety and efficacy preclinical studies are necessary to reduce the likelihood for graft and/or differentiation failure.86Neural stem cells (NSCs) expanded from ESCs and iPSCs have been investigated as an alternative source for neural cell replacement treatment for PD owing to their multipotent and self-renewing properties.87 Elevated neuroinflammatory responses have been implicated in neurodegenerative diseases, such as PD and Alzheimer’s disease.88 These findings have led to increased interest in MSCs as a potential cell source candidate owing to their immunomodulatory effect.89 Indeed, MSCs have demonstrated the capability to differentiate into astrocyte-like cells that secrete neurotrophic factors and ameliorate the motor deficits in a rodent model of PD.90 In addition to immunomodulation, MSCs or MSCs differentiated into a neuron-like phenotype have been reported to migrate across the corpus callosum to a 6-OHDA-lesioned SNpc and striatum when initially transplanted in the contralateral hemisphere.91 However, it is unclear whether the cells are attracted to the lesion or the acute inflammation at the injection site.92MSCs are multipotent, non-hematopoietic stem cells capable of self-renewal that typically arise from bone marrow and differentiate into adipocytes, osteoblasts, and chondroblasts, and have been reported to elicit an anti-inflammatory effect.93 This strategy could theoretically provide autologous dopaminergic neurons reprogrammed from a patient’s own somatic cells, alleviating the ethical and limited tissue availability with fetal cells and minimizing complications associated with allogenic transplants. Although reprogramming has historically used viral vectors, recent advancements in microRNA technology has led to the development of alternative transcription factor delivery methods that have been reported to increase the conversion efficiency in vitro and in vivo from somatic cells into functional dopaminergic neurons and ameliorate motor symptoms in a model of PD.94Direct neuronal reprogramming has recently become an interesting cell source candidate due to the potential for generating dopaminergic neurons from somatic cells by adding transcription factors for cell lineage conversion.53 As noted above, cells implanted into the striatum may create new “factories” for dopamine, but the cells do not receive the full suite of normal inputs that regulate dopaminergic production and delivery in the striatum. However, as striatal innervation is a critical requirement for any potential cell source, “short-circuiting” the nigrostriatal pathway is useful for developing effective stem cell therapies.The next key consideration for development of a successful regenerative strategy is the location of the graft. Based on the success of the early studies, several graft locations have been investigated, such as (1) in the striatum, (2) in the SNPc, or (3) along the entire length of the nigrostriatal pathway. Many cell-based transplantation strategies have primarily focused on using intrastriatal dopaminergic grafts to restore the physiological levels of dopamine within the striatum.99 Despite these improvements, inconsistent functional recovery in animal models and human studies has been reported, likely owing to the variations in graft composition, low dopaminergic neuron survival, poor neurite extension, minimal graft volume, differing immunosuppressant regimes, and/or the inhibitory microenvironment.101Intrastriatal transplantation of dopaminergic neurons in a NHP model of Parkinsonism has been reported to demonstrate extensive neurite outgrowth in the striatum and increased dopamine uptake near the graft, resulting in significant amelioration of the cardinal motor symptoms of PD.103 Despite the development of various approaches to increase neurite outgrowth for targeted reinnervation, these strategies might result in the innervation of aberrant targets and/or only attain sub-centimeter scale growth, thus insufficient to reach what would be required in the human brain .106 For example, a recent study reported that transplanation of human ventral mesencephalic patterened progenitor cells established extensive innervation towards appropriate forebrain targets and reduction of behavioral deficits in rodents.80 Although these findings are promising, off target growth is difficult to predict and/or prevent with increased distance between the graft and intended target, especially in larger animal models and humans.Early intranigral transplantation studies largely failed because long-distance axonal regrowth to the correct target(s) within the CNS is limited.108 Although extremely promising, the scalability of this strategy to centimeter scale axon paths will be paramount, as clinical translation requires consistent and long-distance outgrowth with precise targeted reinnervation to restore the full motor circuit.However, a potentially transformative approach is being advanced to create axon guidance paths via factors secreted by host cells transfected with precise spatial precision (via stereotaxic microinjection) to overexpress growth factors, creating chemotaxic gradients to drive long-distance, targeted axonal outgrowth from endogenous and/or transplanted cells in the CNS.109 Although this strategy appeared promising, in primate studies, the attraction was greatly diminished with increasing distance between the striatal “stepping stone” and the SNpc graft and the preferential attraction to the striatal “stepping stone” prevented outgrowth beyond the graft to the intended host striatum.105 Other “bridging” strategies have been proposed, such as fetal striatal tissue, fibroblast growth factor-4 secreting schwannoma cells, GDNF-secreting Schwann cells, and kidney tissue.101 Bridging the nigrostriatal pathway with growth factors or cells alters the inhibitory microenvironment and provides a supportive conduit for regenerating axons that effectively guides immature axons from the SNpc to the DA-depleted striatum.To address the challenging regenerative distance, early studies reported using embryonic striatal tissue as a “stepping stone” to effectively enable targeted outgrowth from axons grafted in the SNpc toward the graft, and ultimately the host striatum.110 Although kainate administration was shown to increase striatal dopamine release and reduced motor behavioral deficits, kainate is an excitatory neurotoxin, thus significantly limiting its potential regenerative strategy for patients with PD.111 Following this approach, a similar strategy has been investigated using GDNF as an attractant for guiding transplanted axons from the SNpc to the striatum. Administering a track of GDNF from the fetal graft in the SNpc to striatum facilitated outgrowth and improved rotational behavior in lesioned rats.112 This