Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motoneurons and paralysis. The mechanisms underlying neuronal degeneration in ALS are starting to be elucidated, highlighting disturbances in motoneuron proteostasis. Endoplasmic reticulum (ER) stress has emerged as an early pathogenic event underlying motoneuron vulnerability and denervation in ALS. Maintenance of ER proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER-located kinase and endoribonuclease that operates as a major ER stress transducer, mediating the establishment of adaptive and pro-apoptotic programs. Here we discuss current evidence supporting the role of ER stress in motoneuron demise in ALS and build the rational to target IRE1 to ameliorate neurodegeneration. SOD1, TARDBP, FUS and hexanucleotide repeat expansions in C9orf72  is a fatal neurodegenerative disease that affects motoneurons of cerebral cortex, brainstem and spinal cord, leading to muscle weakness, paralysis and premature death within 3–5 years after diagnosis  also leads to neuroprotection associated to autophagy induction, enhancing degradation of mutant huntingtin  brings about the possibility to fine-tune IRE1 activity in vivo (Ghosh et al., The involvement of ER stress and IRE1 signaling in a myriad of diseases has fostered the development of different categories of compounds targeting IRE1 Figure . Most IRIn addition to pathology, the UPR has essential roles in various organs, highlighting specialized secretory cells that require a developed ER for their proper function (Cornejo et al., In summary, the compelling evidence in the literature linking ER stress to ALS pathogenesis and the continuous development of specific small molecules to modulate IRE1 activity may offer the opportunity to investigate novel therapeutic approaches to treat this devastating disease. Further research in mouse models of ALS is needed to address the relative contribution of IRE1 signaling in motoneurons and glia to disease progression, in addition to define its mRNA targets under pathological conditions in the nervous system.DBM performed bibliographical research, and wrote and reviewed the article. JVG and PF performed bibliographical research, prepared figures and reviewed the article. CH performed bibliographical research and wrote and reviewed the article.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.


To do this, we conducted a systematic search for post-mortem, pharmacological, functional magnetic resonance and molecular imaging studies of dopamine function in bipolar disorder. Converging findings from pharmacological and imaging studies support the hypothesis that a state of hyperdopaminergia, specifically elevations in D2/3 receptor availability and a hyperactive reward processing network, underlies mania. In bipolar depression imaging studies show increased dopamine transporter levels, but changes in other aspects of dopaminergic function are inconsistent. Puzzlingly, pharmacological evidence shows that both dopamine agonists and antidopaminergics can improve bipolar depressive symptoms and perhaps actions at other receptors may reconcile these findings. Tentatively, this evidence suggests a model where an elevation in striatal D2/3 receptor availability would lead to increased dopaminergic neurotransmission and mania, whilst increased striatal dopamine transporter (DAT) levels would lead to reduced dopaminergic function and depression. Thus, it can be speculated that a failure of dopamine receptor and transporter homoeostasis might underlie the pathophysiology of this disorder. The limitations of this model include its reliance on pharmacological evidence, as these studies could potentially affect other monoamines, and the scarcity of imaging evidence on dopaminergic function. This model, if confirmed, has implications for developing new treatment strategies such as reducing the dopamine synthesis and/or release in mania and DAT blockade in bipolar depression.Bipolar affective disorder is a common neuropsychiatric disorder. Although its neurobiological underpinnings are incompletely understood, the dopamine hypothesis has been a key theory of the pathophysiology of both manic and depressive phases of the illness for over four decades. The increased use of antidopaminergics in the treatment of this disorder and new  However their modes of action have not been well established. By contrast antidopaminergic drugs do have well-understood class action at D2/3 receptors and have long been used to treat acute manic episodes. However, as will be explained below, newer drugs (particularly olanzapine and quetiapine) have been shown to have antimanic and antidepressant actions in bipolar disorder together with long-term benefit in preventing relapse to either pole of the illness.11 Furthermore drugs may be associated with significant side-effect burden and the risk of toxicity and/or teratogenicity in some instances.18 Hence better understanding of pathophysiology and drug action appears necessary to improve the use of current treatments and develop better alternatives.Despite these advances in treatment, many patients continue to experience high levels of disability.21 Early incarnations focused on mania, and the parallels between the behavioural consequences of amphetamine ingestion and the antimanic actions of antidopaminergic drugs. If hyperdopaminergia underlies the development of manic symptoms, then hypodopaminergia might underlie the depressive phase of the illness. Thus, opposite changes in dopaminergic function were hypothesised to underlie the opposing affective poles of the disorder.21 This theory did not explain how hyper- or hypodopaminergia would arise and subsequent versions proposed an additional component to the hypothesis, where an intrinsic dysregulation in the homoeostatic regulation of dopaminergic function leads to cyclical changes in dopaminergic neurotransmission,23 which would further distinguish the dopamine hypothesis of bipolar from theories of schizophrenia.24 Thus, faulty homoeostatic mechanisms responding to hyperdopaminergia in the manic phase of the illness are proposed to result in an excessive reduction in dopaminergic function, rapidly leading to a hypodopaminergic state and depression. In turn a faulty regulatory response to hypodopaminergia leads, to a switch back to mania.23 Implicit in this model is that a normalisation of dopaminergic function leads to remission and euthymia. Clearly, some kind of dysregulation must be required to account for the onset of episodes, but BD is also characterised by inter-episode mood instability.25 Any satisfactory theory must account for the randomness of much bipolar experience as well as the cyclicity. As current antimanic antipsychotics are all dopamine D2 receptor blockers, their use for mania has long supported the involvement of dopamine in mania, but the increasing use of some antidopaminergic drugs to treat bipolar depression and as maintenance drugs makes it timely to review the dopamine hypothesis of BD. Furthermore, a number of new lines of evidence relevant to the role of dopamine, in particular from molecular and functional neuroimaging, have developed in recent years. Thus, we synthesize evidence from pharmacological, in vivo neuroimaging and post-mortem studies addressing the role of the dopamine system in BD and then consider the treatment and drug development implications.The dopamine hypothesis of BD dates back at least to the 1970s.27 Following this, a dopamine transporter (DAT) knockout rodent model was developed, and dopamine transporter blockers other than amphetamine (such as GBR12909) have been used. Both these approaches have been shown to induce manic-like behaviour, such as hyperlocomotion and increased exploration.29 Subsequently, dopamine receptor stimulation using quinipirole (a dopamine agonist) was shown to induce manic-like behaviour.30 Interestingly, mood stabilisers such as valproate and carbamazepine reversed these effects.30 Recently, Sidor and colleagues demonstrated the impact of alterations in the regulation of circardian rhythm on dopaminergic activity and behaviour.31 In this study, mice with a mutation in a circadian clock gene displayed a manic-like phenotype as measured by hyperlocomotion in the daytime. Moreover, hyperlocomotion was linked to an elevated daytime spike in ventral tegmental area (VTA) dopaminergic activity, as well as increased dopamine synthesis and tyrosine hydroxylase activity.31 This was further validated using sustained optogenetic stimulation of the VTA, which also induced hyperlocomotion behaviour.31 Taken together this evidence suggests that hyperdopaminergia, induced either by increased dopamine release, dopamine transporter blockade or dopamine receptor stimulation, results in a mania-like phenotype in rodents. In contrast, lesions in dopaminergic areas (such as the VTA and substantia nigra) induce depressive behaviour, such as learned helplessness.32 Moreover, optogenetic stimulation of VTA dopaminergic neurons reverses depressive behaviour .33 These models thus provide evidence that hypodopaminergia may induce depressive-like behaviours and hyperdopaminergia may induce manic-like behaviour. Further research is needed to determine the key components of the dopaminergic circuit underlying this, particularly in relation to depressive behaviour.Initial animal models of mania were based on amphetamine-induced hyperlocomotion, and it was shown that lithium reverses this behaviour.35 However, to date only four studies,36 have been conducted, and only two focus on the same region . Thus, this finding still requires replication in other brain regions to determine if there is regional specificity. The literature on other dopaminergic receptors is more limited, with only two studies on the D1 receptor37 and one study on the D5 receptor.38 Another issue for the post-mortem studies is that they are affected by several confounding factors such as medication status, post-mortem interval, substance abuse and cause of death. Antidopaminergic treatment has been shown to increase D2/3 receptor levels in animals,39 and there is evidence this may also occur in schizophrenia.40 This suggests that prior treatment could also have affected the D2/3 receptor findings in BD. Finally, data on phase-specific changes are difficult to characterise as clinical status at the time of death remains unknown. Despite these limitations, post-mortem data supports the notion that an abnormality within the dopaminergic pathways, in particular involving D2/3 receptors, might play a role in the pathogenesis of BD.in vivo imaging studies of mania in 11C]-methylspiperone, when compared with healthy controls (HC) and non-psychotic mania patients,42 although, as this tracer has significant affinity for 5HT2 receptors as well,43 this finding requires replication with more selective tracers. Moreover, no significant difference in the striatal D2/3 density was noted in non-psychotic mania patients compared to HC.44 These studies also explored the relationship between manic symptoms  and dopamine synthesis capacity and D2/3 density, finding no significant correlations between these variables in patients with mania.45 However, in one of these studies D2/3 density was directly correlated with psychosis scores on the present state examination.41 Taken together, these data suggest that psychotic symptoms in mania may be associated with dopaminergic abnormalities, although the same cannot be inferred in non-psychotic mania patients.We have summarised the et al reported an upregulation of the dopamine transporter in drug naive euthymic bipolar