Chronic inflammatory diseases are associated with accelerated atherosclerosis and increased risk of cardiovascular diseases (CVD). As the pathogenesis of atherosclerosis is increasingly recognized as an inflammatory process, similarities between atherosclerosis and systemic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel diseases, lupus, psoriasis, spondyloarthritis and others have become a topic of interest. Endothelial dysfunction represents a key step in the initiation and maintenance of atherosclerosis and may serve as a marker for future risk of cardiovascular events. Patients with chronic inflammatory diseases manifest endothelial dysfunction, often early in the course of the disease. Therefore, mechanisms linking systemic inflammatory diseases and atherosclerosis may be best understood at the level of the endothelium. Multiple factors, including circulating inflammatory cytokines, TNF-α (tumor necrosis factor-α), reactive oxygen species, oxidized LDL (low density lipoprotein), autoantibodies and traditional risk factors directly and indirectly activate endothelial cells, leading to impaired vascular relaxation, increased leukocyte adhesion, increased endothelial permeability and generation of a pro-thrombotic state. Pharmacologic agents directed against TNF-α-mediated inflammation may decrease the risk of endothelial dysfunction and cardiovascular disease in these patients. Understanding the precise mechanisms driving endothelial dysfunction in patients with systemic inflammatory diseases may help elucidate the pathogenesis of atherosclerosis in the general population. An expanding body of evidence demonstrates that chronic autoimmune inflammatory diseases are associated with accelerated atherosclerosis and increased cardiovascular morbidity and mortality compared to the general population ,2. AlthoEndothelial dysfunction has been postulated to represent an initial step in the pathogenesis of atherosclerosis in the general population . AccordiWe queried the PubMed database  using the MESH searches for relevant studies using the following search terms in various combinations: rheumatoid arthritis; systemic lupus erythematosus; psoriasis; seronegative spondyloarthritis; inflammatory bowel disease; endothelial function; endothelial dysfunction; endothelial activation; forearm blood flow; flow-mediated vasodilation; cardiovascular disease (CVD); cardiovascular mortality; myocardial infarction; inflammation. Because of the limited number of relevant studies, there were no defined inclusion or exclusion criteria. Studies were screened informally for size and methodological quality. Studies reviewed ranged over the period of 1982–2014, with preference given to more recent data. Systematic reviews and meta-analyses were incorporated when available.Through its capacity to sense and respond to mechanical and biochemical stimuli, the endothelium plays an active and critical role in the physiologic regulation of vascular tone, cellular adhesion, vascular smooth muscle migration and resistance to thrombosis ,10. EndoEndothelial dysfunction has emerged as an important surrogate endpoint for cardiovascular events. Its role in initiating the cascade of events leading to atherosclerosis and atherothrombosis may position it well for use as an early indicator of disease at a point that may allow for effective risk factor modification or pharmacologic intervention prior to the development of full-blown atherosclerosis. Furthermore, the endothelium is viewed as an integrator of vascular risk: the mechanisms by which epidemiologically proven cardiovascular risk factors lead to atherosclerosis might be interrogated best at the level of the endothelium, where the processing of these pathogenic signals may converge into one or several common pathways in the genesis of advanced atherosclerosis.Endothelial function can be assessed in humans by assaying its capacity to perform its various physiologic functions, including regulation of vasomotor tone, expression of adhesion molecules and maintenance of an anti-thrombotic microenvironment. In contemporary clinical research, endothelial function is typically assessed by measuring changes in vasomotor tone in response to various stimuli. Methods of measuring vascular reactivity have become the standard largely due to their reproducibility and demonstrated correlation with other measures of atherosclerosis. Quantification of soluble cellular adhesion molecule expression has also been widely performed, although the usefulness of this technique has not been well established. The most common methods are reviewed below.Quantification of forearm blood-flow (FBF) by venous occlusion plethysmography in response to intra-arterial infusions of vasodilator substances has been historically used to assess vascular reactivity in various patient populations . In thisN-mono-methyl-l-arginine (l-NMMA), an inhibitor of NOS, leads to marked reduction in brachial artery dilatation [Flow-mediated vasodilation (FMD) is currently the most widely used method for assessment of vascular reactivity due to its non-invasive nature ,13,14. Tlatation ,16,17.There has been some concern that assessment of conduit artery function may not accurately reflect endothelial function in the microcirculation. Assessment of endothelium-dependent vasodilation in the cutaneous microcirculation is typically performed by using laser Doppler imaging to measure responses to infusion of vasodilator substances via iontophoresis . SimilarEfforts to define plasma biomarkers for endothelial dysfunction have largely focused on soluble intercellular adhesion molecules (CAMs), including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), E-selectin and others ,21. ThesRecently, the protein asymmetric dimethylarginine (ADMA), an endogenous eNOS inhibitor, has garnered interest as a potential biomarker for endothelial dysfunction . Plasma Rheumatoid arthritis, systemic lupus erythematosus, the seronegative spondyloarthropathies, psoriasis and inflammatory bowel disease have all been associated clinically with excessive cardiovascular risk ,6,7,8,25It has been known for many years that coronary artery disease is largely responsible for the excess morbidity and mortality in patients with RA. Endothelial dysfunction in RA was first described in a seminal 2002 study demonstrating impaired brachial artery responsiveness to ACh by FBF in patients with early disease . Impaireet al. [Efforts to characterize endothelial function by measuring soluble plasma biomarkers in patients with rheumatoid arthritis have been largely unsuccessful. Littler et al.  first deet al. ,49,50, aet al. . There iet al. . In geneThe excess burden of CVD in patients with SLE is now well established. Similar to RA, endothelial function has been widely used as a surrogate endpoint for CVD in patients with SLE. Impaired FMD was observed in patients with SLE as early as 2002 . MultiplAs with RA, efforts to characterize the expression profile of biomarkers for endothelial dysfunction in patients with SLE have been less successful than vascular reactivity studies. Sfikakis demonstrated increased levels of circulating ICAM-1 in patients with SLE . Tulek aMuch less is known about the cardiovascular risk associated with the seronegative spondyloarthropathies; however, the available evidence suggests that these diseases confer increased risk of cardiovascular morbidity and mortality ,5. Similet al. [i.e., erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), disease severity score) in patients with spondyloarthropathies. Moreover, markedly elevated ICAM-1 levels (>400) were found only in patients with disease. In contrast, Sari and colleagues failed to demonstrate any significant differences in ICAM, VCAM, E-selectin or P-selectin levels in patients with spondyloarthropathies [Less is known about adhesion molecule expression in this population. As with RA and SLE, the available data are discordant. Wendling et al.  demonstropathies . Plasma opathies ,71.Cutaneous psoriasis, even in the absence of joint disease, has been linked to accelerated atherosclerosis. The evidence for this association is less robust than for RA and SLE, though the available data support a significantly increased risk for cardiovascular events and mortality in patients with severe disease. Patients with mild disease may be spared ,25. AccoSimilar to psoriasis, there has been recent interest in exploring the cardiovascular implications of inflammatory bowel diseases (IBD), Crohn’s disease and ulcerative colitis (UC). While individual studies have demonstrated an increased risk of cardiovascular events in patients with IBD, mortality risk is less clear. A 2014 meta-analysis of 9 studies demonstrated that patients with IBD were at significantly increased risk of ischemic heart disease and stroke . MortaliIn 2003 Hatoum and colleagues demonstrated that the intestinal microvessels in patients with IBD show significantly impaired endothelium-dependent vasodilation compared to vessels from healthy control patients . In addiMultiple models of inflammatory arthritis have been described for the study of rheumatoid arthritis and other inflammatory joint diseases in animals. The collagen-induced arthritis (CIA) model in mice is the most widely-used animal model for the study of RA. In this model, intradermal inoculation of mice with type II collagen and Complete Freund’s Adjuvant (CFA) generates an intense inflammatory polyarthritis immunologically similar to RA . He and While it is clear that multiple chronic inflammatory diseases are associated with endothelial dysfunction and cardiovascular morbidity, the