strategy was adapted in a NHP model of PD that utilized viral-vector-mediated overexpresion of GDNF in the striatum, enabling outgrowth from a fetal graft in the SNpc toward the striatum. In this study, only a few grafted dopaminergic axons innervated the striatum, however, these findings demonstrated neurotrophic supplementation could enable targeted long-distance axonal outgrowth.106Alternative strategies have been explored to improve targeted striatal reinnervation from fetal mesencephalic tissue grafted in the substantia nigra, such as injecting kainate between the midbrain and striatum to create a trophic environment for axonal outgrowth.113 Although rodent transplantation studies have reported some improvement in motor functionality, consistent recovery in humans would likely require greater striatal reinnervation.Although the ideal location for cell transplantation is likely the SNpc, successful striatal innervation in humans and NHPs requires significantly more-targeted axonal outgrowth to achieve a regenerative distance on the order of centimeters.115 Extranigral transplants may provide trophic support to otherwise denervated host neurons, preventing widespread degeneration from second-order neuron neurotransmitter depletion, thereby increasing the number of potential targets for regenerating axons from dopaminergic neurons transplanted in the SNpc. Reinnervating the extranigral regions with dopaminergic neurons extending from the SNpc that receive proper local inputs from other cell populations likely closes the motor feedback loop, restoring the ability to generate complex movement patterns.115 In a small open-label pilot study, simultaneous nigral and striatal transplants resulted in significant long-term clinical improvement of motor function in patients.118 Although intriguing as a regenerative strategy for PD, further research is necessary to understand the exact mechanism behind the impressive functional recovery and graft survival in rodents and humans.A promising strategy for reconstruction of the nigrostriatal pathway utilizing multiple transplantations along the basal ganglia circuitry has been reported to enable complex movement patterns in models of PD.65 However, inconsistent functional recovery has been reported across multiple clinical trials: although patients experienced symptomatic relief ranging from nonexistent to significant improvement, ~15% of patients developed off-phase dyskinesia.119 GID is an adverse effect likely caused by runaway dopamine release and excessive outgrowth from the embryonic graft.100 Unlike the typical presentation of dyskinesias in patients with PD caused by chronic l-DOPA administration, GID-induced abnormal motor movements do not resolve with reduction and/or cessation of l-DOPA treatment.120 Clinical findings from these studies revealed an association between the development of GID and the preoperative presentation of l-DOPA-induced dyskinesia (LID); specifically there was a high incidence in patients who reported severe motor fluctuations prior to transplantation.100Another important consideration for development of cell-based strategy for PD is the potential for graft-induced dyskinesia (GID). Clinical trials with human embryonic tissue have demonstrated that dopaminergic neurons can reinnervate the striatum, release dopamine, and functionally integrate with the host brain.121 This hypothesis originated from the clinical observation that GIDs were only reported in patients who either did not receive immunosuppression or only short-term, low-dose immunosuppression after fetal engraftment.122 Other studies have suggested the presence of serotonergic neurons in the graft might play a critical role in the expression of GIDs.123 The mechanism is still poorly understood, but it is likely that serotonergic neurons were transplanted in the graft, which led to a dysregulation of dopamine release. Indeed, in a clinical study without any patients who developed GID, the postmortem analysis revealed a 2:1 ratio of dopaminergic to serotonergic neurons in the graft regions.118 Future clinical trials for fetal engraftment might need to include immunosuppression and avoid patients with established LID to increase the likelihood for successful transplantation, a strategy that has been adopted by various ongoing and upcoming clinical trials.124It has been suggested that biological rejection and persistent low-grade inflammatory response might compromise the tissue graft, leading to neurotransmitter dysregulation and ultimately development of dyskinesias.124As described elsewhere in this review, early human fetal ventral mesencephalic transplantation studies demonstrated graft survival, reinnervation of the striatum, and functional restoration. However, it has been argued that the development of adverse events, such as GIDs, as well as failure of two double-blinded, placebo-controlled studies to show differences between patients transplanted with human fetal ventral mesencephalic tissue and control-treated patients led to diminished interest in cellular therapies for treatment of PD. In the last decade, thorough reanalysis of these clinical observations provided the basis for well-defined criteria in future clinical trials. This approach led to the establishment of a European Union-funded multi-center team (TRANSEURO) for a human ventral mesencephalic tissue transplantation study that has focused on the implementation of well-defined criteria, such as patient selection; tissue dissection, preparation, and storage; grafting technique; immunosuppression protocol; and experimental design. However, the TRANSEURO study was unable to complete enrollment owing to scarcity of human fetal tissue supply, leading to only 20 of the planned 90 patients undergoing transplantation.124 Although human ESC-derived dopaminergic neurons likely have the capability to innervate the putamen, some teams have chosen to use HLA-matched, autologous iPSCs to avoid the need for immunosuppression.Human pluripotent stem cells (hPSCs) derived from ESC and iPSCs are an attractive alternative cell source that avoids the ethical and practical complications with fetal tissue. In 2014, different teams from academic institutions across Europe, the United States, and Japan began a new global initiative, GForce-PD, that aims to advance hPSC-derived dopaminergic neurons to first-in-human-clinical trials.125 Incorporated cell types may include primary, stem, differentiated, genetically engineered, autologous, allogeneic, or heterologous cells. Biomaterials utilized within the constructs often provide structure, protect cells (implanted and/or host), and produce an environment in which cells can adhere, migrate, differentiate, and signal to each other and to the host. Tissue-engineered constructs may possess a