patients, while Anand et al. observed a downregulation in a group consisting of both euthymic bipolar and bipolar depression patients.47 Additionally, one study has assessed the vesicular monoamine transporter protein (VMAT-2), located in presynaptic neurons and involved in the storage of dopamine. There was an increase in VMAT-2 in the thalamus and dorsal brainstem, but no significant alteration in striatum, in currently euthymic patients with a history of psychotic mania compared with HC.48 A positron emission tomography (PET) study using [11C]-SCH23390 found decreased D1 density in the frontal cortex but not in the striatum of bipolar patients compared to HC, suggesting a regional difference in D1 receptor distribution  (including nucleus accumbens) and prefrontal cortex have an important role in reward processing56 Changes in BOLD signal occur in the human nucleus accumbens, striatum and frontal cortex over a time course of seconds in response to anticipated reward.57 Consistent with the animal literature, event-related functional magnetic resonance imaging (fMRI) reward paradigms have demonstrated robust activation of the ventral striatum in relation to both the anticipation and receipt of reward,61 presumably related to dopaminergic neurotransmission.69Investigations in animals have shown that individual neurons signal reward-related information in the midbrain (substantia nigra and VTA), striatum, orbitofrontal cortex, amygdala and associated structures. Most dopamine neurons in the substantia nigra and VTA show brief, phasic responses that reflect the difference in value between received reward and predicted reward. The precise relationship between cell firing and function is still debated, but it would be misleading to think of dopamine neurotransmission simply in terms of a pleasure signal.82 This provides another indirect line of evidence for a dopaminergic abnormality in this disorder. We discuss the evidence in relation to specific mood polarities below.In BD several fMRI studies employing reward tasks support the existence of abnormal reward-related neural activity in the VS and frontal cortex ,81 compared with controls. Reward feedback related activation in the frontal cortex has also variously been reported as elevated,76 decreased (in euthymic and mildly depressed patients)81 or no different77 in bipolar I patients compared with controls. In euthymic bipolar II patients Caseras et al. reported elevated VS and prefrontal cortex activity for cued reward anticipation compared with controls.72 This finding, however, was not replicated in a more recent study of medication-naive bipolar II patients, which instead reported reduced anticipation-related activity in the right dorsal striatum in patients.82 Both studies reported no difference in VS activity during reward feedback in patients vs controls. Increased responses in the anticipatory phase of the monetary incentive delay task have been described in euthymic unipolar patients.83 Careful comparison of unipolar with bipolar cases will be necessary to distinguish network dysfunction associated with (hypo)mania from that with depression. All such studies need to be adequately powered and preferably address pre-specified hypotheses, not always a standard observed in imaging studies.Euthymic patients are of particular interest because they are at risk of mania. In these patients abnormalities in dopaminergic function would then indicate how vulnerability might be mediated. In medicated euthymic bipolar I patients, VS and prefrontal cortex activity to cued reward anticipation has been variously reported as increased,post hoc choice of analysis contrasts between these studies may account for the inconsistencies and non-replication. Further studies are required to explore the suggested differences between euthymic bipolar I, bipolar II and unipolar disorder.In summary, the precise nature of the abnormality of reward-related activity in euthymic bipolar patients remains unclear. However, abnormalities of reward processing are probably not confined to the manic phase of the illness. Important differences in task structure and the per se was increased in left ventrolateral prefrontal cortex in patients with bipolar-1 disorder, compared with healthy controls or patients with unipolar depressive disorder; it may represent a more generalised ‘arousal' response in the bipolar depressed group.73 Ventral striatal and prefrontal activation during reward feedback in bipolar depressive patients has been reported both as similar to controls79 and decreased.78Three studies have compared monetary reward processing in medicated depressed bipolar I patients and HC. Only one study analysed activity during cued reward anticipation; it reported no difference in anticipation-related activity between patients and HC in VS or prefrontal cortex, although there was blunting in the anterior cingulate cortex in depressed patients (bipolar and unipolar). Neural activity related to outcome anticipation 86 as well as major depressive disorder and alcohol addiction.84 In contrast, the fMRI findings in bipolar disorder summarised above do not provide consistent evidence for blunting. It may be important to consider only studies in the manic state, because depressed or even euthymic (often dysthymic) bipolar patients may well show blunting.82 In mania there was increased frontal activation to reward anticipation in two out of three studies. If this finding is confirmed in further studies, including direct comparisons with patients with schizophrenia, it could point to a key difference in the reward network between mania and schizophrenia. To date a small number of studies have compared reward processing in bipolar disorder with schizophrenia70 or major depressive disorder.78 Of these, one study reported blunted VS activation in depressed patients diagnosed with bipolar disorder compared with major depressive disorder, at reward feedback.78 Only one study compared reward processing in schizophrenia and manic patients with controls.70 Patients with schizophrenia and healthy controls showed an activation in the VTA on expectation of monetary rewards and nucleus accumbens activation during receipt vs omission of rewards. Manic patients, however, showed reduced differential activation in the nucleus accumbens on receipt vs omission of rewards compared to the healthy control subjects. Taken together with the findings in the frontal cortex discussed above, this small study does suggest that in mania there is a deficit in prediction error processing not seen in schizophrenia. It may further imply bipolar disorder specific changes in dopaminergic function, although how much of the fMRI response is driven by disrupted dopaminergic function remains to be determined. Further studies investigating differences in reward processing between bipolar disorder and other psychotic and affective disorders, and across illness phases, will shed light on the reward processing abnormalities specific to bipolar disorder.Recent fMRI studies and meta-analyses have demonstrated blunted activation of the reward network in the ventral striatum and frontal cortex in schizophrenia,90 Further, there are several case series of manic and hypomanic episodes in Parkinson's disease patients treated with Levodopa.93 Bromocriptine, another dopamine agonist, has also been shown to induce manic symptoms.95 Similarly, an increased risk of hypomania/mania was observed in bipolar patients who received stimulant  augmentation for bipolar depression.96 Secondly, pharmacological strategies to deplete tyrosine, a dopamine precursor, are known to reduce manic symptoms, although it remains to be established if this is specifically due to reducing dopamine levels.98 Alpha-methyl-p-tyrosine, which induces dopamine depletion by inhibition of tyrosine hydroxylase, also attenuates mania-like symptom in bipolar patients.100The role of dopaminergic abnormalities in BD has been extensively investigated using a variety of pharmacological approaches . Firstly102 In addition, a high prevalence of depression is seen in patients with Parkinson's disease and this has been linked to loss of striatal dopaminergic innervation.104 Finally, depletion of tyrosine has been shown to precipitate depressive symptoms in remitted patients with a history of major depressive disorder.106Reserpine and tetrabenazine depletes synaptic dopamine by irreversibly inhibiting vesicular uptake of monoamines and has long been known to induce depression.110 Olanzapine, risperidone, quetiapine, aripiprazole, asenapine, ziprasidone and cariprazine have been approved by the Food and Drug Administration (FDA) as monotherapy for the treatment of acute mania.111 Pertinently, a network meta-analysis of 68 randomised controlled trials (RCT), involving 16 073 participants, addressed the efficacy of different drugs in the treatment of acute mania. This showed that dopamine antagonists have larger effect sizes  relative to placebo: −0.56 (95% CI −0.69 to −0.43)), risperidone (−0.50 (−0.63 to −0.38)), olanzapine (−0.43 (−0.54 to −0.32)) than mood stabilisers for the treatment of acute mania (lithium) (−0.37 (−0.63 to −0.11)), carbamazepine (–0.36 (–0.60 to −0.11)), valproate (–0.20 (–0.37 to −0.04)).14Dopamine antagonists and partial agonists are increasingly used in the treatment of acute mania, bipolar depression and also as maintenance treatment.24 it is likely that reduced dopamine neurotransmission at least contributes to the clinical efficacy of these drugs. This assumption is supported by the greater efficacy of the cis- but not the trans-isomer of clopenthixol in the treatment of manic symptoms,112 as the cis-isomer is a high affinity D2/3 receptor blocker while the trans-isomer has much lower affinity for D2/3 receptors.112As the blockade of the dopamine D2/3 receptors is their common mechanism of action,111 Although lithium and valproate should be preferred, recent guidelines highlight that dopaminergic drugs have a place in maintenance treatment.113The FDA have approved olanzapine as monotherapy, quetiapine and ziprasidone as adjunctive therapy, and aripiprazole and risperidone long acting injections as both adjunctive and monotherapy for the maintenance treatment of BD.45 In a study consisting of 13 manic patients, 2 weeks of sodium valproate was shown to decrease presynaptic dopamine synthesis capacity,45 although this was not correlated with clinical improvement. Interestingly D2/3 density remained essentially unchanged after valproate treatment,44 despite the reduction in dopamine synthesis capacity with valproate, which is anticipated to alter D2/3 availability. Taken together these two could either suggest valproate blocks the capacity of the D2 receptor to respond to reduced dopamine synthesis, or that the capacity of D2 receptors to respond is intrinsically impaired in bipolar disorder. Although both possibilities are speculative at this stage, the latter is particularly interesting as it suggests that a failure of D2 adaptation could contribute to the pathophysiology of bipolar disorder, making the dopamine system more vulnerable to dopamine transporter driven changes in presynaptic function.Interestingly, it is possible that sodium valproate might exhibit antimanic effect through an action on the dopaminergic system.114 Furthermore, lithium acts on the adenyl cyclase and phospho-inositide, as well as protein kinase C pathways, which are part of the intracellular signalling pathway downstream of dopamine receptors.115 In addition, micro-dialysis studies show a reduction in extracellular dopamine levels in lithium