mechanistic links between inflammatory diseases and CVD have not been fully elucidated. The role of traditional cardiovascular risk factors in patients with RA and SLE has received considerable attention, though traditional factors alone are insufficient to explain the excess burden of CVD in these populations. It seems likely that chronic inflammation, a shared feature of these diseases, is involved in the pathogenesis of accelerated endothelial dysfunction. Several potential mechanisms are explored below .The vascular endothelium is known to be a target of TNF-α. On a cellular level, TNF-α induces the expression of genes associated with inflammation, coagulation and proliferation. Decreased nitric oxide (NO) bioavailability appears to be a common and critical step linking TNF-α to endothelial dysfunction. Multiple groups have shown that eNOS protein expression is reduced via TNF-α- induced inhibition of eNOS promoter activity and mRNA destabilization ,87. NO ain vivo in both animal and human models. Intravenous delivery of TNF-α in rats leads to impaired endothelium-dependent vasodilation [Salmonella typhi vaccination also causes reduced FBF [l-NNMA (l-NG-monomethyl Arginine) [−/−, TNF-α−/−apoE mouse model. Mice deficient in TNF-α develop less atherosclerosis than those with intact TNF-α expression  [The effect of TNF-α on NO availability and subsequent endothelial dysfunction has also been demonstrated dilation . Intra-adilation . Non-speuced FBF . This efrginine) . The downockout) . This isIt is well known that TNF-α plays a critical role in the inflammation associated with RA, SLE, IBD, psoriasis and spondyloarthritis. This common feature is illustrated by the efficacy of anti-TNF-α agents in many of these diseases. Given the central role of TNF-α in the pathogenesis of many chronic inflammatory diseases and its well-characterized effects on the endothelium as described above, it is reasonable to conclude that increased circulating TNF-α is implicated in the induction of endothelial dysfunction and initiation of atherosclerosis in these diseases . This hyin vivo animal models have demonstrated reduced NO bioavailability in the presence of elevated ROS, and reversal of endothelial dysfunction has been achieved via infusion of antioxidants [Chronic inflammatory diseases are generally associated with increased oxidative stress. In RA, reactive oxygen species (ROS) levels from peripheral blood neutrophils correlate positively with disease severity and markers of systemic inflammation . Inflammoxidants .In addition to downregulating NO bioavailability, superoxide and other ROS are capable of inducing NF-κB, a critical step in transforming endothelial cells into an “activated” state characterized in part by increased surface expression of CAMs ,99. As dversus healthy controls, whereas others have described a distinct profile of suppressed LDL and HDL in RA patients with more advanced disease  [The role of traditional cardiovascular risk factors such as dyslipidemia and insulin resistance in the pathogenesis of endothelial dysfunction and atherosclerosis in patients with chronic inflammatory diseases has received significant attention. Although it has been reported that patients with RA and other rheumatic diseases are more likely to have elevated low-density lipoprotein (LDL) and total cholesterol and reduced high-density lipoprotein (HDL) levels, the data are inconsistent. For example, multiple studies have shown no differences in lipid profiles of patients with RA achexia) . Chronicachexia) . TNF-α aachexia) .Dyslipidemia is independently associated with endothelial dysfunction. Elevated LDL and total cholesterol are associated with impaired endothelium-dependent vasodilation, whereas elevated HDL levels correlate with improved endothelial function . ImpaireMany chronic inflammatory diseases are associated with production of autoantibodies, many of which are instrumental in the pathogenesis of the disease. Similarly, autoantibodies directed against normal endothelial or plasma constituents have been detected and implicated in the pathogenesis of endothelial dysfunction and atherosclerosis in the general population. Anti-endothelial cell antibodies (AECA) directed against a variety of endothelial cell structural proteins have been identified in a number of autoimmune diseases, including SLE . These aAntibodies directed against oxidized LDL (anti-oxLDL) have been described in patients with and without chronic inflammatory diseases. In SLE, anti-oxLDL antibodies correlate with disease activity and markers of systemic inflammation . Althougi.e., lupus anticoagulant, anticardiolipin antibody, and anti-B2GPI antibody). Although their contribution to venous and arterial thrombotic events is well known, the relative contribution of aPL to the development of endothelial dysfunction in humans, if any, is currently unclear. The effect of aPL on endothelium-dependent vasodilatation may be reflected in the observation that patients with aPL exhibit impaired FMD and reduced NO bioavailability versus healthy controls [in vitro and in vivo. Efforts to measure circulating levels of soluble adhesion molecules in patient with APS have been less consistent, however [Antiphospholipid antibodies (aPLs) are present in the serum of nearly one third of lupus patients. The antiphospholipid antibody syndrome (APS) is characterized by recurrent venous or arterial thrombosis, pregnancy loss and the presence of antiphospholipid antibodies (controls . aPL hav however . FurtherMethotrexate remains the mainstay of therapy for RA and several other rheumatic diseases . An inhiThe critical role of TNF-α in the generation of severe systemic inflammation in these conditions likely explains the effectiveness of these agents. TNF-α may also be largely responsible for the endothelial dysfunction and accelerated atherosclerosis in these patients, making anti-TNF-α agents attractive therapeutic options for preventing CVD in this population. Numerous studies have demonstrated improved endothelium-dependent vasodilation in patients with RA after initiation of anti-TNF-α therapy. This has been demonstrated in a vessel-specific manner by measuring FBF immediately after intra-brachial infusion of infliximab. In this model Cardillo and colleagues demonstrated that the local effect of infliximab on the brachial artery improved brachial artery endothelial function without altering markers of systemic inflammation . Multiplet al. [Anti-TNF-α agents have also been shown to reduce levels of plasma biomarkers of endothelial dysfunction, although results have been inconsistent. Klimiuk et al.  demonstret al. . Adalimuet al. . These fversus no therapy [Corticosteroids have long been used to manage a variety of inflammatory diseases, but their effects on CVD have been controversial. The association between steroids and insulin resistance and obesity has raised concern for increased cardiovascular risk, while their anti-inflammatory effects might mitigate this risk. Studies addressing the association between long-term steroid use in RA and CVD have yielded variable results. A 2011 systematic review of studies of low-dose steroid use in RA found that corticosteroids are generally associated with mildly increased cardiovascular risk . Studies therapy . This st therapy . These ret al. [Statins exhibit pleiotropic properties influencing the vasculature that are thought to contribute to their clinical benefit beyond the lipid-lowering effect. Although the mechanisms are incompletely characterized, statins have been shown to improve endothelial dysfunction in patients with traditional cardiovascular risk factors. Potential mechanisms include upregulation of eNOS, leading to enhanced bioavailability of NO and improved vasoreactivity . The useet al.  demonstrPatients with chronic inflammatory diseases are at high risk for cardiovascular morbidity and mortality. In many inflammatory diseases, this heightened risk of CVD is reflected in early endothelial dysfunction as assessed by vasoreactivity studies, even in the absence of detectable atherosclerosis. The endothelium therefore represents an integrator of vascular risk and the study of its dysfunction may help elucidate mechanisms driving accelerated atherosclerosis in these populations.There is strong evidence that the mechanisms responsible for accelerated atherosclerosis in patients with inflammatory diseases are related to the high-grade inflammation inherent to the primary disease process. The effects of TNF-α and inflammatory cytokines on induction of endothelial dysfunction are well described and are likely to represent key mediators of endothelial dysfunction and atherosclerosis. Furthermore, the numerous studies demonstrating improved endothelial function after anti-TNF-α therapy highlight the importance of these molecules in the pathogenesis of endothelial dysfunction and may lead the way toward advances in pharmacologic prevention of CVD in these populations. Many other mechanisms, including autoantibodies, oxidative stress and interactions with traditional risk factors such as dyslipidemia and insulin resistance are likely to be involved, and further research is required to elucidate the relative importance of these processes. Finally, current strategies to reduce cardiovascular morbidity and mortality are focused on controlling traditional modifiable cardiovascular risk factors and reducing disease activity. The precise mechanisms driving atherosclerosis are likely to vary between different inflammatory diseases, however, and parsing out these subtleties may help identify unique therapeutic targets for each disease.