defined architecture that not only facilitates integration of the transplanted cells/processes with native tissue, but also maintains their desired organization.126 This architecture may be precisely engineered to match the structure and properties of the tissue for integration: to provide directionality for infiltration from implant to host, vice-versa, or both.129 Biomaterials may be synthesized to promote desired cellular organizations or mechanical properties  that are directionally dependent (anisotropic). Likewise, gradients of factors, such as growth factors and signaling molecules, may be used within tissue-engineered scaffolds to generate anisotropic features. The permissive microenvironment or proper matching of mechanical features created by some biomaterial hydrogels may also improve graft survival by influencing inflammatory reactions and minimizing the foreign body response.130The objective of the field of neural tissue engineering is to utilize biomaterial scaffolds and cell-based strategies in combination to augment endogenous regeneration and/or to provide direct replacement of neural cells and circuitry.132 These strategies most commonly involve biomaterial or cellular scaffolds that provide growth-promoting cues or reduce inhibitory factors.134 Although notable, on their own, biomaterial scaffolds do not address the degeneration of source neuronal population(s). Therefore, most scaffold approaches in PD are aimed at promoting a hospitable environment for implanted cells rather than outgrowth from endogenous neurons.Previous research has indicated that long-distance axonal outgrowth rarely occurs in the mature nervous system, and therefore, to restore the full motor circuitry, alternative methods are needed to repair the long-distance circuits disrupted in PD. Biomaterial scaffolds are an alternative to facilitate long-distance axonal outgrowth as they can aid in reconstructing the nigrostriatal pathway by coaxing long-distance axonal outgrowth from the SNpc to appropriate targets in the striatum (either by endogenous or exogenous cells). Studies attempting to restore long-distance axonal connections typically aimed to create a permissive environment for axonal outgrowth, and/or augment the intrinsic capacity of axons to regenerate.135 In particular, our group is pursuing the creation of tissue-engineered “living scaffolds” for several applications, including to provide regenerative pathways for axonal guidance or neuronal migration, and also to directly replace neurons and axon tracts in order to reconstruct degenerated neural circuits.137 Most relevant to PD, tailored constructs may be developed to structurally and functionally emulate the nigrostriatal pathway, toward the goal of replacing dopaminergic neurons and their long axon tracts. This strategy is premised on the plasticity of endogenous as well as tissue-engineered neural networks, whereby neurons intrinsically have the ability to sense and respond to local activity.139 It has been shown that transplanted neurons are capable of receiving synaptic input from local networks as well as propagating action potentials.140 Once the appropriate synapses are established, preformed anatomically inspired constructs could act as functional relays to transmit signals between populations of previously disconnected cells.An emerging strategy in neural tissue engineering involves the development and application of so-called “living scaffolds”, which are defined as constructs with a preformed, often anisotropic architecture, consisting of living neural cells within a 3-D biomaterial matrix.As such, we are pursuing a novel regenerative medicine solution whereby custom-built microtissue-engineered neural networks (TENNs) are transplanted to physically replace SNpc neurons and their long-distance axonal connections from the SNpc to the striatum Fig. . Micro-T141 Cell source and construct length were evaluated and evoked dopamine release was confirmed in vitro. These constructs were stereotactically implanted en masse to mimic the nigrostriatal pathway in a rodent model. At 1-month post implant, histological data revealed neuronal survival and maintenance of axonal architecture. Further studies investigating the synaptic integration and amelioration of motor deficits in a rodent model of PD are ongoing. If successful, dopaminergic micro-TENNs will be the first strategy to facilitate nervous system repair by simultaneously providing neuronal replacement and physically re-creating long-distance axonal connections in the brain. In addition, we are also establishing the future potential of personalized micro-TENNs—built using neurons derived from a patient’s own stem cells—as a potential solution to restore the dopaminergic nigrostriatal pathway and ameliorate motor symptoms without the need for immunosuppression.Therefore, micro-TENNs may have the capacity to restore the nigrostriatal circuitry lost to PD by replacing the dopaminergic neurons in the SNpc, enabling synaptic integration with inputs to the SNpc, and restoring axonal terminals in the striatum. Importantly, pathway reconstruction approaches such as the micro-TENNs have the promise to restore the aspects of the motor feedback path, a feat not possible with methods such as pharmacological dopamine replacement therapy, DBS, or tissue grafts in the striatum. As such, we are currently assessing the ability of dopaminergic micro-TENNs to replace SNpc neurons and restore dopaminergic inputs to the striatum in rat models. In recent proof-a-concept study, microtissue-engineered neural constructs containing a population of dopaminergic neurons on one end and long axonal projections grown through a hydrogel column were fabricated, mimicking the entire nigrostriatal pathway.19 To date, micro-TENNs have been fabricated using dorsal root ganglia neurons, cerebral cortical neurons , embryonic rodent ventral mesencephalic dopaminergic neurons, and human ESC-derived dopaminergic neurons.144 As our understanding of PD pathophysiology expands, it is possible that multiple micro-TENNs could be transplanted to reconnect different damaged regions, comprised of alternative neuronal phenotypes, such as noradrenergic, serotoninergic, and cholinergic cell types. However, it is difficult to predict how transplant therapies, including either transplanted cells or pathways, such as micro-TENNs, could be used to recapitulate widely dispersed innervation of cerebral cortex from cholinergic, serotonergic, or noradrenergic brainstem nuclei.As discussed in previous sections, treatment of many non-motor symptoms of PD has remained challenging owing to the widespread dysregulation of the circuitry connecting basal ganglia, prefrontal cortex, and limbic systems.145 This approach generally alters the gene expression of