treated animals.118 In summary, there is evidence to suggest that some non-D2/3 blocking mood stabilisers also act to reduce dopamine transmission, either through reducing presynaptic dopamine synthesis capacity in the case of sodium valproate, or post-synaptic dopaminergic signal transduction in the case of lithium. The degree to which these effects explain the therapeutic efficacy of these compounds remains to be determined.Although the exact mechanism by which lithium acts remains unclear, it is known to modulate signalling pathways downstream of dopamine receptors. Preclinical studies have shown that lithium reverses dopamine dependent behaviour by acting through the protein kinase B (AKT)/glycogen synthase kinase 3 signalling cascade.127 Although there have not been any RCTs investigating the efficacy of methylphenidate or amphetamines in bipolar depression, the available open-label and naturalistic studies point towards a benefit of stimulants in a selected group of patients with drowsiness and fatigue.131 Randomised controlled studies and open-label reports with other stimulant like agents such as modafinil and its R-enantiomer, armodafinil also indicate efficacy in bipolar depression although the development programme for armodafinil failed.138 There is evidence to support the potential use of monoamine oxidase inhibitors such as tranylcypromine in bipolar depression.140 It is speculated that tranylcypromine acts as dopamine releaser with about 1/10th the potency of amphetamine.140 There are thus several lines of evidence suggesting that dopaminergic augmentation is beneficial in bipolar depression.Three double-blind placebo-controlled studies have shown that short-term use of pramipexole, a D2/D3 receptor agonist, is efficacious as an augmentation strategy for the treatment of bipolar depression and positive results have also been seen in other open-label studies.15 The FDA has approved the combination of olanzapine and fluoxetine, as well as monotherapy with quetiapine or lurasidone, for the treatment of acute bipolar depression. A recent meta-analysis of 24 placebo-controlled trails (n=7307) revealed the following order of efficacy of drugs for the treatment of bipolar depression: olanzapine+fluoxetine⩾valproate>quetiapine>lurasidone>olanzapine, aripiprazole and carbamazepine.141 The analysis is interesting in showing that antidopaminergics on their own have efficacy in bipolar depression.However, there is also evidence that dopamine antagonists are effective in the treatment of bipolar depression.2A antagonists,142 whilst both lurasidone and aripiprazole have high affinity for 5HT1A receptors.143 Potentially supporting this explanation, there are no clinical trials reporting efficacy of pure D2/3 blockers in bipolar depression. Nevertheless, the doses used in the clinical trials correspond to the dose ranges associated with substantial dopamine D2/3 receptor occupancy,24 so dopamine receptor blockade is certainly compatible with antidepressant efficacy. The final consideration is that there has been a relatively small number of good quality RCTs for antidopaminergic treatment in bipolar depression, certainly in comparison to studies in mania. Studies that can directly test the mechanism underlying the mode of action of these drugs and pramipexole would be of great interest.The evidence thus suggests that both dopaminergic agonists and dopaminergic blockers, are effective in treating bipolar depression. This presents a paradox for understanding the role of dopamine in bipolar depression. However, actions at receptors other than dopamine ones may underlie the efficacy of the dopamine blockers. For example, as well as all being D2/3 receptor blockers, olanzapine and quetiapine are both relatively high affinity 5HTOur main findings for bipolar mania are that (i) there is consistent pharmacological evidence, especially from treatment studies, to support the hypothesis that a state of hyperdopaminergia can lead to mania; (ii) imaging studies support this hypothesis, with several studies reporting elevations in D2/3 receptor availability in psychotic mania and fMRI imaging evidence that identifies hyperactivity of the reward circuit in mania. Dopamine synthesis and receptor density appear to remain unchanged, at least in non-psychotic mania patients compared with HC.For bipolar depression (i) pharmacological evidence shows that dopamine agonists are potentially beneficial for bipolar depression, but the same seems true for dopamine blocking drugs; (ii) the imaging studies show replicated increases in dopamine transporter levels, but there is inconsistency and it is not clear if there are other alterations as well. Finally, post-mortem evidence suggests an upregulation of the D2/3 receptors in bipolar patients, but interpretation is limited by lack of information on phase of illness and medication status at the time of death.144 Tentatively, these studies suggest elevated D2/3 receptor availability and a hyper-responsive reward system in ventral striatum in mania, and an increase in striatal dopamine transporter availability in bipolar depression. Dopamine neurotransmission in the striatum is primarily terminated by reuptake of dopamine into the presynaptic dopamine nerve terminal by dopamine transporters. Thus, an elevation in striatal D2/3 receptor availability in mania would lead to increased dopaminergic neurotransmission whilst increased striatal dopamine transporter levels in depression would lead to reduced dopaminergic function. This suggests a model in which elevated D2/3 receptor levels lead to altered reward processing and the development of mania, which is followed by a compensatory increase in dopamine transporter levels to reduce dopaminergic neurotransmission. However, if, over time, D2/3 receptor levels reduce but dopamine transporter levels do not normalise, this would then lead to reduced dopaminergic transmission, leading to depression and, in turn, a compensatory upregulation of D2/3 receptor levels, precipitating a further phase switch. One can see how a failure of homoeostatic regulation of the dopaminergic system could lead to cyclical periods of elevated and blunted dopaminergic neurotransmission. This model might apply most precisely to rapid cycling bipolar disorder. However it could be a component of all manic episodes, with other systems acting to disrupt the regular cyclicity that is so obvious in a substantial number of patients.More than four decades on from the early conceptualisations of the dopamine hypothesis of BD, it has stood the test of time, and the evidence for elements have strengthened. This is particularly the case for mania where the strength of the clinical trial evidence for the benefit of dopamine antagonists and partial agonists, supported by meta-analysis, would require an improbably large number of negative studies for reversal. Added to this there is now molecular and fMRI imaging evidence in bipolar disorder. Further, preclinical studies using optogenetic methods and knockout mice have shown that dopaminergic neuron activation leads to manic-like behaviour and it is linked to circadian gene expression.per se may drive dopaminergic changes.145 It is worth noting that, none of the studies have investigated dopamine function in mixed states. As approximately two thirds of depressed patients have concomitant manic symptoms,146 any theory needs to explain mixed states as well as mania and depression. It would be hard to account for mixed states by dysfunction in dopaminergic function alone. Interestingly, asenapine seems to be more effective than olanzapine for mixed episodes.147 As asenapine has higher affinity for 5HT2A than D2/3 receptors, which could suggest a role of serotonergic system in mixed episode states. Finally elevations in D2/3 receptor availability would be predicted to increase dopaminergic neurotransmission via the indirect pathway, and reduce response to cues,148 while the fMRI findings in mania do not seem to fit with this. However, the majority of the patients in these studies were treated with dopamine antagonists and mood stabilisers .The key limitation of the dopamine hypothesis remains that its strongest supporting evidence comes from pharmacological studies, which offer an indirect and sometimes imprecise approach to studying dopaminergic function. Reserpine, alpha-methyl-para-tyrosine, amphetamine and 50 this is not consistent with the finding of elevated DAT in a euthymic state.47 It also remains unclear whether the dopamine hypothesis can completely explain bipolar depression and how dopaminergic dysregulation will be linked to the involvement of other neurotransmitter systems.Although our model suggests euthymia is a state of normalised dopamine, based on a study demonstrating no significant alteration in the dopamine release paradigm in euthymic patients compared to controls,115 it is not established if this is their mechanism of action. Determining whether dopaminergic mechanisms are common across classes of medication would be an important advance in understanding. Finally, it remains unclear if hyperdopaminergic activity is specific to mania or psychosis because many of the manic patients in the studies had psychotic symptoms as well. It is interesting to note that in schizophrenia molecular imaging studies indicate there is an elevation in dopamine synthesis and release capacity, but unaltered dopamine transporter and D2/3 receptor availability in striatum.40 PET studies have also demonstrated elevated striatal dopamine turnover, but blunted cortical and midbrain dopamine release in schizophrenia.150 In addition, blunting of the fMRI signal during reward tasks is observed in schizophrenia.85 In contrast, studies in bipolar disorder suggest dopamine synthesis capacity is unaltered, at least in non-psychotic mania, but there is elevated D2/3 receptor availability in psychotic mania. However, direct comparisons of dopaminergic function in psychotic mania and schizophrenia as well as longitudinal studies of dopaminergic activity across various phases of illness are needed to determine if there are differences between mania and schizophrenia, and between mania and bipolar depression (A critical limitation is the lack of longitudinal studies that investigate changes across phases of illness, including mixed states. Ideally future studies should focus on elucidating phase-related dopamine dysfunction by studying patients longitudinally in euthymic, manic and depressive episodes to determine the direction of causality. Clearly this will be difficult, although a focus on rapid cycling patients might make it feasible. Cross-sectional studies are more feasible and could test key elements of the dopamine hypothesis if well designed. Another key issue is why some antidopaminergics are effective for bipolar depression. Molecular imaging studies are needed to determine whether dopaminergic, serotonergic or other systems are involved in their mode of action. Finally, although studies have found an effect of valproate and lithium on dopaminergic function,pression .The dopamine hypothesis of bipolar disorder proposes that faulty homoeostasis between dopamine transporter and receptors underlies depressive and manic phases of the illness. The available evidence suggests elevated D2/3 receptor availability and a hyper-responsive reward system in mania, and possibly increased dopamine transporter availability in bipolar depression. Future longitudinal studies are needed to elucidate the precise phase-related changes in dopaminergic function and the specificity of alterations to mania over psychosis.