Oncocytoma or oxyphilic adenoma is uncommon salivary gland tumour, occurs predominantly in the in patients older than 60 years of age. Clinically oncocytoma resemble other salivary tumours while histology is typically consisting of oncocytes with many hyperplastic mitochondria. It usually occurs in the parotid gland. Because the features of oncocytoma are similar to those of other benign and low-grade malignant salivary tumours, clinical diagnosis is often challenging.This report presents the pathologic and imaging findings of an oncocytoma arising in the deep lobe of the right parotid gland in a 74-year-old male. Oncocytoma was diagnosed on the basis of histological, magnetic resonance imaging (MRI), scintigraphic findings and fine needle aspiration cytology (FNAC).This case was unique because in the literature there are few articles about the rare presentation and deep lobe location of this type of parotid oncocytoma. Oncocytoma or oxyphilic adenoma is a rare benign salivary gland tumor, which mostly occurs in the parotid glands, few affect the submandibular and rarely affect the minor salivary glands -4. OncocThe clinical presentation of oncocytomas is usually identical to other benign salivary tumors. The image modalities of choice to identify the well-defined homogeneous mass are CT Scan MRI, ultrasonography, radionuclide salivary scintigraphy and positron emission tomography CT scans , 12.Complete surgical removal with superficial or total parotidectomy is the treatment of choice, whereas radiation therapy is not indicated because oncocytes are radioresistant . RecurreLong-term follow-up using imaging refinements is recommended to evaluate the patient’s progress. Here we are presenting the case of a 74-years-old male patient with right oncocytoma of the deep lobe of the parotid gland extending into para pharyngeal space.A 74-year-old man underwent a computed tomographic (CT) scan to determine the status of cervical lymph node involvement and discrete swelling of the right parotid gland. One year before, the patient had undergone partial resection of soft palate for mucoepidermoid carcinoma. Neck lymph node involvement was negative, but 32 x 24 mm well-circumscribed mass was incidentally detected in the deep lobe of the right parotid gland . MRI of The mass showed low-signal intensity to parotid gland parenchyma on T1- and T2-weighted images and homogeneous enhancement after intravenous gadolinium-based contrast medium administration. On the basis of these findings, the lesion was diagnosed as a deep lobe parotid tumour. FNAC with screening performance was applied immediately prior to operative treatment. A parotidectomy was performed with mandibular osteotomy, selectively excising a portion of a deep lobe of the right parotid gland . PostopePostoperative period was uneventful. The patient was treated with antibiotics and analgesics for five days. There was no clinical evidence of neurological deficit and was discharged with good health on the third day after surgery. The patient was comfortable at review after two months.Oncocytoma accounts for less than 1 - 2.2 % of all salivary gland tumours , 8-10. TThe parotid gland is a preferred site of oncocytoma. The tumours are generally noncystic, encapsulated, and rarely exceed 5.0 cm in diameter. Their location at onset is usually unilateral, but sometimes multiple or bilateral (7%) oncocytomas are seen , 11, 12.The growth grade rate is typically slow. Although malignant transformation and local recurrence of this tumour are unusual and uncommon, clinical follow-up is important because malignant oncocytoma may not be correctly diagnosed owing to histological similarities with benign oncocytoma . DiagnosChakrabarti  reportedWe have evaluated the present case with scintigraphy and supplementary MRI. Adenolymphoma -Warthin’s tumour and oncocytomas reveal intense uptake with marked retention of activity after stimulation on salivary radionuclide imaging while other primary or metastatic malignancies appear cold on radionuclide scintigraphy with technetium-99m-pertechneate. In general, MR images of Warthin’s tumours show decreased signal intensity on T1-weighted images and increased signal intensity on T2-weighted images.In our patient, oncocytoma showed decreased signal intensity on both T1- and T2-weighted images. This difference made it possible to distinguish oncocytoma from Warthin’s tumour. The decreased signal intensity on both T1- and T2-weighted images is attributed to the high cellularity and low free water content of oncocytoma.Fine-needle aspiration cytology (FNAC) is another fairly accurate preoperative procedure for the diagnosis of parotid tumours. Capone et al.,  reportedBecause the scintigraphic findings of the present case were not clearly compatible with Warthin’s tumours or oncocytoma, preoperative FNAC analysis under ultrasonography guidance was performed. The postoperative histological examination was performed for final evaluation of the tumour.In conclusion, oncocytoma of the deep lobe of the parotid gland is extremely rare but have distinctive imaging characteristics that have not been described for other benign or malignant parotid gland tumors. The other salivary gland tumors where oncocytes are found can usually be differentiated on the basis of their more typical cytologic details.Imaging findings are helpful in preoperative assessment to distinguish these benign lesions from other parotid gland tumors in different clinical circumstances. Through this case, we highlight the importance of histopathological examination in the positive diagnosis of parotid oncocytoma as well as in its differential diagnosis and also the importance of FNAC.


The immune system has a key role to play in controlling cancer initiation and progression. T cell activation, which is central to anti-tumor immune responses, coincides with changes in cellular metabolism. Naïve T cells predominantly require an ATP generating metabolic profile, whereas proliferating effector T cells require anabolic metabolic profiles that promote rapid growth and proliferation. Furthermore, specific T cell subsets require distinct energetic and biosynthetic pathways to match their functional requirements. The often hostile tumor microenvironment can affect T cell immune responses by altering the resulting cellular metabolism. Tailoring immune responses by manipulating cellular metabolic pathways may provide an exciting new option for cancer immunotherapy. T cell responses might also be skewed via metabolic manipulation to treat the complications of obesity-associated inflammation, which is a rapidly growing global health problem and a major risk factor for many malignancies. In this review, the diverse metabolic requirements of T cells in anti-tumor immunity are discussed, as well as the profound influence of the tumor microenvironment and the possible avenues for manipulation to enhance anti-tumor immunity. EFF), which must rapidly increase cell mass and elicit effector functions through cytokine production and cytotoxicity (MEM) poses a different metabolic demand and to facilitate this need, TMEM cells utilize fatty acid oxidation (FAO) as their main energy source , which allows homing to and retention in secondary lymphoid organs , where they play a pathological role in driving obesity-associated inflammation  (MEM cells unlike naïve and TEFF cells have a greater spare respiratory capacity (SRC) in their mitochondria  . TMEM cechondria  -β. The expression of HIF-1α plays an important role in regulating the balance of Th17/TREG responses as it inhibits differentiation of TREG cells by targeting Foxp3 for proteasomal degradation , which results in the production of high levels of lactate which promotes aerobic glycolysis. Treatment with the PDK-1 inhibitor dichloroacetate (DCA) inhibited the proliferation of T cells and reduced inflammatory cytokine production by switching the metabolism back to OxPhos. In mouse models this resulted in reduced airway inflammation , an inhibitor of hexokinase-2 (Hk2), was used to block glycolysis (Figure MEM cell development, such as AMPK which negatively regulated mTOR and Foxo1, and resulted in enhanced anti-tumor activity of CD8+ T cells (MEM cells by the modulation of fatty acid metabolism have also been investigated. Treatment with metformin to activate AMPK promoted TMEM cell generation and was able to significantly improve the efficacy of an experimental anti-cancer vaccine in vivo (MEM cell activity still requires further investigation (MEM cells (MEM cells include the inhibition of the Th1 promoting transcription factor T-bet and the stabilization of the expression of the related transcription factor eomesodermin (EFF cells to virus specific TMEM cells without inhibiting the ability of the infected animal to clear the antigen (MEM cells is to enhance the efficacy of cancer vaccines. The main goal of any vaccination is to induce an effective TMEM cell response (MEM cell generation have a better recall response and are more protective following re-challenge (Increasing the number of long-lived tumor specific T T cells . Other m in vivo  (Figure tigation . Many metigation  and novetigation . RapamycEM cells  (Figure sodermin . Treatme antigen . Due to  antigen . Like me antigen  and inhi antigen . Unlike  antigen . Althoug antigen . Anotherresponse , many ofhallenge .+ and CD8+ T cells are significantly enriched in obese VAT, while others have found that frequencies of TREG cells are decreased (+ T cell proliferation in a mouse model of arthritis (EFF cells as key players in adipose tissue inflammation, ERK and mTOR inhibitors might elicit a similar anti-inflammatory effect in obesity (Obesity induces a state of tumor-promoting chronic inflammation, marked by elevated levels of free fatty acids, abnormal cytokine production, and activation of inflammatory signaling pathways , 119. Thecreased , 124. Thrthritis . This st obesity . Treatme obesity . In addi obesity . The sma obesity . The inh obesity . From thREG and TMEM cells favor FAO, which allows these cells to survive long term, when glucose and growth factors are limited. This ensures the control of inappropriate immune responses and long term immunity. Extensive metabolic reprograming not only fulfils the extra biosynthetic demands of activated T cells, but it is also integrated with signaling pathways that determine T cell fate, thus enhancing our understanding of distinct T cell subset function and regulation. The different metabolic pathways may also provide a new opportunity for modulating the immune response during tumor development and progression. One of the hallmarks of cancer is evasion of the immune system (EFF and TMEM cell-mediated responses are enhanced, while those of TREG cells are decreased, ultimately providing a therapy that might “switch” back on the immune system to target the tumor cell. Reactivating exhausted T cells by adjusting their metabolism also holds potential as a treatment for cancer, however more research needs to be carried out to fully elucidate the role of cellular metabolism in T cell exhaustion. Due to the similarities between tumor cells and activated T cell’s metabolic programing, any therapies designed to modulate tumor cell metabolism must take into account their effects on T cell metabolism in the immune response to cancer.It is clear that T cell survival, function, and differentiation are intimately linked to T cell metabolism. The increase in glycolysis and glutaminolysis facilitate the rapid proliferation of T cells required during activation to allow fast clearance of pathogens or tumor cells. Te system  and in tEFF cells are found to be enriched in the VAT of cancer patients, it is likely that they play pathological roles in driving obesity-associated inflammation. Attenuation of this inflammation might be achieved via metabolic manipulation of inflammatory TEFF cell differentiation and activation. Suppression of excessive pro-inflammatory T cell responses through inhibition of metabolic pathways has already been demonstrated across a spectrum of chronic inflammatory disorders in mice and could represent a viable therapeutic target for obesity-associated cancers.Since the WHO now estimates that obesity is directly attributable for as many as 41% of malignancies and with the rates of obesity continuing to increase dramatically, it is clear that novel therapeutic options are urgently required. Since activated inflammatory TThe possibilities of modulating anti-tumor immune responses through targeting T cell metabolic pathways hold huge promise and clearly warrant further investigation.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.