astrocytes—an abundant glial cell type in the area of degeneration—to convert them into neurons as a means for localized neuronal replacement. Direct reprogramming of striatal astrocytes to dopaminergic neurons in a rodent model of PD has been reported to improve dopamine tone and reduce motor deficits.94 Although extremely promising, conversion of striatal astrocytes would not reproduce the nigrostriatal circuit, and conversion of nigral astrocytes would require coupling with an axonal pathfinding strategy as described above to elicit target connections to the striatum.Promising recent work has utilized viral vectors to re-program endogenous non-neuronal cells to become neurons.146 However, cell-based or tissue-engineered constructs  may eventually succumb to the underlying pathology and degenerate similar to native tissue. Indeed, recent studies have hypothesized that dysfunction of iron metabolism in the substantia nigra might be implicated in the degeneration of dopaminergic neurons and ultimately the progression of PD.147 Although the susceptibility of transplanted cellular constructs to the typical progression of PD pathology is still unclear, dopaminergic neurons from tissue grafts can survive up to 24 years in vivo despite ongoing degeneration of the native dopaminergic system.52 However, in some patients with long-term survival of fetal mesencephalic grafts, α-synuclein-positive Lewy bodies were found in 1–5% of transplanted dopaminergic neurons at 12–22 years and 12% in one patient at 24 years post transplantation. Thus, the development of PD-resistant neurons might be necessary to overcome this vulnerability, improve the construct lifespan, and prevent α-synuclein aggregation by augmenting the genes implicated in autophagy, α-synuclein clearance, or decreased iron accumulation in the substantia nigra.149 Furthermore, genetically enhanced dopaminergic neurons engineered to increase dopamine production and/or release could minimize the number of surviving cells necessary to restore dopamine levels in the striatum, thus maximizing the likelihood for successful innervation of the striatum, and potentially halting the evolution of PD symptoms.Cellular engineering strategies may also be advantageous for cell transplants, with such genetic manipulation being employed in vitro before in vivo implantation. For instance, specific genetic alterations may be beneficial to improve survival, integration, and efficacy. One type of modification could be to limit immunogenicity and thereby improve the survival and integration of the cells.38 In addition, dopaminergic input to the striatum requires continuous modulation and feedback from other neural structures via SNpc inputs to alleviate potential runaway dopamine excess and dystonia, a potential side effect from cell transplants into the striatum.The classic motor symptoms of PD result from selective degeneration of dopaminergic neurons in the SNpc, and subsequently the disruption of key motor control circuits. This circuit disruption results from loss of the finely tuned dopamine delivery to the striatum via long-distance axonal projections. Current treatments for PD, including the use of dopamine replacement strategies and DBS, are aimed at treating symptoms rather than the underlying neurodegeneration. Newer treatments have focused on neuroprotection, but to date, no therapies have been shown to clearly slow progression of neuronal degeneration and hence motor symptoms.To address these gaps in treatment, emerging regenerative medicine solutions are being pursued to replace lost neurons and axonal circuitry in PD. Indeed, the ultimate goal is to replace the nigrostriatal pathway—dopaminergic neurons in the SNpc and their axonal projections to the striatum—thereby allowing implanted cells/tissue to be subject to the normal cellular regulation that dopaminergic SNpc neurons are subject to in order to “close the loop” and restore this crucial circuit for motor control feedback. Reconstructing the pathway from the SNpc to the striatum is the most likely means to provide dopaminergic inputs that can be tuned and controlled by natural feedback mechanisms present within the brain. On this front, our research team is pursuing a tissue engineering-based strategy to accomplish this goal, as we are developing preformed, implantable column-like micro-constructs that mimic the architecture of the nigrostriatal pathway: a discrete population of dopaminergic neurons with unidirectional, long-projecting axonal tracts. The method may be uniquely suited to simultaneously replace lost dopaminergic neurons within the SNpc and recapitulate the full nigrostriatal pathway—spanning several centimeters—to provide naturally regulated dopaminergic inputs to the striatum. Unlike DBS, which attempts to disrupt pathologic activity in the indirect pathway, our preformed micro-constructs are themselves an auxiliary pathway. In general, such precisely engineered constructs may be highly controllable, where the number of neurons and generation of dopamine can be known prior to implantation, thus potentially ameliorating the inconsistency historically seen in fetal tissue grafts.82 Combined with tissue engineering methodology, such autologous stem cell sources may enable the construction of patient-specific constructs tailored to their particular extent of degeneration. Such next-generation constructs may provide a transformative and scalable solution to directly replace SNpc neurons, restore axon-based dopaminergic levels in the striatum, and thereby alleviating the cause of motor symptoms in PD. However, similar to the current limitations to treatments for PD, potential benefits for cell-based neurorestorative strategies would likely be dependent on the duration of disease and thus degree of neurodegeneration. In the coming era of restorative neurosurgery, emerging regenerative medicine therapies may revolutionize PD treatment by permanently reconstructing lost neuroanatomy and markedly improve outcomes for millions of patients worldwide afflicted by PD.In order to supply the requisite number of healthy dopaminergic neurons needed for functional improvement in humans , cell sources will need to be significantly scaled up prior to clinical trials. Stem cells and other expandable, self-renewing cell sources—whether allogeneic or autologous—are promising to address these needs. Such methods, while presenting challenges in controlling differentiation, heterogeneity, and maturity, among others, will avoid the limitations of fetal tissue grafts, specifically ethical considerations and dearth of fetal source material. In particular, using emerging iPSC methodology, a patient’s own cells could be efficiently differentiated into dopaminergic neurons, thereby eliminating the need for immunosuppression.