Lung cancer is a devastating disease with overall bleak prognosis. Current methods to diagnose lung cancer are rather invasive and are inadequate to detect the disease at an early stage when treatment is likely to be most effective. In this study, a shotgun sequencing approach was used to study the microRNA (miRNA) cargo of serum-derived exosomes of small cell lung cancer (SCLC) (n=9) and non-small cell lung cancer (NSCLC) (n=11) patients, and healthy controls (n=10). The study has identified 17 miRNA species that are differentially expressed in cancer patients and control subjects. Furthermore, within the patient groups, a set of miRNAs were differentially expressed in exosomal samples obtained before and after chemotherapy treatment. This manuscript demonstrates the potential of exosomal miRNAs for developing noninvasive tests for disease differentiation and treatment monitoring in lung cancer patients. Liquid biopsy, or the test of blood samples to look for biological material shed from tumors, is being used increasingly for cancer diagnostics, treatment planning, and monitoring treatment response. Among the entities targeted by liquid biopsy are tumor cells, cell-free DNA and extracellular vesicles, specifically exosomes , 2. ExosLung cancer is a devastating disease with 228,190 newly diagnosed cases and 159,480 cancer-related deaths in the US in 2016 . Lung caRecently established methods for plasma DNA analysis have advanced targeted therapies for early-stage NSCLC by detecting biomarkers indicative of actionable mutations, such as EGFR and ALK mutations . Liquid The number of sequences successfully aligned to the human genome and the number of mature miRNA detected in different disease and treatment groups are presented in Table Unsupervised hierarchical clustering was used to analyze the differentially expressed exosomal miRNA. As shown in Figure The differentially expressed miRNAs were examined to determine their capacity to differentiate case and control samples or to mark therapy progress. ROC analysis was conducted for 18 miRNAs differentially present between NSCLC and control groups, 16 miRNAs that distinguish SCLC and control, and 2 and 17 miRNAs, respectively, that can discern NSCLC and SCLC groups before and after treatment. A separate test was conducted to examine 24 miRNAs differentially expressed between non-treated SCLC and NSCLC specimens. Features with an area under curve >0.8, signifying good and excellent accuracy of prediction, were selected and are presented in Table Based on the differences observed in exosomal miRNA content between NSCLC and SCLC patients, we hypothesized that the exosomal miRNA could be indicative of the biological differences existing within different types of lung cancer. To gain new insight into the disease biology, we employed the Ingenuity Pathway Analysis (IPA) platform Table . In bothA similar analysis applied to the set of SCLC exosomal miRNAs affected by treatment revealed biological alterations associated with the therapeutic intervention. The changes in the list of top five Diseases and Disorders categories included a switch of “Organismal Injury and Abnormalities” and “Cancer” between the first and second place and appearance of Inflammatory Response within the list of top five categories. On the other hand, the list describing Molecular and Cellular Functions underscored the strong effect of treatment; the top categories affected by treatment were Cellular Growth and Proliferation, Cellular Development, Cell Death and Survival, Cell Cycle, Cellular Movement. The top Canonical Pathways affected by SCLC treatment were Molecular Mechanisms of Cancer, Glioblastoma Multiforme Signaling, Glioblastoma Signaling, PI3K/AKT Signaling, and Glucocorticoid Receptor Signaling. In summary, the analysis suggests that the treatment affects the functional profile of tumor and causes the rise of inflammatory processes.The hypothesis that the exosomal miRNA cargo reflects the patterns of gene expression in tumor tissue was tested further using the Molecular Activity Prediction algorithm implemented in IPA software. The assumption was made that elevated miRNA leads to suppression of target genes and that decrease of miRNA facilitates expression of target genes. The activity prediction algorithm was then implemented for the regulatory networks of six important lung cancer oncogenes , EPHA2, AXL). Of them, focal adhesion kinase (FAK) is a non-receptor tyrosine kinase, upregulated in NSCLCs, and involved in neoplastic transformation, invasion, and metastases, such as cell adhesion, migration and apoptosis ; PXN is In summary Table , the preThe prediction was verified in Oncomine using the relevant NCBI GEO dataset GSE3398; gene expression was compared between Lung Adenocarcinoma and Small Cell Lung Carcinoma cases. According to the algorithm, the levels of AXL, EPHA2, MET and PXN expression were greater in NSCLC, the expression of RON was greater in SCLC, and the model predicted no difference in levels of FAK expression between NSCLC and SCLC. Consistent with the prediction, with the exception of AXL and RON, gene expression data from Oncomine confirmed the expression patterns for EPHA2, MET, PXN, and FAK, indicating that the model delivers accurate prediction for 2/3 of the genes.To understand biological functions, miRNAs exhibiting differential expression above 2 logs fold of magnitude were selected. The development of NSCLC was associated with an increase of molecules associated with endoplasmic reticulum (ER) stress  - hsa-miIn SCLC, several molecules were elevated: the marker of breast cancer chemotherapy resistance and self-renewal capability - hsa-miR-4508 (up 2.07 folds) , serum-bIn the case of SCLC, treatment lead to elevation of hsa-miR-1228-5p (up 5.17 folds), hsa-miR-483-5p (up 3.45 folds), and the implicated in breast cancer chemotherapy resistance  - hsa-miThe treatment of NSCLC was not associated with any significant changes in exosomal miRNA content. Two of the most affected miRNA molecules, hsa-miR-423-5p, known as a positive regulator of autophagy in hepatocellular carcinoma  and the The cargo of exosomes reflects biological processes taking place inside the cells of origin  and the in silico modeling could be higher if verified against matching biopsy tissues rather than a contradictory Oncomine reference set.The disagreement between predicted and observed behavior of several genes deserves a special comment. Recent studies demonstrate overexpression of the receptor tyrosine kinase AXL in lung adenocarcinoma tumor tissues compared with adjacent lung tissues  and stroThe utility of miRNA signatures for lung cancer diagnosis and prognosis is well established –53. ThermiRNA profiles have also been utilized as a prognostic biomarker for lung cancer progression. For instance, a group of 34 miRNAs detected in the serum can determine asymptomatic individuals with early-stage lung cancer at risk for progression to an advanced stage . FurtherIn contrast to NSCLC, there are currently very few studies that have identified miRNA specific for SCLC using peripheral blood. For example, Nishikawa et al. reported elevated miR-375  in lung Of note, none of the molecules seen in the present study featured as lung cancer biomarkers in the large report of Inamura . Of the In this study, the miRNA species from blood-derived exosomes were studied in lung cancer patients (NSCLC and SCLC) and a group of healthy control individuals. We have demonstrated that exosomal cargo is different between patients with different types of cancer as well as between tumor-bearing and control individuals. We have demonstrated the change in exosomal miRNA profiles of patients who underwent chemotherapy treatment—the effect was stronger in the SCLC cohort. Our analysis suggests that exosomal miRNA could serve as a potential marker of biological processes within a tumor, can differentiate patients, and mark chemotherapy response in lung cancer patients.The study cohort comprised 30 subjects Table  that incExosomal RNA was extracted from 500 μl of serum using exoRNeasy Serum/Plasma RNA kit for purification of RNA from exosomes and other extracellular vesicles out of serum or plasma samples  , 72 accoSequence data analysis and statistical comparisons were carried out using Bioconductor packages and an in-house developed analysis pipeline using R statistical environment. After mapping the deep sequencing data onto the human genome and counting the reads for the mature miRNAs in the miRBase database, raw miRNA expression data were normalized, and differential expression analysis was performed by Bioconductor package “edgeR.” Significant miRNAs were selected when fold change was more than 2 or less than 0.5, and FDR ≤ 0.05. Heatmaps were generated using Cluster v3.0.Receiver operator characteristic (ROC) analysis was conducted in STATA v12 using miRNA counts standardized to the count of mature miRNA in the sample. The models of gene signaling networks and molecular activity prediction were created using IPA software.


In vitro and in vivo studies and evidence from human tumors have long implicated Rho GTPase signaling in the formation and dissemination of a range of cancers. Recently next generation sequencing has identified direct mutations of Rho GTPases in human cancers. Moreover, the effects of ablating genes encoding Rho GTPases and their regulators in mouse models, or through pharmacological inhibition, strongly suggests that targeting Rho GTPase signaling could constitute an effective treatment. In this review we will explore the various ways in which Rho signaling can be deregulated in human cancers. Rho GTPases bind to a wide range of effector proteins and play central roles in the regulation of the actin and microtubule cytoskeletons and gene transcription.Rho GTPases are molecular switches which cycle between an inactive GDP-bound form and an active GTP-bound form see . The GTPConversely, GAPs activate the weak intrinsic GTPase activity of Rho proteins leading to the hydrolysis of bound GTP, switching the GTPase to an inactive conformation .7 The abGuanine nucleotide dissociation inhibitors (GDIs) are a third class of regulators of Rho proteins. They sequester inactive GTPases in the cytoplasm by masking their C-terminal lipid moieties that mediate plasma membrane localization, which can inhibit their activationAt the simplest conceptual level, anything which increases the abundance of the active form should increase signaling, while anything decreasing the abundance of the active state, or actively stabilizing the inactive state, should decrease signaling. Disruption of this balance—by direct activation of Rho GTPases or indirectly through changes in regulators as described above—is increasingly being linked to oncogenesis see . In thisBefore the finding of direct mutations of Rho GTPases, the main way they were thought to be misregulated in cancer was through changes in expression levels see . IncreasRac1 has been found to be overexpressed in testicular, breast and prostate cancer, as well as gastric and lung cancers.Animal experiments support a requirement for Rac1 in tumor formation and growth in many different tumor models. Mice with Rac1 deletion specifically from keratinocytes are resistant to developing Ras-induced skin cancerA splice variant of Rac1, Rac1b, was found at elevated levels in colon and breast tumors.Rac2, which shares a high degree of sequence conservation with Rac1, is restricted to expression in the haematapoetic cell lineage. Although no aberrations of Rac2 have been directly linked to oncogenesis, Rac2 is emerging as a therapeutic target, as abrogation of Rac2 signaling slows the growth of AML and CML tumors , may help to reverse some of this historical bias.An alternative mechanism by which many tumors upregulate Rho GTPase signaling is by changing the levels or activities of GTPase regulators, including GEFs, GAPs and Rho GDIs .44,45 WhΤhe GEFs Ect2, MyoGEF, P-Rex1, Tiam1, LARG, Dock180, Vav1, Vav2, Vav3 and β-PIX are overexpressed in a variety of human tumors.in vitro showing that P-Rex1 can regulate invasion.This data from human tumors