Although the blood–brain barrier (BBB) was thought to protect the brain from the effects of the immune system, immune cells can nevertheless migrate from the blood to the brain, either as a cause or as a consequence of central nervous system (CNS) diseases, thus contributing to their evolution and outcome. Accordingly, as the interface between the CNS and the peripheral immune system, the BBB is critical during neuroinflammatory processes. In particular, endothelial cells are involved in the brain response to systemic or local inflammatory stimuli by regulating the cellular movement between the circulation and the brain parenchyma. While neuropathological conditions differ in etiology and in the way in which the inflammatory response is mounted and resolved, cellular mechanisms of neuroinflammation are probably similar. Accordingly, neuroinflammation is a hallmark and a decisive player of many CNS diseases. Thus, molecular magnetic resonance imaging (MRI) of inflammatory processes is a central theme of research in several neurological disorders focusing on a set of molecules expressed by endothelial cells, such as adhesion molecules , which emerge as therapeutic targets and biomarkers for neurological diseases. In this review, we will present the most recent advances in the field of preclinical molecular MRI. Moreover, we will discuss the possible translation of molecular MRI to the clinical setting with a particular emphasis on myeloperoxidase imaging, autologous cell tracking, and targeted iron oxide particles . Brain–immune interactions play a central role in acute neurological disorders including ischemic stroke, intracranial hemorrhage and traumatic brain injury . Given tBrain–immune interactions occur all over the time course of acute neurological diseases. Upon injury, endothelial cells of the cerebrovasculature become activated and release the content of their Weibel-Palade bodies. This leads to P-selectin exposure on their surface and subsequent adhesion of neutrophils through P-selectin – P-selectin Glycoprotein Ligand-1 (PSGL-1) interactions . In paraImprovement in our general knowledge of the brain–immune interactions taking place during acute neurological disorders revealed new potential therapeutic targets. Nevertheless, the successes and failures of immunomodulatory treatments for acute brain injury remind us that precise characterization of the interplay between leukocytes, endothelial adhesion molecules, glial, and neuronal cells is mandatory for efficient design of therapeutic strategies targeting brain–immune interactions. For instance, Natalizumab, a monoclonal antibody targeting the interaction between very late antigen 4 (VLA-4) and VCAM-1, has been successfully developed as a treatment for multiple sclerosis . In contFigure 1).In this context, molecular MRI appears highly promising to select patient candidates for immunomodulatory treatments, monitor treatment efficiency, and more generally improve our knowledge on brain–immune interactions in human patients. The purpose of this review is to present recent advances in the field of molecular MRI of brain–immune interactions. After presentation of the theoretical bases of molecular MRI and of the brain-specific issues of this non-invasive method, we will present recent studies relevant to brain–immune interactions after acute cerebral injury with a particular focus on MPO imaging, autologous cell tracking, unlabeled ultrasmall superparamagnetic particles of iron oxide (USPIOs), targeted USPIOs, and targeted microsized particles of iron oxide . New methods of molecular MRI have been recently developed  but they still present a limited sensitivity compared to more conventional methods. Their development remains however of great interest since such contrast agents could allow imaging multiple targets simultaneously  from biological tissues. Basically, two different classes of MRI contrast agents are available: T1 contrast agents increase the signal on T1-weighted images by shortening the spin-lattice relaxation time (T1 constant). T2 and T2* contrast agents decrease the signal on T2- and T2*-weighted images by both shortening the spin–spin relaxation time (T2 constant) and dephasing adjacent protons (by modification of their precession angular velocity). Therefore, depending on the contrast material injected, a molecular target can be revealed by an increase  or a decrease  in the MRI signal  to a protein of interest using a targeting moiety (pharmacophore). This targeting moiety can be either a specific ligand  or a substrate of an enzyme which can accumulate in the regions where its corresponding enzyme is overexpressed/active. For instance, VCAM-1 imaging can be performed using microparticles of iron oxide  coupled to monoclonal antibodies targeting VCAM-1 (the pharmacophore). After intravenous injection, this agent binds to endothelial VCAM-1 and induces T2* effects leading to decreased signal on T2*-weighted images  in regions presenting activated endothelial cells .+ emitting atoms at picomolar concentration, MRI using classical Gadolinium-based contrast agent (GBCA) presents a sensitivity in the micromolar range. To overcome this limitation, contrast agent carrying large payload of gadolinium or iron oxide have been developed, including paramagnetic liposomes and iron oxide particles . In this regard, MPIOs (1–4 μm in diameter) are particularly interesting since even a single particle can be detected by high-field MRI at high resolution  effects induce signal voids on T2*-weighted images which are comparable to MPIO-induced signal void and could therefore lead to false positive results. To overcome this, we demonstrated in a recent study in mice that a short preparation time (30 min) of normobaric hyperoxia can be sufficient to transform deoxyhemoglobin (paramagnetic) into oxyhemoglobin (diamagnetic) by increasing the tissular concentration of O2 . Thus, tThe presence of the BBB represents a challenge for molecular imaging of the brain. Indeed, to reach parenchymal targets, contrast agents should be able to cross the BBB. To date, there is no contrast agent able to cross the BBB in sufficiently large quantities to induce significant changes in MRI signal. Therefore molecular imaging of the brain is limited to endothelial proteins , to diseases with compromised BBB  and to cells capable of crossing the BBB (such as monocytes). The purpose of this paragraph is to present the main issues related to each imaging targets that should be kept in mind when performing molecular MRI of brain–immune interactions.Endothelial proteins are targets easily accessible to large contrast-carrying particles and have been accordingly successfully imaged using different contrast agents . The first issue related to imaging of endothelial targets is the requirement of a high affinity of the pharmacophore for its target because of the force exerted by the blood flow. These shear forces promote detachment of the contrast agent from the endothelial wall and thus limit the time window for imaging . MoreoveIssues related to imaging of intraparenchymal targets in conditions of injured BBB are numerous. First, the molecular weight of the contrast agent should be low to maximize its passive extravasation through the BBB. The drawback is that this limits the quantity of contrast material carried by contrast agent molecules. In fact, only GBCA have been used for this purpose. Second, the specificity of this method is limited: even in the absence of the molecular target, the contrast agent accumulates in an unspecific manner in the brain regions with BBB leakage. Activatable contrast agents such as bis-5HT-DTPA(Gd) (a substrate for MPO) might partially overcome this limitation . Enzyme Figure 4.Imaging of leukocyte diapedesis is the third strategy developed for molecular MRI of the brain. Accumulation of leucocytes  in the brain is a specific feature of neuroinflammation and then represents an attractive target for molecular imaging. However, this is a relatively delayed event after injury and requires a delay between injection of the contrast agent and imaging in order to allow its uptake and accumulation of leukocytes within the parenchyma. The prototypic contrast agents used to label leukocytes are unlabeled ultrasmall particles of iron oxide USPIO; . These sTable 1).In the next paragraphs, we will present the main studies reporting the use of molecular MRI for studying brain–immune interactions after acute cerebral injury  is associated with a Gadolinium-based contrastophore which is responsible for the paramagnetic effect. In the presence of MPO, the 5-hydroxytryptamide moiety of bis-5HT-DTPA(Gd) is oxidized and radicalized by hydrogen peroxide. The radicalized bis-5HT-DTPA(Gd) molecule can react with another radicalized bis-5HT-DTPA(Gd) molecule to form a polymer of up to five subunits leading to an amplification of the enzymatic reaction. In mice, the activated agent can also bind to proteins within inflammatory sites, trapping the agent, further increasing its molecular weight, and increasing the signal intensity on T1-weighted MRI . ConvertIn experimental autoimmune encephalomyelitis (EAE) induced with synthetic proteolipid protein, a model of multiple sclerosis, MPO imaging has been tested for its ability to detect active plaques before clinical symptoms appearance in mice. The authors found that bis-5HT-DTPA allows detection of MPO, as a surrogate marker of the appearance and size of active plaques, and was correlated to clinical grading . In the Bis-5HT-DTPA has also been used in a model of transient focal stroke . In this study, MPO imaging correlated with infarct size, and the authors hypothesized that MPO imaging could be used to select and stratify patients for clinical trials of therapies targeting inflammation . In an eMyeloperoxidase imaging has thus proven relevant to diagnose, predict, or evaluate therapeutic efficiencies in several models of CNS disorders. However, as already mentioned, it is widely recognized that these MRI techniques have limitations because contrast enhancement reflects breakdown of the BBB with leakage of paramagnetic chelates rather than active inflammation, and the two may not always correspond. Another potential limitation is that MPO imaging does not discriminate between MPO secreted from resident microglia, infiltrated macrophages or neutrophils.Finally, the activated MPO sensor is cleared from the brain within 6 h after administration; the protein-bound, activated MPO agents are likely digested and released by proteases that are present at sites of inflammation .Therefore, enzymatic imaging targeting MPO points to an interesting technology for non-invasive confirmation of active inflammatory lesions in brain disease. This could potentially not only improve disease diagnosis and treatment assessment in the clinical setting, but may also lead to a better evaluation of drug development and clinical trials of new therapies. This method is, however, impaired by its lack of specificity due to passive extravasation and accumulation of the unprocessed contrast agent, which contributes significantly to MRI signal changes.in vivo labeling of circulating cells using untargeted USPIO, this method has the advantage of a higher specificity since the contrast agent particles are clustered inside immune cells and are thus less susceptible to reach the brain in an unspecific