Aspergillus and Cryptococcus and are highly relevant for developing countries. An Aspergillus galactomannan lateral flow assay is also now available. Real-time PCR remains difficult to standardize and is not widespread in countries with limited resources. Isothermal and conventional PCR-based amplification assays may be alternative solutions. The combination of real-time PCR and serological assays can significantly increase diagnostic efficiency. However, this approach is too expensive for medical institutions in developing countries. Further advances in next-generation sequencing and other innovative technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tools may lead to efficient, alternate methods that can be used in point-of-care assays, which may supplement or replace some of the current technologies and improve the diagnostics of fungal infections in developing countries.Extensive advances in technology offer a vast variety of diagnostic methods that save time and costs, but identification of fungal species causing human infections remains challenging in developing countries. Since the echinocandins, antifungals widely used to treat invasive mycoses, are still unavailable in developing countries where a considerable number of problematic fungal species are present, rapid and reliable identification is of paramount importance. Unaffordability, large footprints, lack of skilled personnel, and high costs associated with maintenance and infrastructure are the main factors precluding the establishment of high-precision technologies that can replace inexpensive yet time-consuming and inaccurate phenotypic methods. In addition, point-of-care lateral flow assay tests are available for the diagnosis of  Cryptococcus infections in sub-Saharan countries ), and RNase H [didiasis ,101. Thedidiasis  and to hdidiasis ,103. A cdidiasis ,105. ComSince its discovery and subsequent commercialization, PCR has gained vast popularity and has become an indispensable identification tool with a plethora of applications in medical microbiology. Owing to its affordability and reproducibility, PCR is recommended by the WHO as a reliable diagnostic tool to be used in developing countries . Recent Conventional PCR-based assays utilize two primers to amplify targets in singleplex or multiplex reactions with subsequent visualization by gel electrophoresis. Variations in the length and banding patterns of PCR products along with restriction digestion profiles enable differentiation among various target fungal species. Conventional PCR-based assays are categorized as simple PCR, restriction fragment length polymorphism (RFLP)-PCR, and nested-PCR.Candida species [Histoplasma capsulatum [Mucorales [Aspergillus [This method is a combination of PCR and restriction digestion. PCR-amplified products obtained with sets of universal primers are checked by gel electrophoresis and digested by restriction endonucleases. The resulting specific fragmentation patterns visualized by gel electrophoresis are used for identification of various fungal species. PCR-RFLP is applied to analyze DNA obtained from both pure cultures and clinical samples and can detect a range of pathogenic fungi, including  species , Histoplpsulatum , Mucoralucorales ,112, andergillus . Howeverergillus ). MoreovCandida species [Aspergillus [Simple PCR assays are relatively easier and more straightforward than PCR-RFLP assays and differentiation of target species mainly relies on specific primers and the length of the amplified fragments. Ease of primer design, straightforward processing, and low-cost account for the wide use of this assay in the identification of a broad range of clinically relevant fungal species, including the most common pathogenic yeasts , cryptic species , onychom species , and Aspergillus ,118. Mulergillus . Althougergillus ,121 or cergillus ,118. In Aspergillus and Mucor in paraffin wax-embedded tissues [Candida species in blood samples [Mucorales species [Candida species [The method is based on two consecutive PCR rounds. Fragments are amplified with outer primers in the first round and used as a template for inner primers in the second round. This strategy (two primer sets and 60–70 rounds of PCR runs) addresses the sensitivity and specificity issues of PCR-RFLP and simple PCR and is suitable for direct detection of low numbers of fungal cells in clinical samples, thus enabling timely administration of an appropriate antifungal . Nested  tissues  and Cand samples ,125. Mor species . However species ,125 and  species  by neste species , and abiEase of application, low cost, and high specificity for the chosen targets demonstrated by conventional PCR, together with the increasing number of sequenced fungal genomes, which facilitates the design of specific primers, make this assay an important stand-alone diagnostic method in some parts of the world. However, the requirements for quantitative analysis and monitoring of amplification in real-time have led to the development of quantitative real-time PCR. Utilization of short amplicons (100–200 bp) resulting in time reduction, easy multiplexing due to availability of a wide range of reporters, recording and storage of quantitative data, and the high-throughput platform are the other advantages of qRT-PCR over conventional PCR.ACT), elongation factor 1 alpha (TEF1α), RNA-polymerase II (RPB2) [Aspergillus fumigatus detection. Taqman probes  and Molecular Beacons  are among the most popular probes used for diagnostic purposes [In qRT-PCR, amplicons are detected using DNA-binding dyes and probes. SYBR Green is the most common DNA-binding dye, however, it can inhibit amplification at higher concentrations. Therefore, other dyes such as Eva Green, which is less inhibitory and produces more distinct melting curves, have been developed . HoweverI (RPB2) , which aI (RPB2) , found tpurposes , howeverCandida spp. and their use for identification of the other fungi is still limited. One of the toughest challenges is DNA template preparation and standardization, which is due to the low number of target fungal cells present in circulation [It should be noted that despite extensive application of different PCR-based detection methods in clinical practice in general, in diagnostic mycology their use is not yet so widespread since they have been slow to enter the market and to be approved by the Food and Drug Administration in the United States for a number of reasons . Most PCculation  as well culation . The choculation . DNA tarculation .Aspergillus PCR initiative. Through these efforts, more assays should enter clinical practice as approved methods for sensitive detection of fungal species in clinical samples.Finally, because of the high sensitivity of PCR, particular care must be taken to perform reactions under clean conditions to prevent contamination and to include adequate controls. Template preparation and PCR setup require dedicated areas separated from that of electrophoresis to avoid contamination due to a large number of target copies present in a single band in the gel. PCR workstations, including a hood with light and UV decontamination, dedicated pipettors, barrier tips, and tubes are common in laboratories performing high throughput diagnostic assays. PCR assays need multiple controls for reagent contamination, internal amplification control for inhibitory substances, as well as negative and positive controls. Although these requirements can be challenging, various groups have been working to improve PCR for application in fungal diagnostics, including the International Society for Human and Animal Mycoses and the European ERG11) in yeast species [cyp5A1) in A. fumigatus [FKS1 and/or FKS2 [A. fumigatus strains in pure cultures and clinical samples. Conventional and qRT-PCR assays have been developed to identify azole- and echinocandin-resistant Candida isolates [Studies on susceptibility to antifungal agents revealed that resistance to azole compounds is predominantly caused by the acquisition of