is supported by evidence from transgenic mouse models highlighting the importance of a number of GEFs in oncogenic progression. Tiam1 has been shown to be important for Ras-mediated skinMice deficient for the Rac1/Cdc42 GEFs Asef1 and Asef2, which are downstream of APC and are overexpressed in colorectal tumors, show reduced spontaneous formation of intestinal adenomas.The GAP DLC1 (deleted in liver cancer) is a tumor suppressor frequently downregulated in many cancer types either by deletion or epigenetic silencing. Loss of DLC1 leads to an activation of RhoA, and cooperates with oncogenic Myc in a mouse model of liver cancer.The picture for Rho GDIs is relatively complex, possibly due to their ability to target multiple Rho GTPases and their roles in regulating Rho GTPase activity, stability and trafficking.As discussed earlier, Rho GTPases are regulated by a whole host of post-translational modifications, many of which are now being linked to inappropriate Rho GTPase function in human cancers and a few of which we will discuss here as illustrative examples. Ubiquitylation of Rac1, RhoA and Cdc42 can be deregulated in cancer cell lines, a fact that could indicate a link between Rho GTPase protein ubiquitylation and cancer.http://www.cbioportal.org/), a database that collects cancer genomics data sets from tumor samples across cancer studies,https://www.intogen.org/search), which assesses mutational data across multiple tumor types to identify potential driver mutations.Early studies had identified mutations in RhoH such as the rearrangement of RhoH/TTF gene and the mutation of the 5′-UTR of RhoH gene in some haematopoietic malignancies.One early study aiming to detect Rac1 mutations in human brain tumors identified deletions, frame shift and point mutations in 12 out of 45 samples from human patients with brain tumors,A serious clinical problem in the treatment of melanoma is the swift development of resistance to the front line treatments of RAF and MEK inhibition. A 2014 study revealed that Rac1 P29S expression in melanoma cell lines and in mouse tumor models conferred resistance to RAF and MEK inhibitorsIn the 2012 study conducted by Hodis et al., a homolog to the Rac1 P29 residue was found to be mutated in Rac2, substituting Proline (P) with Leucine (L) (P29L mutation).As with Rac1, no mutations in RhoA had been detected in human cancers until very recently. RhoA mutations were identified by several groups in 2014,Another study conducted with gastric adenocarcinoma samplesRhoB has been found to be mutated in 5% of bladder cancer cases from a sample of 131 high grade tumors not treated with chemotherapy  making it one of 9 genes mutated in this disease.Two other family members, RhoC and RhoT1, present a number of published mutations in cancer samples and cell lines, with the S73 residue a hotspot in RhoC, while mutations in RhoT1 include a P30L mutation, which by homology may have similar effects to the Rac1 P29S mutation. Deletion of RhoC from mice has been observed to reduce the frequency and growth of tumors,in vivo evidence, such as deletion of Cdc42 from hepatocytes which lead to spontaneous tumor formation,The classical activating mutation G12D  has been found in Cdc42 in melanoma cells in the same study which identified the Rac1 P29S mutation,in vitro and in vivo data showing Rho protein involvement in malignant transformation, observed changes in Rho protein expression levels or changes in their regulators and post-translational modifications, and now direct mutation of Rho GTPases, in human cancers, targeting Rho protein signaling is an increasingly attractive target for new cancer therapeutics. Small molecule inhibitors of many Rho proteins are currently being developed and tested.Given the long-standing Two different small molecule inhibitors of Rac are currently in use, utilizing 2 different strategies for inhibition. NSC23766 works by inhibiting the interaction between Rac1 and its GEF Tiam1, reducing the activation of Rac1.NSC23766 can halt the proliferation, anchorage-independent growth and invasion of prostate cancer cells.in vitro studies to target prostate cancer cells.Given that Rac1 and Cdc42 are highly expressed and active in ovarian cancer,An additional 3 inhibitors of Cdc42 have been developed. Secramine has been identified as a small molecule inhibitor that perturbs Cdc42 activity in a RhoGDI1-dependent manner,Reducing signaling through the Rho pathway is often achieved by targeting the Rho target ROCK.It is likely that further structural modification of these compounds, or further high-throughput compound-screening, will lead to more specific inhibitors, and that as we further our understanding of both normal and abnormal Rho GTPase signaling we will be better placed to deploy them therapeutically.In conclusion, Rho GTPase signaling is frequently seen to be modified in human cancers through a variety of mechanisms, and work is continuing to understand the consequences of this aberrant signaling. Understanding the wider landscape of Rho GTPase signaling in a tumor type is likely to be important for making the correct, clinically-relevant interventions. Modifications occur from the level of mutation of the GTPases to under or overexpression of their regulating proteins, which both generates a highly complex signaling network that needs further work to be untangled and also suggests many fertile avenues for therapeutic intervention.


A new study reveals comprehensive and unique epigenetic properties of glioma stem cells, leading to novel molecular insights and therapeutic potentials toward glioblastoma multiforme treatment. Glioblastoma multiforme (GBM), also known as glioblastoma, is one of the most aggressive and infiltrative tumors in the central nervous system (CNS). The 5-year survival rate of patients with GBM is less than 5%, and they have a poor prognosis for recovery . Therefo, cultured glioma stem cells (GSCs) that were derived from primary GBM cells possess many features of NSCs and remarkably have characteristics resembling those of GBM cells, such as genetic aberrations, global transcriptome profiles, and tumorigenic potentials [Neurogenesis generates various functional neural cell types from multipotent neural stem cells (NSCs) in the mammalian CNS, which participate in specific neural activities and responses . The contentials . TherefoIn this issue of Genome Biology, Zhou et al. take on this vital challenge to generate comprehensive genome-wide epigenomic profiling for various forms of cytosine modifications of DNA at single-base resolution and signature histone modifications of enhancers and transcriptomes of GSCs isolated from patient-derived xenografts, with NSCs isolated from fetal brains as controls .Covalent modifications of DNA at the 5-carbon position of cytosine, such as 5-methylcytosine (5mC), play significant epigenetic roles in the mammalian brain. 5mC is thought to be a static and irreversible modification. However, the recent discovery that Ten-eleven translocation (TET) proteins, viewed as 5mC “erasers,” catalyze the conversion of 5mC to 5-hydroxymethylcytosine (5hmC), providing a new perspective on the plasticity of cytosine modification. Subsequent studies revealed that TET proteins further oxidize 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which are converted to cytosine through DNA repair pathways by thymine-DNA glycosylase (TDG). Cytosine modifications are dynamically and precisely regulated during neurodevelopment and neuronal functions, to spatially and temporally control key gene expression. Thus their dysregulations are linked to many brain disorders, including GBM , 8.In this study, the authors first focused on connecting the expression of DNA modification machinery and the global levels of these modifications in GSCs. There was a notable depletion of certain DNA modifications, such as 5hmC and 5fC, compared with NSCs, indicating a global DNA modification perturbation in GSCs. Interestingly, they found a significant negative correlation of 5mC modification with TET2 and TDG expression, and a positive correlation between TET2 expression and 5fC modification, in GSCs. On the basis of the findings by Zhou et al., it is plausible that TET2 sits at the center stage of maintaining DNA modification homeostasis in normal brain function, and its dysregulation could result in the profound alteration of neuroepigenetic landscaping, contributing to GBM tumorigenesis.Transcriptome profiling of GSCs by Zhou et al. identified the upregulation of HOX genes, which potentially account for the GSC proliferation, and the downregulation of genes involved in apoptosis, growth inhibition, and neural development. Also, several highly conserved downregulated miRNAs were observed and potentially linked to patient survival. These findings at the molecular level identify the characteristics of GSCs and provide solid foundations for future mechanistic exploration.cis-regulatory regions to influence tumorigenic transcription ectopically. In addition, global loss of 5mC and 5hmC accompanied by gain of 5fC or 5caC were found in GSCs, which could contribute to gene misregulation by recruiting a distinct set of “readers.” A recent observation suggests that TET2 plays a critical role in further converting 5hmC to 5fC/5caC, as depletion of TET2 results in the accumulation of 5hmC in promoters and gene bodies [The authors also performed systematic, integrated analyses of genome-wide DNA modification dynamics, enhancer activities, and gene expression changes to define the epigenetic mechanisms that account for the transcriptome alteration in GSCs. While GSCs share a significant portion of enhancers with NSCs, as defined by their histone marks, many GSC-specific active enhancers were identified that potentially link to gene misregulation. These enhancer-shifting patterns indicate that GSCs could lose a set of NSC-specific enhancer footprints and gain a new set of e bodies . The uprAsymmetrical differentiation of GSCs could contribute to the tumor mass, and terminal differentiation of these GSCs could eliminate these cell populations for ideal therapeutic outcomes. Epigenetic changes of GSC and NSC differentiation were profiled in parallel, to understand the precise epigenetic alterations during GSC differentiation relative to that of NSCs. While NSC differentiation acquires developmentally programmed DNA modifications during differentiation, the DNA modifications in the GSC genome appear to be more loosely or randomly controlled, which may explain the fact that GSCs possess greater plasticity regarding various endogenous and extracellular differentiation cues for tumorigenesis. Consistent with this, the GSCs with differential TET2 or TET3 expression levels clearly displayed distinctive responses to chemotherapeutic agents, providing important mechanistic insights of current therapeutic challenges. Given the central role of TET proteins in tumorigenesis, it is possible that they may serve as therapeutic targets for GBM patients. Owing to technical challenges and poor antibody quality, the genome-wide distributions of TET proteins in GSCs are still unknown, and it would be important to correlate the binding dynamics of epigenetic machinery with their marks to gain an improved understanding of their molecular mechanisms in order to further therapeutic efforts.The present study presents the first detailed characterization of the GSC epigenome, including DNA methylation/demethylation and histone modifications associated with distal regulatory elements. However, the GSCs used in this study were isolated from GBM patient-derived xenografts. The development of single-cell-transcriptome and epigenome analyses will enable the further delineation of epigenomic changes associated with primary GBM tumors and GSCs, as well as tumor organoids. These results will provide further insight into epigenomic alterations associated with GBM and GSCs. More importantly, it will be critical to determine the functionality of causal loci with epigenetic alterations associated with GBM, which may lead to identification of novel therapeutic targets and pathway for GBM.