manner (such as passive extravasation through a leaky BBB). Moreover, it allows direct imaging of brain–immune interactions as a whole, taking into account all the processes involved in the regulation of endothelial binding and blood to brain diapedesis of autologous cells. The basis of this method is to harvest autologous cells (most frequently leukocytes) and to label them with a high payload of contrast agent without impairing their ability to reach the CNS. Several labeling methods have been performed, based either on phagocytic uptake of contrast agent particles or on direct transfection of the cells with iron particles or Gadolinium using permeating agents.Administration of exogenously labeled cells and subsequent tracking by non-invasive imaging has encountered numerous successes in molecular imaging of brain immune interactions. Unlike One of the first studies investigating the use of systemically injected autologous labeled cells for molecular imaging demonstrated that rat bone marrow stromal cells were able to invade a photochemically induced brain lesion 7 days after intravenous injection . Later, ex vivo using polylysine and ferumoxide and injected the labeled cells intraperitoneally in recipient mice. The authors were able to detect both ex vivo and in vivo the labeled cells in the lumbar spinal cord of EAE mice after the onset of clinical signs. Interestingly, the primary location of the encephalitogenic T-cells revealed by molecular MRI in this study was later confirmed by A similar method has been used by in situ by lipopolysaccharide injection. They found that brain delivery was dramatically enhanced when the injected cells and the brain expressed VLA-4 and VCAM-1, respectively. This kind of clever experiment can be performed using various types of circulating cells to improve our knowledge of brain–immune interactions.Besides these proof-of-concept studies, molecular imaging of exogenously labeled cells allows to easily investigate the mechanisms driving the interactions between circulating cells and the brain endothelium. For instance, The main limitation of this method is its reduced applicability for diagnostic purposes in humans. Although radiolabeling of autologous cells has been used successfully for imaging inflammation using SPECT and PET , the lim3O4), and are usually encased in polysaccharide, synthetic polymers, or monomer coatings (Unlabeled USPIOs are virus-sized molecules with a very long blood clearance time (over 24 h). Iron oxide agents have greater contrast sensitivity compared to GBCA, generating signal voids on MRI, due to shortening of T2 and T2* relaxation times. A large range of sizes exist, from nano- to micron-sized iron oxide particles. USPIOs are among the most famous MR contrast agents, with a range in size from 10–50 nm, and should be differentiated from the larger SPIOs and MPIOs with diameters about 50–300 nm and 0.9–5 μm, respectively. Iron oxide particles are based on magnetite  was initially approved for iron-replacement therapy in patients with chronic renal failure. However, ferumoxytol has also been investigated in humans for various CNS imaging applications  was injected intravenously 5 h post-onset  whereas for USPIOs SHU 555 C , it is below 8 pg iron/cell (®) with a long blood half-life compared to larger ferumoxides, such as dextran-  or anionic-carboxydextran- (Resovist®) coated SPIOs . This would avoid non-specific free iron particles leakage over the BBB and/or entrapment in the vasculature , as suggested by experimental  rabbits  and in h rabbits . These g rabbits . Using tin vivo imaging. Moreover, USPIOs conjugated to a VCAM-1 specific cyclic peptide  have recently been developed for in vivo detection of inflamed vessels in early and advanced atherosclerotic plaques by ultra-high field strength MRI ) on the MPIOs surface.Micro-sized particles of iron oxide have singular properties for endothelial cell-specific molecular imaging. First, their micron size range allows endovascular specificity unlike USPIOs agents which are susceptible to passive diffusion within the brain parenchyma, passive accumulation in atherosclerotic plaques or even non-specific macrophage uptake. Second, MPIOs convey a large payload of iron oxide , which is an order of magnitude larger than USPIOs contrast agents, resulting in strong hypointense contrast effects that may extend up to 50 times the physical diameter of the particle. This phenomenon, known as “blooming effect,” provides high sensitivity articles . Third, articles , allowinarticles . FinallyLimitations of these agents for CNS imaging are related to their non-biodegradable coat and their potential iron toxicity. Indeed, MPIOs’ coated-sheath includes inert polymers giving them a non-biodegradable nature. Micro-sized particles may therefore accumulate in the reticulo-endothelial system , 2008. TMicrosized particles of iron oxides have been applied for imaging of inflammation in many experimental studies, including various animal models of CNS disorders. Due to its properties mentioned earlier, VCAM-1 is an interesting biomarker in the field of molecular imaging and has been thoroughly studied over the last years. For instance, MPIOs targeting VCAM-1 has been studied in mouse models of acute cerebral inflammation , 2012, cin vivo detection of cerebrovascular inflammation in pre-clinical models in mice . In a model of acute inflammation in mice receiving a microinjection of pro-inflammatory cytokine tumor necrosis factor (TNF) into the right striatum, while the left striatum was not injected and served as internal control, we reported that in vivo T2*-weighted MRI reveals a potent hypointense contrast effect in the TNF injected hemisphere after MPIOs-αVCAM-1 injection 24 h post-onset. No contrast effects were seen in the non-injected hemisphere or in animals injected with control MPIOs-IgG. Similarly, although pre-contrast MRI images failed to reveal the ongoing pathology, contrast-enhanced MRI using MPIOs-αVCAM-1 revealed hypoperfusion-triggered CNS injury in vascular dementia, unmasked amyloid-induced cerebrovascular activation in Alzheimer’s disease and allowed monitoring of disease activity during EAE  and used them for non-invasive and high-sensitive  in mice . They pr in mice , and arering EAE .in vitro using similar dual antibody-conjugated MPIOs, targeting both VCAM-1 and E-selectin (Other vascular biomarkers (mostly cell adhesion molecules) have been studied over the last few years as potential targets for MR molecular imaging of inflammation. ICAM-1, unlike VCAM-1, is constitutively expressed in endothelial cells. ICAM-1 is nevertheless up-regulated during an inflammatory response. selectin .Microsized particles of iron oxides are well-tolerated in mice, with no short-term ill effects. Clearance experiments showed that MPIOs are sequestered by the liver and spleen 24 h after injection, with no evidence of adverse effects such as tissue infarction, inflammation, or hemorrhage. The MPIOs used so far in experimental studies are non-biodegradable, due to their inert coat. However, the basic iron contrast mechanism is potentially transferable to humans with suitable adaptation of the surface coat.Their size similar to circulating platelets, their short blood half-life and the exceptionally potent T2* MRI contrast effects of targeted MPIOs, together with advancement of biocompatible MPIOs, provide translational imaging opportunities for improved diagnosis and treatment of endovascular inflammation in various CNS disorders. However, extensive toxicological profiling and synthesis of sterile, clinical-grade targeted MPIOs according to good manufacturing practice will be required before clinical translation is feasible.To date, molecular MRI studies consisted primarily on proof-of-concept studies reporting the feasibility to image a particular target. Very few studies used molecular MRI as a tool to investigate brain–immune interactions. Reasons for this are numerous including the lack of reliability of the imaging procedures with the already mentioned false negative (mainly due to a lack of sensitivity of most molecular MRI procedures) and false positive results . In particular, most of the MRI contrast agents are described in unique studies and no confirmatory results in other models or by other groups are presented. Altogether, these drawbacks limited the confidence of the molecular imaging community in molecular MRI and finally raised doubts about its feasibility. Of note, the recent use of MPIO-based contrast agents dramatically improved the reliability of molecular MRI and the latest studies were able to provide new insights into brain immune interactions.Figure 6). This pathophysiological concept is supported by other studies demonstrating the critical role of VCAM-1 dependent T-cells diapedesis in delayed lesion growth after ischemic stroke  not only in the ischemic lesion, but also in intact brain regions . In partc stroke . In our c stroke . Experimc stroke . The excin vitro study demonstrated that MPIOs labeled with VCAM-1 targeting antibodies do not promote endothelial inflammation, in contrast to monocytes  and on the spatial resolution of the MRI acquisition. This last parameter would be crucial to optimize for clinical translation since the resolution achieved in preclinical study is below 80 μm (isotropic) and allows single MPIO imaging whereas the highest resolution of 3D T2*-weighted images on clinical magnet is around 500 μm. This lower resolution, because of partial volume effects, will lower the sensitivity of the MRI acquisition to detect MPIOs. To overcome this issue, longer acquisition times to increase the signal to noise ratio and strengthen the T2*-weighting will be necessary and could limit the clinical availability of this method. Interestingly, a whole brain 3D T2*-weighted image with an isotropic resolution of 500 μm can be acquired using a 7 Tesla clinical magnet in 6 min with a high signal to noise ratio using echo planar imaging . Such imWhile several years ago the feasibility of rapid and reliable molecular imaging of brain–immune interactions using MRI was elusive, the mini-revolution represented by the development of MPIO-based contrast agent has changed this view. Although molecular MRI using targeted MPIOs still remains restricted to preclinical imaging, studies aiming at closing the translational gap are ongoing. Once available, such contrast agents will undoubtedly help to improve our knowledge on the brain–immune interactions taking place in the human brain after acute neurological injury. Besides, they will represent potent tools to select patient for anti-inflammatory treatment and subsequently monitor the therapeutic response. Nevertheless, molecular MRI using targeted MPIOs is limited to imaging of endothelial targets and does not allow long term investigation of the fate of leukocyte once in the brain parenchyma. To this aim, exogenous labeling of autologous cells and subsequent cell tracking using MRI remains the method of choice. These two imaging strategies  appear today as the most promising technologies to non-invasively study immune cells trafficking in both preclinical and clinical studies. These tools will help to determine whether molecular MRI of brain–immune interactions can improve the management of patients presenting acute brain injury.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.