various universal and species-specific mutations in the genes encoding lanosterol-alpha demethylase  technology allowed the development of an identification tool called Specific Highly Sensitive Enzymatic Reporter UnLOCKING (SHERLOCK), which could successfully identify target nucleic acids in attomolar concentrations, differentiate closely-related viruses, and genotype single base-pair differences . New-genThe DNA sequencing of both genomes and specific targets has become an increasingly important diagnostic tool for clinical microbiologists. However, sequencing systems such as Sanger sequencing are out of reach for most small clinical laboratories in developing countries, with the rare exception of regional reference laboratories , becauseStaphylococcus aureus alpha-hemolysin upon application of an electric field [https://www.pacb.com/> and Oxford Nanopore Technologies  <https://nanoporetech.com/>, successfully released their products into the market. Oxford Nanopore Technologies has developed multiple devices with very small space requirements, including one the size of a computer memory stick called the MinION [Nanopore sequencing became possible after the first description of DNA translocation across a lipid bilayer membrane through a pore created by ic field . This stic field . Two come MinION . The smaCandida in positive blood culture bottles and compared its performance with Sanger sequencing. Wurzbacher et al. [Although the technology is very recent, it has already been applied to fungal detection in a number of studies. Ashikawa et al.  used nanr et al.  used nanr et al. . For funOne of the most advantageous features of nanopore sequencing is rapid optimization of instrumentation and reagents. This system can be used to create true point-of-care devices, which could be easily deployed in the clinic or field and facilitate rapid and reliable diagnosis. In combination with companion platforms for automatic DNA template preparation, nanopore sequencing potentially enables sample-to-answer sequencing and can provide advanced molecular diagnostics in the most remote areas of the world without significant capital investments. The comprehensive review by Gabaldon et al. can be consulted to obtain more comprehensive information regarding diagnostic application of various molecular assays ranging from PCR to next-generation (NGS) platforms .www.theyeasts.org, may allow timely institution of appropriate antifungal drugs and avoiding expensive and time-consuming phenotypic antifungal susceptibility testing.As the number of fungal species causing human infections increases, the lack of accurate and rapid diagnostic tools poses a challenge for epidemiological studies in resource-poor and developing countries, whose financial resources are limited to obtain expensive diagnostic instrumentation. To circumvent this problem, alternative affordable diagnostic/identification tools should be provided or grants and free services offered to encourage the installation of MALDI-TOF MS platforms in laboratories of these countries. On the other hand, although some medical mycological groups launched important initiatives to find solutions and presented meaningful and accurate data about invasive fungal infections, collaborations among developing countries should be extended in order to define diagnostic limitations and devise comprehensive strategies to improve diagnostics and treatment of fungal infections. Availability of inexpensive and portable sequencing machines applicable in the field along with the abundance of online sequencing data of mutations conferring resistance to azoles and echinocandins from online databases, such as


Randomized controlled trials (RCTs) provide the highest level of evidence informing clinical practice; however, their implementation in surgical oncology remains challenging.The findings of this study demonstrate that crossover in surgical oncology RCTs is common, affecting one in eight patients. Moreover, its incidence was shown to reduce with increasing surgeon experience and decreasing patient comorbidity . Importantly, the clinical consequences of this crossover within surgical oncology RCTs included increases in 30-day mortality, anastomotic leak rate, and complications as demonstrated by meta-regression.5Future RCTs must develop and implement strategies that include pretrial phases and surgeon credentialing by volume and/or video assessment to reduce the incidence of crossover and thus maintain randomized homogenous groups to adequately test the hypothesis. Moreover, given its independence as a predictor of crossover, as well as its potential effect on complications, it may be advisable to only include low-comorbidity patients in RCTs when initially comparing novel MIS techniques with open surgery. More complex statistical methods developed to account for the crossover effect may also need to be considered. A more pragmatic approach could involve reporting endpoints in three groups rather than two, i.e. the (completed) MIS, open, and converted (crossover) trial arms in addition to the traditional intention-to-treat analysis. This type of analysis will also allow the evaluation of factors associated with crossover, and thus try to predict which patients would not constitute good candidates for MIS due to their high risk of conversion and thus complications.


RNA interference (RNAi), which is mediated by small interfering RNAs (siRNAs) derived from viral genome or its replicative intermediates, is a natural antiviral defense in plants, fungi, and invertebrates. Whether RNAi naturally protects humans from viral invasion is still a matter of debate. Nevertheless, exogenous siRNAs are able to halt viral infection in mammals. The current review critically evaluates the production of antiviral siRNAs, delivery techniques to the infection sites, as well as provides an overview of antiviral siRNAs in clinical trials. The term RNA interference (RNAi) is used to describe gene silencing at the mRNA level guided by small complementary non-coding RNA species. There are several classes of RNAi mediators, one of which, namely small interfering RNAs (siRNAs), facilitates antiviral immunity in plants, fungi, and invertebrates  chemical synthesis; (2) enzymatic production; and (3) Molecules of ssRNA are produced by automated solid-phase synthesis employing 2′-hydroxyl protecting groups that provide ribonucleoside phosphoramitides  to synthesize a complementary RNA strand starting from the very 3′ end  transcribed in a cell from an expression cassette containing a polymerase III promoter (U6 or H1), a DNA template of desired shRNA sequence, and transcription stop signal  vector is the first choice since it has been proved to be non-toxic, non-pathogenic, easy to produce, and it does not integrate into human genome  and RSV in animal models  thymidine overhangs on both 3′ ends to prevent its nuclease degradation  polymerase, viral proteins 24, and 35 completely protected rhesus macaques from lethal infection (Geisbert et al., Two clinical trials have been initiated to assess the efficacy and safety of shRNA-based TT-034 therapeutics, which was created for chronic hepatitis C treatment and delivered to hepatocytes by AAV vector. TT-034 was shown to be safe and well-tolerated. However, in February 2016 Benitec Biopharma decided to discontinue hepatitis C program due to low commercial opportunitiesIn conclusion, the development of RNAi-based therapeutics is still in its early stage and has experienced numerous pitfalls. Nonetheless, it has already been demonstrated that siRNAs can effectively inhibit the replication of various viruses despite different mechanisms they evolved to resist the pressure imposed by immune system and antiviral drugs. However, the possibility of generation of escape mutants, recently discovered inhibitors of RNAi in human viruses (Fabozzi et al., AL and MMP conceptualization, AL manuscript draft, AL and MMP manuscript revision.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.