Core deficits in social functioning are associated with various neuropsychiatric and neurodevelopmental disorders, yet biomarker identification and the development of effective pharmacological interventions has been limited. Recent data suggest the intriguing possibility that endogenous cannabinoids, a class of lipid neuromodulators generally implicated in the regulation of neurotransmitter release, may contribute to species-typical social functioning. Systematic study of the endogenous cannabinoid signaling could, therefore, yield novel approaches to understand the neurobiological underpinnings of atypical social functioning. This article provides a critical review of the major components of the endogenous cannabinoid system  and the contributions of cannabinoid signaling to social functioning. Data are evaluated in the context of Research Domain Criteria constructs  to enable interrogation of endogenous cannabinoid signaling in social functioning across diagnostic categories. The empirical evidence reviewed strongly supports the role for dysregulated cannabinoid signaling in the pathophysiology of social functioning deficits observed in brain disorders, such as autism spectrum disorder, schizophrenia, major depressive disorder, posttraumatic stress disorder and bipolar disorder. Moreover, these findings indicate that the endogenous cannabinoid system holds exceptional promise as a biological marker of, and potential treatment target for, neuropsychiatric and neurodevelopmental disorders characterized by impairments in social functioning. Clinicians categorize patients phenomenologically using the DSM, then researchers attempt to identify pathophysiological mechanisms in patient-participants. However, congruence in phenomenology between patients does not necessarily signify similarity in endophenotypes or genotypes, confounding explicit links to pathology.4 To address the disparity in our current understanding of brain dysfunction with clinical phenomenology, a paradigm shift towards a dimensional ‘bottom-up' design in research has been adopted. The Research Domain Criteria (RDoC) is a translational research approach that utilizes a matrix framework to facilitate studies that cut across diagnostic boundaries by probing functional constructs associated with neural circuits. The RDoC provides a comprehensive infrastructure to interrogate social functioning impairments and strongly emphasizes the appropriate experimental classification of patients to support direct translation of research findings for clinical use.2 Social competence is an emergent property, inter- and intra-dependent on functional integrity of three of the RDoC domains: negative valence systems, positive valence systems and cognitive systems. Initial stages of social interaction require overcoming negative valence systems  to initiate the interaction and are reinforced by positive valence systems . Cognitive Systems  guide the exchange after social interaction has commenced. Social process systems  exert supramodal control to coordinate germane practices. Dysfunction in one construct intrinsically affects social information processing and impacts the ability to function typically. In addition, the RDoC framework encourages experimental inquiry at multiple levels of analysis  to foster research within and across constructs, which fundamentally highlights neuromodulatory systems that operate in multiple constructs, such as the cannabinoid system. The functional heterogeneity of the endogenous cannabinoid system recapitulates the diversity of social functioning abilities in neuropsychiatric and neurodevelopmental disorder patients and parallels dimensional analysis of the RDoC.Social functioning impairments are frequently cited as core features of neuropsychiatric and neurodevelopmental disorders, yet progress in biomarker identification and development of targeted pharmacotherapies has been extremely limited. Delays in progress are attributed to social deficit heterogeneity and use of the Diagnostic and Statistical Manual of Mental Disorders (DSM) for investigational research.7 Dr. Moreau noted similarities from experiences in healthy humans after ingesting North African hashish (which contains ‘very high concentration of THC [Δ9-tetrahydrocannabinol]'5) with the diversity of behaviors in neurological dysfunction.6 Moreover, first-person reports from hashish users detail traits that neatly map onto RDoC constructs: for example, ‘fluctuations of emotions' , extreme ‘happiness, excitement' , ‘errors of time and space…and illusions and hallucinations'6 , ‘irresistible impulses…and dissociation of ideas' (cognitive domain).9 However, current research is focused on the use of inhibitors of endogenous cannabinoid degradation  in the study of reward-related processing in social interactions.14 The present article reviews the basic biology of the endogenous cannabinoid system and the roles of its relevant components across each RDoC construct as it pertains to social functioning. Data reviewed within each RDoC construct are applicable to multiple neuropsychiatric and neurodevelopmental disorders, the most notable of which are highlighted as exemplars at the conclusion of each section. With development of diagnostic assessments and treatment options for social functioning deficits limited due to inherent phenotypic heterogeneity, methodical research on the endogenous cannabinoid system has high potential to provide inroads in identifying underlying mechanisms shared across disorders.The first reports on cannabinoid involvement in social functioning are from the nineteenth century psychiatrist Dr. Jacques Moreau de Tours for the treatment of distributed ‘neurological dysregulation' and ‘social alienation'.15 which are distributed throughout the central nervous system16  synapses, mediating GABA suppression or glutamate release and reuptake,19 thereby contributing to the homeostatic maintenance of the brain's excitatory-inhibitory balance.21 CB1R activation also suppresses release of serotonin, dopamine, acetylcholine and noradrenaline,21 thereby mediating the characteristic cognitive and antidepressant effects.18 CB2Rs, in contrast, were initially considered a peripheral nervous system receptor as they were originally from immune cells and gene expression levels were highest in inflammatory responses.22 The presence of brain CB2Rs was first detected after harm, insult or neuroinflammation, which breaks down the blood–brain barrier and allows for non-neuronal immunocytes to infiltrate the central nervous system, thereby increasing CB2R expression.23 Accordingly, expression levels of brain CB2R represent brain health,23 as very low expression indicates a fit brain.24 At homeostasis, low CB2R expression25 is observed in cell bodies and dendrites of neural progenitor cells, neurons, oligodendrocytes, astrocytes26 and stimulated microglia—‘resident macrophages of the central nervous system'.22 Activation of CB2Rs upregulates cell-surface factors in regeneration and degeneration, functions in migration and proliferation, as well as neural cell maturation and survival,27 but the underlying mechanisms remain unclear.Cannabinergic effects is chiefly mediated by stimulation of cannabinoid receptors, type 1 and 2 (CB1R and CB2R),em16 see . Both ty66 Each cannabinoid class and type is distinct in mechanisms of action, behavioral profiles and action in neural circuits. Phytocannabinoids are produced in secretory cells of glandular trichomes at high concentrations in unfertilized female Cannabis flowers. THC is a low potency partial agonist at CB1R and CB2R.65 The non-psychoactive analog of THC, cannabidiol (CBD), is of burgeoning clinical interest for its very low efficacy and partial agonism at CB1R and CB2R. At low molar concentrations, CBD acts as an antagonist or inverse agonist (particularly at CB2R) to limit THC effects and contributes to the upregulation of endogenous cannabinoid signaling.67 Clinically, CBD is a neuroprotectant used as an anti-inflammatory, anticonvulsant, antiepileptic and antipsychotic.8 CBD's effects are attributed to the full agonism it exhibits for non-cannabinoid G-protein-coupled receptors  and interactions with serotonergic, adenosinergic and vanilloid systems.15 Notably, the most efficacious therapeutics are observed with formulations combining THC and CBD (two to four times greater than single cannabinoid preparations68) as CBD enhances the therapeutic action of THC by potentiating its psychotropic effects, augmenting THC tolerability, and widening the therapeutic window.67 Enhanced efficacy is attributed to the entourage effect,68 the synergistic action of the more than 480 biologically active and inactive compounds in Cannabis. It is likely that the as-yet identified major and minor cannabinoids in plant extracts may further enhance the therapeutic benefit observed with THC and CBD through improved stimulation of the endogenous cannabinoid system.65Elucidation of the delta-9 double bond in plant-derived cannabinoid (phytocannabinoid), Δ9-tetrahydrocannabinol (THC), in the early 1960s instigated the search for endogenously synthesized cannabinoids (endocannabinoids) and led to the detection of anandamide (AEA) and 2-arachidonoylglycerol (2-AG).de novo synthesis from membrane phospholipids in response to increased intracellular Ca2+ via depolarization,69 eCBs act presynaptically to inhibit GABA and glutamate release.17 The physiological endpoints of AEA and 2-AG are comparable to THC,69 but are functionally and temporally distinct from one another. AEA has high binding affinity ,70 but low efficacy for cannabinoid receptors. It is most active during steady-state conditions and regulates basal synaptic neurotransmission. Data from rodent autism spectrum disorder (ASD) models demonstrate that genetic mutations in the neuroligin gene disrupt tonic eCB signaling71 and intimate the importance of eCB signaling mode (tonic or phasic) in neurodevelopment and neuropsychiatric disorders. 2-AG is the more prevalent eCB (~4 nmol g−1 in brain tissue vs AEA at <100 pmol g−1)70 and regulates synaptic plasticity.69 It is representative of ‘phasic' signaling evoked in sustained depolarization. The greater overall efficacy and binding affinity of 2-AG ,70 particularly at CB2R,72 is likely related to the enhanced native tone and role in adaptive response. Inactivation of AEA and 2-AG is primarily through enzymatic degradation. AEA is transported across the transmembrane proteins73 and hydrolyzed via fatty acid amide hydrolase (FAAH),47 whereas 2-AG can be hydrolyzed by FAAH or monoacylglycerol lipase.74Endocannabinoids (eCBs) are small (<400 Da) lipophilic activity-dependent retrograde messengers in the brain. Produced post-synaptically ‘on demand' through The endocannabinoid system provides a consummate model to examine social functioning deficits across multiple clinical populations. The distribution of eCB components is congruent with affected neuroanatomy in neuropsychiatric and neurodevelopmental disorders with social functioning impairments see . Changes29 Anxiety is elicited by putatively dangerous, uncertain, imminent situations or events that lead to an acute behavioral response in preparation for threats to individual integrity that is disproportional in intensity or chronicity.29 Anxiety is physiologically manifested as an exaggerated startle response, increased muscle tension, decreased motion, avoidance behaviors and autonomic hyperactivity. With regard to social functioning, pathological anxiety can impede competent functioning in the absence of triggering stimuli. Major components of the neurocircuitry mediating anxiety, such as the prefrontal cortex (PFC), hippocampus, amygdala and hypothalamus are rich in CB1R and CB2R expression , but further study is needed to clarify how best to leverage neurocircuitry modulation with precision to ensure anxiolytic function of cannabinergic pharmacologic interventions and the potential restoration of homeostatic eCB signaling.Anxiety, fear, and stress as terms are often semantically interchangeable, though each is independently characterized by the nature of the eliciting event and magnitude of response.sion see . In anim79 Across development, protracted exposure to stressors imposes a progressive pattern of dysfunction in social functioning, beginning as asociality and culminating in antisociality.79 Persistent activation  of stress neurocircuitry breaks down homeostatic balance33 and creates a 'hypocannabinergic state' through downregulation of CB1R expression in the hippocampus, hypothalamus, striatum and dorsal root ganglion.34 No significant change in CB2R expression was observed in three separate mouse lines, following exposure