However, prior to employing neurons as a model in a dish, they need to be evaluated for their electrophysiological properties, including both passive and active membrane properties, dynamics of neurotransmitter release, and capacity to undergo synaptic plasticity. In this review, we survey recent attempts to study these issues in human induced pluripotent stem cell-derived neurons. Although progress has been made, there are still many hurdles to overcome before human induced pluripotent stem cell-derived neurons can fully recapitulate all of the above physiological properties of adult mature neurons. Moreover, proper integration of neurons into pre-existing circuitry still needs to be achieved. Nevertheless, in vitro neuronal stem cell-derived models hold great promise for clinical application in neurological diseases in the future.In order to understand and find therapeutic strategies for neurological disorders, disease models that recapitulate the connectivity and circuitry of patients’ brain are needed. Owing to many limitations of animal disease models,  In vitro models using patient-derived cells are currently emerging to study neuropathologies and test possible treatments, as the in vitro system is more scalable, controllable and cheaper. In particular, recently developed techniques to generate human induced pluripotent stem cells (iPSCs) [The complexity of the human central nervous system and its inaccessibility to direct studies make its modeling necessary in order to investigate physiological and pathological processes occurring in it. Animal disease models have been introduced to study pathophysiological processes and eventually develop new treatments. However, the use of animal models has drawbacks, including high costs of maintenance and difficulties to fully mimic the characteristics of a human neurological disease.  (iPSCs)  allow th (iPSCs)  and the  (iPSCs) , 3. SuchIn this review, we will first discuss the characteristics of the development of both basic electrophysiological properties in maturing neurons and their synaptic activity, as well as integration of individual neurons into synaptic circuitry. The passive and active membrane properties and the presence of spontaneous postsynaptic currents are strong indicators of neuronal maturation and can be used to evaluate the potential therapeutic viability of the different protocols. Next, we will evaluate the derangement of synaptic properties underlying disease processes. Finally, we will discuss recent studies on stem cell-derived human neurons and how they recapitulate physiopathological features of brain neurons.in), membrane capacitance (Cm), and the membrane time constant (τ) as well as the resting membrane potential (RMP). With progressive neuronal development, Rin and τ values have been found to decrease whereas Cm values increase and the RMP shows a negative shift [in and τ values together with depolarized RMPs enable AP generation in response to weak membrane currents. Thereby, the electrophysiological profile of immature neurons might function to compensate for the rather low frequencies of synaptic neurotransmission in early developing networks by increasing the chance of AP generation upon presynaptic transmitter release. Similar to passive membrane properties, measurement of active membrane properties underlying AP formation and propagation allows for analysis of the electrophysiological profile of developing neurons and is particularly helpful in distinguishing pyramidal glutamatergic from inhibitory interneurons via their distinct AP shapes and firing patterns [A central characteristic of neurons is their ability to send and receive signals by means of action potential (AP) formation and propagation with subsequent synaptic neurotransmission. The underlying neuronal properties permitting intercellular signaling are progressively changing during early network formation as well as during differentiation of stem cells into neurons. Indeed, the developmental stage of neurons can be assessed electrophysiologically by measuring their passive and active membrane properties as well as synaptic currents. Passive membrane properties commonly investigated in studies monitoring neuronal development include input resistance , the occurrence of giant depolarizing potentials (GDPs) and progressively increasing frequencies of both GABAergic and glutamatergic spontaneous neurotransmission, indicative of developmental synaptogenesis . ImportaBoth in the developing and the adult central nervous system, glial cells facilitate differentiation and maturation of cells into neurons  and playN-methyl-d-aspartate receptors (NMDARs) and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors located at the postsynaptic membrane. While during the resting state NMDARs are blocked by Mg2+ ions, when a strong depolarization is induced by glutamate binding to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors the Mg2+ block is removed and Ca2+ ions enter the cell via NMDARs. The calcium influx activates a cascade of second messenger events that trigger nuclear transcription factors followed by gene transcription, protein synthesis underlying synaptic strengthening. NMDAR-dependent LTP thus requires presynaptic glutamate release and postsynaptic depolarization promoting structural presynaptic and postsynaptic changes [Strong, repetitive synaptic activity leads to synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) are two forms of synaptic plasticity that have been widely investigated and are linked to learning and memory (reviewed in ). During changes . Convers changes .In summary, important factors that control synaptic potentiation or depression are the intensity of presynaptic activation, postsynaptic depolarization and the amount of calcium flowing through the NMDARs . BecauseIn order to investigate the functionality of the neurons, different methodologies have been developed over the years. The method of choice depends on many factors: the level of resolution needed, the possibility to study neuronal circuitry or intact systems, the necessity to manipulate the experimental conditions, the interest in studying synaptic events or sensory responses and, no less important, the cost, time and difficulty of using the technique. Most of the time, complete understanding of a phenomenon requires the use of more than one methodology. In this chapter, we present the methods most frequently used by neuroscientists to analyze neuronal properties. A summary of the characteristics of the methods described in this section is presented in Table in vitro and in vivo can be studied by a series of techniques including both intracellular and extracellular recordings, as well as imaging techniques allowing investigation of neurotransmitter release. The method of choice depends on the required level of detail and the specific scientific question to be answered. At a cellular level, intracellular recordings are used to measure voltages or currents across the cell membrane [Electrophysiological properties of neurons membrane . The patmembrane  allows tin vitro multi-electrode array methods have been developed [At a multicellular level, extracellular recordings of local field potentials are used to study the collective activity of many cells by monitoring the signals in the extracellular space of the brain, in order to investigate the synaptic connectivity of neuronal circuits in specific areas. More recently, eveloped  and are eveloped .in vivo recordings, where the same techniques explained above are used in live animals. With this method, one can study brain regions or neurons in their intact state with their normal complement of inputs and targets. The cells being studied usually have not been severed or damaged, in contrast to studies in brain slices, and have developed normally in the intact organism, in contrast to the culture preparation. Importantly, this technique allows for the investigation of neuronal responses to sensory stimuli.Another important category of electrophysiological analysis is represented by 2+ influx triggers exocytosis of synaptic vesicles containing neurotransmitters [2+ level in dendritic spines is essential for the induction of activity-dependent synaptic plasticity [Calcium is an essential intracellular messenger in mammalian neurons. At rest, most neurons have an intracellular calcium concentration of about 50 to 100 nM that can rise transiently during electrical activity to levels that are 10 to 100 times higher . Use of smitters . Postsynasticity .2+ ions by changing their fluorescence properties, and detecting Ca2+ transients using a fluorescence microscope and a charge-coupled device camera. Images are then analyzed by measuring fluorescence intensity changes, and the derived fluorescence intensities are plotted against calibrated values for known calcium levels to learn the Ca2+ concentration.Briefly, the calcium imaging technique consists of two steps: loading the cells with fluorescent molecules that can respond to the binding of Ca2+ signals in neuronal processes in vivo using the genetically encoded calcium indicator GCaMP [in vitro differentiated neurons following transplantation in future studies.There are two main classes of calcium indicators: chemical indicators and protein-based genetically encoded calcium indicators. Among the chemical indicators, the most recently developed Oregon Green BAPTA and fluo-4 dye families  are wideor GCaMP . This meRecently, a new technology has been developed to control, either by exciting or inhibiting, the activity of targeted individual neurons: optogenetics. An August 2005 report described how neurons became precisely responsive to light upon introduction of a microbial opsin gene . In the in vitro differentiated cells. Furthermore, a recent study transplanted ESC-derived motor neurons that formed functional neuromuscular connections allowing for optogenetic control of muscle contraction in vivo [in vitro differentiation methods thus provides an attractive prospect for future patient-specific treatments.Importantly, optogenetic approaches have been used to confirm functional integration into pre-existing networks of neurons and NPCs differentiated from iPSCs and embryonic stem cells (ESCs) –52. By s in vivo . The useNeurotransmitter release is often investigated though FM dye visualization during vesicle cycling . These dThe study of neuronal electrophysiological properties in a clinical setting has been mostly permitted by electroencephalography (EEG) and evoked potentials. Nowadays, the combination of scalp EEG recording with functional magnetic resonance imaging (fMRI) allows an in-depth understanding of phenomena underlying EEG activity. By combining the two techniques, one can determine which brain areas change their neuronal activity during an epileptic discharge, and consequently understand the anatomical location and physiological activation of neuronal networks generating normal and abnormal EEG signals. For instance, in temporal lobe epilepsy, temporal localized spiking on EEG has been found to be accompanied by a widespread activation in distant regions on fMRI, especially in the contralateral homologous regions, suggesting that the neuronal network involved in temporal spiking is much wider than what appears on scalp EEG . FurtherNowadays, clinical investigation