Melanoma is one of the most fatal cancers, and its incidence is increasing worldwide. Thanks to the better understanding of the molecular mechanisms involved in the pathogenesis of melanoma, recently new targeted agents have been developed. In this article, we review the current state of knowledge of clinical presentation, mechanisms, and management of the most common cutaneous side effects observed during treatment with targeted and immunological therapies approved for advanced melanoma. We include discussion of BRAF/MEK inhibitors and immune-checkpoint inhibitors, notably CTLA-4 and PD-1 inhibitors. Melanoma is one of the most fatal cancers, and its incidence is increasing worldwide. Metastatic melanoma has a poor prognosis representing about 90% of skin cancer mortality. In the recent past, the therapy of metastatic melanoma was based only on dacarbazine, an alkylating chemotherapy agent, which has not shown improvement of overall survival . ClassicGenetic alterations resulting in a constitutive activation of mitogen-activated protein kinase (MAPK) pathway occur in almost all cases of melanoma. The most frequent mutation is the BRAF mutation which has been found in about 50% of melanomas –4. BRAF The first selective BRAF inhibitor approved by FDA and EMA in 2011 for the treatment of patients with metastatic or unresectable melanoma was vemurafenib , followeCutaneous side effects of BRAF inhibitors are very common and usually occur within days of the undergoing treatment. Skin toxicity induced by BRAF inhibitors is similar to that observed with EGFR inhibitors as BRAF represents a downstream mediator of EGFR signalling which is a critical regulator of epidermal homeostasis . AlthougBiological results demonstrate that BRAF inhibitors promote paradoxical activation of the MAPK pathway in cells that do not carry BRAF mutation leading to keratinocyte proliferation and hyperproliferative skin lesions which are the most common side effect of BRAF inhibitors . A concuThe profile of skin toxicity associated with MEK inhibitors differs from that occurring during BRAF inhibitors therapy. Squamoproliferative skin lesions developed in patients treated with BRAF inhibitors have not been reported during treatment with MEK inhibitors. The typical rash (papulopustular or acneiform) observed during MEK inhibitors agents is different from the rash (hyperkeratotic maculopapular) caused by BRAF inhibitors .Skin rash has been reported in patients treated both with BRAF and MEK inhibitors. Skin rash is mostly seen in patients under treatment with BRAF inhibitors (64–71% with vemurafenib and up to 18% with dabrafenib) . It usuaPapulopustular rashes can be treated with topical (clindamycin and erythromycin) and oral antibiotics (doxycycline and monocycline), topical (prednicarbate) and oral steroids (prednisone), oral antihistamines, and oral isotretinoin , 22.Occasionally, keratosis pilaris-like eruptions may appear, and they can be managed with mild keratolytics such as urea cream or salicylic acid and topical steroids .It has also been reported a nonspecific rash similar both clinically and histologically to idiopathic Grover's disease. This benign acantholytic dermatosis presents as scattered erythematous papules with or without crusting usually asymptomatic or only slightly pruritic. Lesions involve the trunk, the upper arms, and legs. It can be treated with moisturisers, topical steroids, and oral antihistamines or oral prednisone and acitretin in severe cases , 23.Serious cutaneous rashes such as Stevens–Johnson syndrome and toxic epidermal necrolysis have been rarely reported .Photosensitivity reaction represents one of the main adverse effects related to BRAF inhibitors, and it is experienced more frequently with the use of vemurafenib (22–67%) compared to dabrafenib (3–33%) . PhotoseLocalized hyperkeratosis appeared as painful hyperkeratotic areas on points of pressure or friction mostly on the soles and less frequently on the palms . It is oThe term verrucal keratosis is used to describe keratotic lesions as warts, keratoacanthomas, or nonspecific hyperkeratotic papules induced by BRAF inhibitors. These keratotic proliferative lesions develop as single lesions or a diffuse eruption at various anatomical sites, on both sun-damaged and non-sun-damaged skin with a median time to presentation of 11 weeks. Although verrucal keratoses are benign lesions, they should be monitored for changes such as rapid growth, pain, and erythema which are indicative signs of evolution into cutaneous SCC. Cryotherapy can be useful for small lesions. The use of acitrein as a chemopreventive drug has been beneficial , 23.One of the most relevant adverse events reported in patients receiving BRAF inhibitors is the development of cutaneous tumors in the form of well-differentiated and keratoacanthoma-type squamous cell carcinomas. Clinically, these lesions appear as hyperkeratotic crateriform papules in various anatomical sites. SCCs have been reported to occur early during BRAF inhibitors therapy with a median time to onset of approximately 8 weeks. The reported incidence is 4–31% in patients treated with vemurafenib and 6–11% in patients treated with dabrafenib . The treSeveral changes affecting the hair follicle have been observed in patients during BRAF inhibitors treatment including alopecia, slower and thinner scalp hair growth, and structural changes in shape (from straight to curly) and in color (turn gray) of hair. These hair abnormalities are temporary, and they could spontaneously regress without therapy modifications. Although alopecia is a reversible effect, it has a strong impact on patient quality of life and may also lead to voluntary treatment disruption. Minoxidil solution until 3–6 months after therapy termination can be considered as a treatment for alopecia , 22.Panniculitis on the lower extremities has been described with both vemurafenib and dabrafenib. It occurs as a painful, erythematous to livid, subcutaneous nodules, often accompanied by fever, chills, and arthralgia. Panniculitis responds to regular nonsteroidal anti-inflammatory drugs, and oral steroids (prednisolone) can also be considered