to chronic stressors, but without analogous data in humans, few conclusions can be made about CB2R function under chronic stress.25 Alterations in CB1R expression are concurrent with changes in eCB signaling in the hippocampus, striatum, dorsal raphe nucleus, hypothalamus, nucleus accumbens (NAc), PFC  and the amygdala. In the absence of stressors, AEA tone suppresses activity of the hypothalamic–pituitary–adrenal (HPA) axis (the chief modulator of the stress response81) and disruption of this suppression initiates HPA-axis activation.81 Chronic stressor exposure impairs AEA signaling and elevates 2-AG content within the amygdala (and increases glucocorticoid hormone secretion), hippocampus, hypothalamus and PFC,33 likely due to altered FAAH and monoacylglycerol lipase-mediated hydrolysis, as the synthesis of these eCBs is unimpaired.82 Glucocorticoid feedback inhibition of HPA-axis activity is suggested to occur on two temporal scales, rapidly via the PFC and 2-AG tone enhancement, or less rapidly via negative feedback inhibition at the paraventricular nucleus of the hypothalamus.82 Glucocorticoids then normalize the amygdalar AEA levels to support basal HPA function.34 The enhancement of 2-AG signaling is perceived as an attempt to habituate to the chronic stress exposure and may be crucial in understanding how to properly engage eCB signaling in the modulation of the HPA axis to guarantee a positive adaptive response.34 However, the presence of a mechanism to examine maladaptive responses to chronic stress exposure highlights novel areas for social deficits in which exposure to a chronic stressor is unavoidable .Stress is an adaptive response to a specific internal or external stressor in preparation for injury or threat. Definitions of stress emphasize the physiological and emotional consequences to relate acute parallel activation of autonomic and endocrine systems. Chronic stress is a response to persistent stressors, like social threat or defeat, which reduces social motivation and social interactions by intensifying fear towards  and avoidance (anxiogenic behavior) of unknown conspecifics.43 Recent data linking reward and social neuropeptides with eCBs highlight a novel, potential intervention specific to social functioning deficits.13 Adaptive reinforcement of social interactions requires long-term synaptic plasticity at excitatory synapses of the NAc and is dependent on oxytocin (OT), a neuropeptide that regulates prosocial behavior and its dysregulation has been implicated in social impairments.84 In the examination of neural circuits of socialization, Wei et al.13 demonstrated an obligatory role of AEA in a socially isolated rodent model. Salience and reward were modulated by AEA mobilization in the NAc and hippocampus via activation of OT receptors by endogenously released OT following social contact. Additional experiments to control for the anxiogenic induction of AEA release during stressful social situations found the observed effects were independent of the stress response and specific to social reward.13 Data suggest that OT acts as a social reinforcement signal to induce long-term depression in medium spiny neurons and requires specific activation of CB1Rs by AEA to regulate social incentive salience. These preclinical data provide insight into the mechanisms associated with OT in social functioning in clinical populations. If improvement in social functioning requires OT-evoked AEA signaling as data suggest, then enhancement of OT to improve social deficits must be sufficient to engage AEA mobilization for proper functioning of the neural circuit. Researchers have advocated that these findings are of particular relevance to ASD, although these results can also be broadly applied to neuropsychiatric disorders characterized by dysregulated OT and impaired social functioning .Reward attainment is one of the only RDoC constructs to explicitly detail eCBs as candidate modulators of reward learning, valuation and processing.37 The inability to separate motivation from social interest has contributed to the assumption that deficits in social functioning are a cause of disrupted social interest, as opposed to a consequence of them.37 Motivation is necessary to attend to social information, seek and engage in social interaction, as well as foster and maintain social bonds, whereas interest does not necessitate action. Individual differences in social motivation are associated with alterations in the activity of neurocircuitry that overlap with reward learning; high social motivation is correlated with enhanced activation of the amygdala and orbital frontal cortex, whereas weaker activation is related to lower social motivation. Opioid and dopaminergic neurons in the ventral tegmental area have complementary roles in motivated behaviors and require eCB signaling to fine-tune dopamine release in incentive-related reward learning.52 Concomitantly, enhanced motivation is observed following CB1R stimulation and with increased AEA signaling in the amygdala, NAc and dorsal striatum and 2-AG in the NAc.42 Diminished motivation is associated with CB1R blockade.39 Wei et al.87 suggest increased AEA content in the forebrain in animal models of ASD can ameliorate social motivation deficits. Extending previous results in rats in which modulation of CB1R or genetic removal of FAAH negatively impacted social interactions,88 researchers examined the influence of AEA on social behaviors via FAAH modulation.13 A social approach task and elevated plus maze were used to assess the effects of increased AEA (via FAAH inhibition) on social motivation in BTBR mice  and Fragile X Syndrome mice . Increased AEA signaling in the forebrains of ASD mouse models correlated with increased time in the social chamber and preferential interaction with novel animals compared with untreated and control mice.87 Effects were not associated with decreased anxiogenic responses, as no change in behavior on the elevated plus maze was observed. Improved prosocial behaviors were ‘generalizable'87 and suggest common neural circuitry of social motivation between idiopathic and syndromic ASD.Social motivation leads to enhancement of social development by promoting social interactions. Abnormal motivation precludes proficient social interaction and has negative consequences on the acquisition of higher-order social abilities.44) supports social interactions by providing biographical and episodic recall.49 It functions as a store of experience-outcomes and integrates previous conclusions with new input, as well as emotion, motivation and perception.44 Stimulation of cannabinoid receptors in hippocampal circuits diminishes glutamate release to below-threshold levels, inhibiting long-term potentiation necessary for encoding.90 In animal models, stimulation of receptors before or after learning induces impaired performance on water maze, contextual fear conditioning and object recognition memory assessments.91 In humans, deficits were not observed in the retrieval of previously stored information, and learning impairments were transient as after a 3-month abstinence from phytocannabinoids, deficits were no longer observed.92 Importantly, cannabinergic modulation of cognitive effects can vary on the basis of the use of exogenous or endogenous cannabinoids. AEA and 2-AG are both robust modulators of early-stage acquisition, consolidation and extinction,90 but it is the enhancement of 2-AG that is correlated with disrupted encoding in spatial memory.94 An abundance of evidence demonstrates transient, dose-dependent THC-induced memory impairments (with a tolerance effect in heavy users) and the contrasting absence of memory deficits following CBD administration. Data also suggest that CBD is protective against THC-induced impairments in episodic and spatial memory.92 Explicit control of eCB signaling in declarative memory for social functioning deficits must account for individual differences in abilities. Brain disorders characterized by difficulties in uncontrolled recall  may benefit from control over extinction, whereas disrupting short-term memory consolidation may be advantageous for other disorders .Declarative memory  and inhibitory control via inhibition of PFC glutamatergic activity, whereas CB1R agonists increased impulsive behaviors. A neuroimaging study90 suggests that THC impacts activity in cerebral inhibition response circuits causing increased hyperactivity in the PFC and anterior cingulate cortices. Acute administration of THC reduces response inhibition89  and causes hyperactivity at dopaminergic synapses in the PFC.90 Data from animal models suggest that cannabinoid signaling interacts with dopaminergic, GABAergic and glutamatergic pathways to mediate behavioral changes. Data in FAAH knockout mice demonstrate that AEA-biased tone improves acquisition in working memory tasks, but the effects are transient and do not persist into later trials.66 This evidence suggests that strategies to improve components of working memory  should focus on control of AEA tone and local modulation of 2-AG signaling.More immediate information processing involves working memory, which actively maintains and updates relevant information, but is capacity-limited.57 Mouse pups that receive intensive maternal care demonstrate enhanced maternal behavior in adulthood demonstrating the enduring effects of maternal care on adult social functioning.96 Disruption of the mother–infant bond has lasting consequences on neuroendocrine and cognitive functioning, increasing the risk for subsequent psychopathology.56 OT is a primary regulator of social behavior,84 particularly in maternal care,54 and is associated with eCB signaling.97 Schechter et al.95 performed several studies examining eCB and OT signaling in maternal care and demonstrated that genetic ablation of CB1R negatively affected maternal care. This impairment correlated with decreased hippocampal OT receptor expression and increased hippocampal levels of 2-AG.95 Results are consistent with observations from socially isolated animals. These studies demonstrated that region-specific CB1R expression in the supraoptic nucleus of socially isolated animals can reduce social play and social interaction compared with those that were pair-housed and handled daily.55 Changes in receptor density affect GABAergic and glutamatergic input to OT-synthesizing neurons for mobilization of OT release,57 which is necessary for social bonding. Valproate-exposed rats, another animal model of social deficits used in ASD research, demonstrate similarly reduced sociability, but changes in CB1R expression were also correlated with reduced hippocampal 2-AG expression.98 Moreover, after social play, increased AEA and 2-AG levels in the NAc and the amygdala were observed.88 These data demonstrate that perturbation of eCB signaling is an elegant potential causal effector in the regulation of maternal care and affiliative behaviors. These results are pertinent to better understanding how cannabinoid modulation contributes to the neurocircuitry of psychiatric disorders related to early-life adversity, such as depression, bipolar disorder and schizophrenia.96Maternal care is a newborn's first social experience and variation in maternal care has been shown to dramatically influence social development.18 which suggests a role for eCB signaling in social communication. However, current findings only supports a role for eCB signaling in nonverbal motor-related aspects of social communication . Genetic variants  in CB1Rs are correlated with greater gaze duration to happy faces and are considered a putative endophenotype for communication deficits, particularly in ASD research.99 Animal models of social communication impairments demonstrate a direct connection to cannabinergic function, which interacts with the forkhead box (FOXP) protein family.58 Interestingly, FOXP are instrumental in ultrasonic vocalizations (USVs) production and vocal learning.100 In rodents, USVs serve a communicatory function to elicit social interaction62 or to share socially relevant information with known conspecifics.63 Moreover, USVs are developmental- and context-dependent62 relating identity, emotionality,63 receptivity to affiliative and sexual behaviors, and changes in the environment.56 Persistent activation of CB1R, via agonists or inhibitors of enzymatic degradation, decrease emission of USVs in rodent pups. Cannabinoids modulate USVs induced by maternal separation in pups,61 whereas in adult rodents, cannabinoids increase the emission of USVs when exposed to anxiogenic stimuli.63 Moreover, CB1R knockout mice have low USV emission throughout development,56 which parallels the communication