also uses techniques that have been developed especially for animal studies. For instance, at the level of single neurons, epileptic activity is investigated on brain specimens resected during epilepsy surgery using the patch clamp technique. These studies have shown a major role for ion channels in the generation of excessive and hypersynchronous neuronal discharges , a hallmark of the epileptic activity. In fact, the abovementioned discharges originate from alterations of neuronal excitability or synaptic transmission induced by spontaneous or experimental modifications of the properties of voltage-dependent or receptor-activated ion channels .Integration of stem cell-derived neurons is not yet used in clinical practice. However, implants of iPSC-derived neuronal precursor cells in embryonic or adult mouse brains are currently explored. Thus, one can envision a time at which EEG, fMRI and evoked potentials will be utilized to assess functionality of stem cell-derived neuronal grafts in humans.Dysfunctional synaptic circuits have been found in neurodevelopmental disorders such as autism spectrum disorders, psychiatric disorders such as schizophrenia, and neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD) and spinal muscular atrophy (SMA) –66. BothIn AD amyloid precursor protein transgenic mice there is a reduction of sodium current density, which results in aberrant EEG activity and impaired performance with the Morris water maze memory test . ElectroOne of the most common features of neurodegenerative diseases is the progressive and region-specific accumulation of proteins. These aggregated proteins induce neurons in the affected regions to adapt to their immediate environment and affect synaptic plasticity . For insWith respect to PD, cortical Lewy bodies are found in the soma of nigral dopamine neurons. These Lewy bodies are composed of protofibrils of aggregated α-synuclein . In animWith regard to SMA, a deficiency of survival motor neuron protein is associated with sensory-motor network dysfunction and muscle deterioration. Importantly, in mice lacking survival motor neuron protein a reduction of proprioceptive synaptic inputs onto motor neurons and spinal reflex deficits have been observed .Nevertheless, animal models cannot fully recapitulate the key aspects of human models . RegardiNeuronal differentiation of stem cells is commonly monitored through immunocytochemical methods confirming the expression of specific neuronal markers . For insRelying on immunocytochemical methods alone to identify synaptic connections might have significant pitfalls. Postsynaptic receptors have been shown without corresponding markers for the presynaptic active zone . SimilarVarious studies characterized the presynaptic expression of neurotransmitters as well as the postsynaptic expression of the respective neurotransmitter receptors, such as glutamate receptors and GABA receptors , 87–90. in vitro differentiated neurons.Recent iPSC studies are using additional approaches, such as microarray analysis, deep RNA sequencing, and quantitative PCR arrays, to determine the status of neuronal differentiation . MicroarTerminally differentiated cells can be converted into neurons either by first generating iPSCs  and subsin vivo has been well characterized in rodent neurons of various brain structures [in values higher than 1 GΩ, with a rapid decline over the course of the first postnatal month to around 100 MΩ. Similarly, τ values are usually high (>30 milliseconds) around the perinatal period, indicating slow adaptation of the membrane potential to membrane currents. τ values also decrease rapidly in the first postnatal month to around 10 to 20 milliseconds. Cm values have been shown to be below 50 pF at the time of birth and progressively increase over the first 2 weeks after birth. Consistently, APs of perinatal rodent neurons are characterized by small amplitudes of 30 to 40 mV, broad full widths at half-maximum (FWHMs) of more than 3 milliseconds and depolarized threshold potentials of around −30 mV. Similar to passive membrane properties, AP properties mature equally rapid over the first postnatal month, with AP amplitudes increasing to >90 mV, FWHMs becoming faster than 1.5 milliseconds and AP thresholds reaching more hyperpolarized values at around −40 mV, depending on the specific brain area and cell type. In addition to intrinsic electrophysiological properties, the development of synaptic neurotransmission has been equally well described in various studies using developing rodent neurons. It was shown that neurotransmission events occur already in the immediate neonatal period at low frequency (<0.5 Hz) and that the frequency of spontaneous neurotransmission increases progressively with development [In the past three decades, the development of electrophysiological properties ructures , 107–110elopment , 112. AnIn contrast to rodents, there are only a few studies on the development of electrophysiological properties in primate neurons. The stage of rodent brain development is thought to be equivalent to the developmental stage of the human brain around the time of the third trimester of pregnancy . Consistin values matured similarly from 1 to 3 GΩ 1 month after differentiation to 0.5 to 1 GΩ after 2 months. Other passive membrane properties followed the same path. Thus, passive membrane properties experienced similar developmental shifts as AP properties. Importantly, electrophysiological properties matured faster in human iPSC-derived neurons co-cultured with astrocytes, showing Rin values below 1 GΩ after 2 months [Neurons derived from either rodent or human iPSCs have been shown to fire a single AP in response to depolarizing current injections as early as 3 weeks after differentiation, and the number of generated APs rises with increased culture durations , 10, 106in vitro differentiated neurons. Electrophysiologically, the measurement of spontaneous neurotransmission provides information about the network. In neurons derived from human iPSCs, spontaneous neurotransmission occurs between 3 and 6 weeks after differentiation [The formation of synaptic connections is of central importance in the estimation of functional quality of ntiation , 10, 104ntiation . When coin vivo and transplantation of human ESC-derived and iPSC-derived neurons into rodent brains has been performed in few studies [in vivo maturation of iPSC-derived neurons is thus required.There are limited electrophysiological data available on the maturation of iPSC-derived neurons  studies , 116. Hu studies , 115. Siin values of human iN cells in the GΩ range, Cm values around −30 pF and an RMP of −40 mV 1 month after transfection, reflecting incomplete neuronal development [The neurophysiological development of the direct conversion of mouse-derived or neonatal human-derived somatic cells appears to follow a similar path to iPSC-derived neurons, with reports of AP firing cells as early as 8 days after transduction , 117. Hoelopment . Importaelopment , 119. CuSpontaneous neurotransmission has been reported for iN cells derived from direct neuronal conversion of mouse and fetal human somatic cells , 117. HoRecently, a new approach has been developed to combine direct conversion with ESC and iPSC methods, termed ESC-iN and iPSC-iN . In theiin vitro differentiation. Several studies have investigated the extent of neuronal differentiation of human iPSCs using whole genome analysis [Over the course of neuronal development, various genes are time dependently expressed and can be analyzed to determine the state of neuronal maturation. Following early neuronal lineage determination of ESCs, NPCs express nestin . During analysis , 124. Branalysis . In the Several studies have recently characterized the functional properties of the neuronal cultures derived from human stem cells. These studies have demonstrated that neurons derived from mouse and human stem cells display neuronal-like characteristics, including voltage-gated currents and the ability to fire APs. However, the maturation of human iPSC-derived neurons appears incomplete, as indicated by their immature passive and active membrane properties . Importain vitro studies [Several studies have transplanted ESC-derived neurons into animal models of neurological diseases. ESC-derived neurons were found to be integrated within brain circuits and to differentiate into mature neurons when implanted into mouse embryos . These n studies , 10, and studies .in vitro and in vivo, and tumorigenicity [With regard to the application of iPSCs for regenerative medicine, the use of iPSCs may eliminate the chances of immune rejection as patient-specific cells may be used for transplantation . There agenicity .Table 4CCuring neurological diseases is a challenging task in experimental therapeutics. The incidence of neurodegenerative diseases has enormously increased due to a rise in life expectancy. However, there are only symptomatic treatments available for several reasons: a lack of knowledge about disease pathogeneses; limited access to tissue samples for early stages of neurodegeneration; and the inability of animal models to recapitulate all aspects of human diseases. The development of stem cell technology provides a powerful tool in neurobiology that might help solve these problems. The availability of human ESCs, iPSCs and iN cells enables the investigation of diseases in unprecedented depth. Human ESCs can be genetically modified to harbor a desired mutation and develop a phenotype of disease. However, the incomplete penetrance and variability among phenotypes of genetic disease can be an obstacle for accurate disease modeling. Bioethics and limited availability are also of concern. On the other hand, human iPSCs or iN cells might be the best methods available to reprogram patient cells into neurons. They allocate the opportunity to turn back the clock or to uncover the mechanisms underlying cause and progression of sporadic neurodegenerative diseases whose cause has yet to be identified . There iin vitro is still a big scientific challenge. Although we can model neuronal circuits in a dish and confirm the expression of neuronal markers and electrophysiological functionality, we cannot yet conclude that these circuits represent the actual connectivity and mature circuitry present throughout the central nervous system. There is thus the need to refine techniques aimed to produce neurons in vitro that properly integrate into pre-existing circuits to assume physiological functions [Electrophysiological techniques are not the only approaches to investigate the phenotype and maturity of stem cell-derived neurons. Genome editing technologies are also being used to demonstrate the relationship between genotype and phenotype. Genome-wide association studies might uncover causative or predisposing genetic loci contributing to multifactorial disease. Another benefit of using stem cell-derived neurons is in large-scale screening of pharmacological agents preventing or curing the disease. The opportunity for personalized medicine is also a very promising application of stem cell technologies in which neurons can be derived from the same individual for whom the therapy is tailored. In summary, recapitulation of human neurological disease following stem cell-derived neuronal differentiation unctions .