for treatment , 22.Although melanocytic proliferations are less frequent than squamoproliferative lesions, changes in preexisting nevi, new melanocytic nevi, and new primary melanomas have been reported in patients receiving BRAF inhibitors. The melanocytic proliferation under BRAF inhibitors treatment seems to be due to paradoxical activation of wild-type BRAF cells. However, it cannot be excluded that the onset of a new primary melanoma is related to the increased risk of developing a second primary melanoma among patients with a personal history of melanoma, rather than therapy. Therefore, a dermoscopic careful monitoring should be included in the follow-up of these patients .Rare cases of basal cell carcinoma have been also described in patients under BRAF inhibitors treatment .Immunotherapy through immune-checkpoint inhibitors is an established treatment for advanced melanoma. The main immune-checkpoint-targeted molecules are CTLA-4 and PD-1, which are expressed on activated T-cells and are involved in the inhibition of the immune system. CTLA-4 is involved in the interaction between T-cells and antigen presenting cells (activation phase in lymphatic organs), whereas PD-1 mediates the interaction between T-cells and tumor cells   Figure .Currently, there are three monoclonal antibodies approved for the treatment of patients affected by metastatic melanoma: ipilimumab, which is an anti-CTLA-4 antibody, approved by FDA and EMA in 2011, and pembrolizumab and nivolumab, which are both anti-PD-1 antibodies, approved by FDA in 2014 and EMA in 2015. In 2016, FDA and EMA also approved the combination therapy of ipilimumab and nivolumab for the treatment of unresectable or metastatic melanoma .Ipilimumab blocks the interaction of CTLA-4 with its ligands, CD80/CD86, allowing T-cell activation through reestablishment of CD28 to CD80/CD86 between the T-cell and antigen presenting cell. The inhibition of CTLA-4 signalling, through the activation of cytotoxic T-lymphocytes against cancer cells and the general increase in T-cell responsiveness, enhances the patient's antitumor immune response .Pembrolizumab and nivolumab inhibit the interaction between PD-1 and PD-L1 enhancing antitumor responses, delaying tumor growth and facilitating tumor rejection , 29.The alteration of the immune system induced by CTLA-4 and PD-1 inhibitors results in the development of various autoimmune manifestations referred as immune-related adverse events (irAEs). Dermatologic toxicity is very common, and it is potentially mediated by a shared antigen coexpressed by the dermoepidermal junction and tumor cells. Rash and pruritus are the most reported cutaneous side effects both in patients under treatment with ipilimumab and in patients treated with PD-1 inhibitors –34.Cutaneous adverse events occurring during anti-PD-1 therapy are usually less severe and develop later than those seen with anti-CTLA-4 therapy (4–10 months compared with 3–6 weeks) .Other common dermatologic adverse effects are lichenoid eruption, eczema, and vitiligo which are all mediated by lymphocyte damage confirming the immune-related mechanism.Cutaneous side effects are mostly low grade and usually can be managed with symptomatic treatment that includes medium-to-high potency topical corticosteroids and oral antihistamines, while therapy interruption is rarely necessary .Interestingly, skin immune-related events may be useful as visible clinical parameters of antimelanoma immunity and clinical response to checkpoint inhibitors. The development of vitiligo seems to be an indicator of antimelanoma immunity and improved survival .Rash has been frequently reported in patients under treatment with immune-checkpoint inhibitors with an incidence of 15–40% . The mosOther common subtypes of rash include lichenoid eruption and prurigo nodularis.Lichenoid eruption has also been described in association with PD-1 inhibitor therapy. It predominantly appears on the chest and back as multiple erythematous and sometimes violaceous papules and plaques. Sometimes, lichenoid oral lesions may appear .Eczematous eruption is an adverse effect typically seen with anti-PD-1 therapy (up to 17% of patients) . It occuIt is well known that the development of vitiligo in patients affected by advanced melanoma (stages III and IV) is associated with tumor regression and prolonged survival , 41. FurSome cases of psoriasis exacerbation have been reported in patients under treatment with nivolumab , 44. T-cSeveral cases of bullous pemphigoid and bullous pemphigoid-like skin lesions have been described in patients under treatment with anti-PD-1 agents –48, wherBullous pemphigoid has also been reported as a paraneoplastic manifestation of melanoma. The exact mechanism of the paraneoplastic phenomenon remains poorly understood, but it has been described that BP180 is expressed in melanoma cells and not in normal melanocytes . While mLess common cutaneous adverse events afflicting patients during immunotherapy include xerosis, photosensitivity reactions, pyoderma gangrenosum-like ulcerations, Sweet syndrome, cutaneous sarcoidosis, alopecia, actinic keratosis, squamous cell carcinomas, seborrheic keratosis, toxic epidermal necrolysis, and severe drug rash with systemic symptoms and eosinophilia , 35.Targeted therapies have a crucial role in patients with advanced melanoma. They have significant benefits in the prognosis, but they are frequently associated with cutaneous side effects that may affect quality of life of patients. Cutaneous adverse events are usually low grade and manageable. Understanding and managing skin toxicity could improve the quality of life and prevent the interruption of the tumor therapy leading to a better clinical outcome. Although current targeted therapies have been shown to reduce melanoma mortality, advanced melanoma is still a significant therapeutic challenge to clinicians. New therapeutic approaches are currently being developed, and they will probably include combination therapies as the association of BRAF inhibitors and immune-checkpoint inhibitors. New potential side effects will likely emerge introducing these novel combination therapies.