impairments associated with disorders such as ASD.62 For clinical populations with impairments in language acquisition, especially nonverbal patients with social deficits, cannabinergic modulation of USVs offers a novel and viable area of biomedical intervention in support of communication acquisition and development.100In humans, high concentrations of cannabinoid receptors are found in the left hemisphere cortical regions that are associated with verbal language function,in vivo, the lack of evidence on direct endocannabinoid (AEA or 2-AG)66 highlights the need for further research. Moreover, in the pursuit of precision health,2 although each component of the eCB system can be targeted for therapeutic development, appropriate targeting with respect to the intersection of the eCB system and a disorder's neurocircuitry must be carefully considered to achieve optimal outcomes. It is clear, though, that the eCB system holds exceptional promise as a biological marker of, and treatment target6 for, neuropsychiatric and neurodevelopmental disorders characterized by pronounced abnormalities in social functioning.The diversity of social functioning deficits present in neuropsychiatric and neurodevelopmental disorders confounds the efficacy and specificity of treatment for affected individuals. However, the recent shift in psychiatric research towards examining the underlying dysregulated neural circuits of brain disorders has allowed consideration herein of novel aspects of the functionally diverse eCB signaling system. This article reviewed the eCB signaling system and the role of its components  in varied, but convergent, RDoC domains of social functioning. From a molecular level of analysis, the cannabinoid system is implicated in negative and positive valence systems to restore homeostatic balance, increase the salience of reward learning and motivate social interactions. Behavioral and physiological effects of the eCB system are observed in cognitive systems. Social process systems show evidence of cannabinergic modulation, and as an emergent domain, rely on appropriate eCB function in the aforementioned RDoC domains. Although further work is needed to clarify the discrete roles of cannabinoid signaling in each RDoC domain, the extant evidence strongly supports the contribution of dysregulated cannabinoid signaling to the pathophysiology of social functioning impairments. With regard to the development of pharmacological interventions, although there is a diverse set of methods to enhance endogenous signaling or receptor stimulation


The increasing demand for liver transplantation and the decline in donor organs has highlighted the need for alternative novel therapies to prevent chronic active hepatitis, which eventually leads to liver cirrhosis and liver cancer. Liver histology of chronic hepatitis is composed of both effector and regulatory lymphocytes. The human liver contains different subsets of effector lymphocytes that are kept in check by a subpopulation of T cells known as Regulatory T cells (Treg). The balance of effector and regulatory lymphocytes generally determines the outcome of hepatic inflammation: resolution, fulminant hepatitis, or chronic active hepatitis. Thus, maintaining and adjusting this balance is crucial in immunological manipulation of liver diseases. One of the options to restore this balance is to enrich Treg in the liver disease patients. Advances in the knowledge of Treg biology and development of clinical grade isolation reagents, cell sorting equipment, and good manufacturing practice facilities have paved the way to apply Treg cells as a potential therapy to restore peripheral self-tolerance in autoimmune liver diseases (AILD), chronic rejection, and posttransplantation. Past and on-going studies have applied Treg in type-1 diabetes mellitus, systemic lupus erythematosus, graft versus host diseases, and solid organ transplantations. There have not been any new therapies for the AILD for more than three decades; thus, the clinical potential for the application of autologous Treg cell therapy to treat autoimmune liver disease is an attractive and novel option. However, it is fundamental to understand the deep immunology, genetic profiles, biology, homing behavior, and microenvironment of Treg before applying the cells to the patients. Depleting the CD25+CD4+ T cells from a T cell inoculum increased the rate at which graft versus host disease (GVHD) and features of autoimmune diseases developed in the recipient strain  and peripheral Treg (previously labeled as adaptive Treg) (Regulatory T cells (Treg) are critical regulators of immune tolerance . Regulatnduction . CD4+CD2  .+CD25highCD127low Treg frequency has been described in patients with alcoholic hepatitis  cells were noted in the peripheral blood of patients compared with controls and in patients who had recovered from a previous episode of HBV infection  inhibiting the function or maturation of antigen-presenting cells (APCs), (ii) destroying target cells by inducing apoptosis, (iii) causing metabolic disruption via the adenosine pathway, and (iv) by secreting immunosuppressive cytokines, transforming growth factor beta (TGF-β), and IL-10 or competitive consumption of survival cytokines in particular IL-2 , thereby removing their availability to act as costimulatory ligands through CD28  protein expression on Treg plays an important role in the suppressor function of Treg . In partugh CD28  -1α that acts to increase the frequency and suppressive properties of thymic Treg , 61. Indvia its portal venous flow. It is clear that the microbiome has a strong influence on the immune system. For example, breast-fed infants develop robust populations of memory T cells as well as T helper 17 (Th17) cells within the memory pool, whereas bottle-fed infants do not, and this may partly explain the variation in human susceptibility to conditions with an immune basis, as well as the variable protection against certain infectious diseases  suppressed effector immune response which results in unwanted Treg activity in cancer, (2) impaired immune-regulatory function in autoimmune or inflammatory diseases, and (3) the deleterious consequences of life-long immunosuppression therapy following organ transplantation, the prospect to control disease progression through targeting the regulatory cells in settings of cancer, solid organ and hematopoietic cell transplantation, transplant rejection, and autoimmune diseases has been an attractive option for clinicians over many years.The cytokine IL-2 is essential for the function and expansion of Treg . Thus, tRegulatory T cell therapy” is that administering a concentrated source of a desired population of Treg can tip the balance of the patient’s immune system to enhance its regulatory capacity. It builds on the knowledge that a lack of Treg function due to mutation of the Treg lineage-defining transcription factor FOXP3 leads to the X-linked autoimmune syndrome immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome (IPEX) (+CD25+ Treg could prevent autoimmune disease (The establishment over recent years of good manufacturing practice (GMP)-compliant reagents and equipment that can allow the isolation of cells according to their expression profile of certain surface proteins has made it possible to isolate Treg from the peripheral circulation as a cell immunotherapy. The concept underlying this “e (IPEX) , and, upTo be used therapeutically, a cellular product must be deemed GMP-compliant. This has massive implications upon its production compared with a basic laboratory reagent as the final product and all the reagents and equipment used in its manufacture must exceed a standard clinical quality (purity) and sterility. Accordingly, all staff involved in its preparation must be highly skilled in aseptic procedures and must receive regular re-training. There must be a detailed log of the production and characterization of the final product. Altogether, this imposes extremely high overheads to the production of cellular therapies and with regard to Treg cell therapy has further limited the precise phenotypic character that can be achieved both for practical and for financial reasons. Consequently, the Treg used in trials until now have differed.+CD25+); second generation, bone fide Treg (CD4+CD25+CD127low/−), and third generation naive Treg (CD4+CD25+CD127low/−CD45Ra+)  now exists that can facilitate the isolation and expansion of a highly pure second (CD4+CD25+CD127low/−) and third generation (CD4+CD25+CD127low/−CD45Ra+) GMP Treg preparation straight from a leukapheresis product within a closed system, reducing the risk of loss of sterility and optimizing the purity of the preparation from the start. Moreover, by including Rapamycin and Retinoic acid in the culture cocktail during subsequent expansion, it is possible to ensure that loss of purity through the outgrowth of any contaminating effector T cells is not of concern. Rapamycin precludes the expansion of effector but not regulatory T cells via differential downstream signaling from the IL-2R  antigenic activation of the donor immune system against the host that if not restrained leads to GVHD and (ii) antigenic activation of the host immune system toward the graft, which can lead to graft rejection. GVHD is a major complication in the setting of bone marrow transplantation, owing to the preponderance of T cells in bone marrow, but in solid organ transplant it is generally of lesser concern. Moreover, the tolerogenic nature of the liver and its ability to regenerate  means th+CD25+CD127low/− (second generation) Treg to combat the difficulty in treating GVHD, following allogeneic bone marrow or stem cell transplantation  success , the grade of the individual’s disease, and whether the patient’s disease is in remission  or in relapse . Given that Cyclosporine reduces Treg number, immunosuppression therapies used together with Treg therapy should be tailored to a rapamycin-based regimen along with Tacrolimus, which increases Treg survival. Patients with active viral infection and previous history of malignancy should be excluded from any Treg clinical trial as Treg would have a negative impact on the disease process.It seems likely that isolated cells will have to be expanded in order to be of adequate number to be used in therapy. Hence, one caveat will, therefore, be the reliable expansion of autologous Treg cells for all patients since most studies, to date, have reported a few subjects who were enrolled but failed to receive treatment or treatment at the intended dose due to an inadequate cell yield.in vivo using a humanized mouse model of human skin graft immune rejection. Allogeneic Treg expanded on myeloid DCs from the skin were more effective at limiting dermal and inflammatory phenotypes of skin rejection by infusions of allogeneic T effector cells than polyclonal Treg . With a view to optimize Treg for transplantation therapy, polyclonal and alloantigen-specific Treg have been compared. Alloantigen-specific Treg represent those Treg that are activated by donor-specific antigens and can be expanded from polyclonal Treg populations in mixed lymphocyte reactions with donor APCs. By contrast polyclonal Treg are expanded with anti-CD3/anti-CD28-coated expander beads. Alloantigen-specific T cells can be identified and selected for further expansion based on their induction of activation markers such as CD69 and CD71. The fact that the therapeutic product created by Todo et al. through mixed lymphocyte reaction had greater suppressive ability over recipient T cells activated by donor as opposed to third-party stimulation was clear evidence of underlying allospecific regulatory activity having been raised within the expanded culture, and, as such, using a system to generate greater donor specificity might be advantageous and should allow considerably reduced numbers of Treg to be used . The valnal Treg . Various B cells  activate B cells . A numbe B cells  and PBC  B cells  are know B cells .www.ClinicalTrials.gov and currently recruiting are summarized in Table Clinical trials of Treg therapy for solid organ transplantation and autoimmune diseases that are listed on Overall, Treg hold promise as personalized therapy in the treatment of liver diseases and, in the future, may be applied in the non-transplant as well as post-transplant setting to overcome or minimize the use of broad spectrum immunosuppressant medications that have unfavorable side effects . Their mAll authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication. The views expressed in this paper are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health.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.