Information about the feasibility, barriers and facilitators of antimicrobial stewardship (AMS) in residential aged care facilities (RACFs) has been scant. Exploring the prevailing perceptions and attitudes of key healthcare providers towards antibiotic prescribing behaviour, antibiotic resistance and AMS in the RACF setting is imperative to guide AMS interventions.Semi-structured interviews and focus groups were conducted with key RACF healthcare providers until saturation of themes occurred. Participants were recruited using purposive and snowball sampling. The framework approach was applied for data analysis.A total of 40 nurses, 15 general practitioners (GPs) and 6 pharmacists from 12 RACFs were recruited. Five major themes emerged; perceptions of current antibiotic prescribing behaviour, perceptions of antibiotic resistance, attitude towards and understanding of AMS, perceived barriers to and facilitators of AMS implementation, and feasible AMS interventions. A higher proportion of GPs and pharmacists compared with nurses felt there was over-prescribing of antibiotics in the RACF setting. Antibiotic resistance was generally perceived as an issue for infection control rather than impacting clinical decisions. All key stakeholders were supportive of AMS implementation in RACFs; however, they recognized barriers related to workload and logistical issues. A range of practical AMS interventions were identified, with nursing-based education, aged-care specific antibiotic guidelines and regular antibiotic surveillance deemed most useful and feasible.Areas of antibiotic over-prescribing have been identified from different healthcare providers’ perspectives. However, concern about the clinical impact of antibiotic resistance was generally lacking. Importantly, information gathered about feasibility, barriers and facilitators of various AMS interventions will provide important insights to guide development of AMS programs in the RACF setting.The online version of this article (doi:10.1186/1471-2334-14-410) contains supplementary material, which is available to authorized users. There is increasing evidence that the elderly population in residential aged care facilities (RACFs) serves as an important reservoir for multidrug-resistant (MDR) organisms , 2, withEssentially, the need for AMS and the potential areas for AMS interventions are reliant upon existing antibiotic use and resistance patterns. For example, a study in Australian RACFs has shown less frequent use of broad-spectrum antibiotics such as fluoroquinolones  comparedInternational guidelines for infection control and prevention have strongly recommended initiation of AMS programs in the RACF setting ; howeverThis is part of a larger study exploring key healthcare providers’ views about improving antibiotic use in the RACF setting. The study targeted primarily high-level care RACFs (i.e. nursing homes) affiliated with four major public healthcare service networks within metropolitan and regional Victoria, Australia. These residential care facilities deliver 24-hour nursing care to elderly residents requiring significant assistance in their activities of daily living. There was no institutional policy for antibiotic prescribing in any of the RACFs; however, intravenous antibiotic therapy when required is normally delivered via specialized support from hospitals.Three major groups of healthcare providers servicing the participating RACFs were invited to participate, namely general practitioners (GPs), nurses and pharmacists. At these RACFs, the medical care is provided by off-site GPs  from different practices, who visit residents periodically or upon request. There are significant roles for nursing staff in daily care of residents, including ringing GPs to request medical assessment. Prescription medicines, including antibiotics, require an order from the GPs and are supplied by external community pharmacies. Medication review for individual residents [known as Residential Medication Management Review (RMMR)] is normally performed on an annual basis by consultant pharmacists, however this does not involve audit of short-term antibiotic use.Institutional ethics approvals from the human research ethics committees of all participating healthcare service networks and Monash University were obtained prior to participant recruitment. A combination of purposive and snowball sampling strategies were used for recruitment of different healthcare providers . The afoNursing staff in different clinical positions  were involved in either one-on-one interviews or focus groups. All NUMs and RNs were involved in daily care of residents, whilst the executive nursing staff were responsible at the policy-making level for quality improvement of resident care. Face-to-face or phone interviews were conducted with the GPs and pharmacists, depending on their preference. We used several triangulation strategies; we sought information from different stakeholders’ perspectives (i.e. data triangulation), performed onsite observation on the organizational workflow and documentations related to antibiotic prescribing , and explored views of participants from RACFs in different locations .All interviews were conducted using a semi-structured interview guide, which was tailored to different healthcare providers , and 21 registered nurses (RNs)], 15 GPs and six pharmacists consented to participate in the study. The majority of participants were interviewed individually, with 15 RNs participating in three focus groups (range 4–6 RNs per focus group). All except four interviews were conducted face-to-face with participants. The demographic characteristics of the participants are described in Table There were mixed views about existing antibiotic prescribing behavior Table . SeveralThere were also mixed perceptions about antibiotic resistance Table . SeveralThe majority of participating nursing staff were unaware of the concept of AMS. In comparison, more GPs and pharmacists were aware of AMS, although they generally felt that AMS was relatively new in the RACF setting. AMS refers to integrated activities that help to optimize antibiotic therapy, ensuring the best clinical outcomes whilst minimizing the risk for the emergence of antimicrobial resistance. When this concept of AMS was explained, in general, all key stakeholders were supportive of AMS programs in RACFs. Amongst GPs, the main value of AMS was thought to be in promoting evidence-based practices for antibiotic prescribing in this setting Table , Q1. AllA number of perceived barriers and facilitators in relation to implementation of AMS interventions were raised. From the GPs’ perspective, several raised concerns about the potential for doctor autonomy to hinder acceptance of institutional policies and guidelines. Heterogeneity in prescribing practices amongst GPs from different practices pose another barrier, thus having fewer GPs with greater patient loads working in each facility would promote more consistent prescribing practices Table , Q7-Q8. Three major areas of AMS interventions emerged as potentially useful and practical in the RACF setting Table . The mosTo our knowledge, this is the first study exploring the perceptions and attitudes of a range of healthcare providers towards antibiotic prescribing behaviour, antibiotic resistance, and AMS implementation in the RACF setting. A greater proportion of GPs and pharmacists than nursing staff felt that there was over-prescribing of antibiotics, suggesting a lack of awareness amongst nurses about potential antibiotic misuse among this population. Antibiotic resistance and the emergence of MDR organisms were perceived as more of a concern from the infection control perspective as opposed to impacting empiric antibiotic selections. Additionally, this study has highlighted the prevailing attitudes amongst key healthcare providers that AMS interventions were needed and deemed useful in the RACF setting. A number of perceived barriers to AMS programs were identified, in particular, nursing staff workload and the logistical issues of off-site GPs and pharmacists. A range of potential AMS interventions have been suggested to provide insights into feasible AMS model for the RACF setting.et al. has explored the views of the physicians and nurses about antibiotic prescribing in Canadian RACFs but it focused specifically on treatment for asymptomatic bacteriuria, reporting that education about asymptomatic bacteriuria was viewed as an important priority by both physicians and nurses [Published data describing the key stakeholders’ views of antibiotic prescribing and antibiotic resistance have mainly focused on the general practice setting, with limited information from the RACF setting , 19. A sd nurses . In addid nurses , 8.Previous studies have reported that increasing the awareness about antibiotic resistance would potentially influence GPs’ decisions in selecting antibiotics, underlining the importance of knowledge about MDR organisms in assisting clinical decisions , 21, 22.AMS interventions in the RACF setting have been few despite mounting evidence of inappropriate antibiotic prescribing among this elderly population , 24. ExiThis study has predominantly involved high-level care hospital-affiliated RACFs. There may be differences in antibiotic prescribing behaviour in comparison to low-level care or private RACFs, and thus this study could be replicated more widely to other RACF setting for further exploration. Given the differences in long-term healthcare delivery models between different countries , the finIn summary, AMS interventions have been deemed applicable and useful by the key stakeholder groups involved in the provision of healthcare in RACFs. Potential barriers and facilitators for AMS interventions have been highlighted, providing important information to guide allocation of future AMS resources. More importantly, the major areas of AMS deemed most needed and practical in the RACF setting have been identified to guide the development of a feasible AMS model.Caroline Marshall and David CM Kong are senior authors.Additional file 1: Semi-structured interview guide for general practitioners.(PDF 216 KB)Additional file 2: Semi-structured interview guide for nursing staff.(PDF 209 KB)Additional file 3: Semi-structured interview guide for pharmacists.(PDF 155 KB)