Chronic inflammation is a major characteristic feature of Parkinson’s disease (PD). Studies in PD patients show evidence of augmented levels of potent pro-inflammatory molecules e.g., TNF-α, iNOS, IL-1β whereas in experimental Parkinsonism it has been consistently demonstrated that dopaminergic neurons are particularly vulnerable to activated glia releasing these toxic factors. Recent genetic studies point to the role of immune system in the etiology of PD, thus in combination with environmental factors, both peripheral and CNS-mediated immune responses could play important roles in onset and progression of PD. Whereas microglia, astrocytes and infiltrating T cells are known to mediate chronic inflammation, the roles of other immune-competent cells are less well understood. Inflammation is a tightly controlled process. One major effector system of regulation is HPA axis. Glucocorticoids (GCs) released from adrenal glands upon stimulation of HPA axis, in response to either cell injury or presence of pathogen, activate their receptor, GR. GR regulates inflammation both through direct transcriptional action on target genes and by indirectly inhibiting transcriptional activities of transcriptional factors such as NF-κB, AP-1 or interferon regulatory factors. In PD patients, the HPA axis is unbalanced and the cortisol levels are significantly increased, implying a deregulation of GR function in immune cells. In experimental Parkinsonism, the activation of microglial GR has a crucial effect in diminishing microglial cell activation and reducing dopaminergic degeneration. Moreover, GCs are also known to regulate human brain vasculature as well as blood brain barrier (BBB) permeability, any dysfunction in their actions may influence infiltration of cytotoxic molecules resulting in increased vulnerability of dopamine neurons in PD. Overall, deregulation of glucocorticoid receptor actions is likely important in dopamine neuron degeneration through establishment of chronic inflammation. Parkinson’s disease (PD) is a common age-related neurodegenerative disorder characterized by cardinal motor symptoms that include bradykinesia with resting tremor, rigidity and gait disturbance. These motor symptoms become evident when already 70–80% of nigrostriatal terminals have degenerated. At present, therapeutic treatments, for example, levodopa, mostly address motor symptoms. However, a wide spectrum of non-motor clinical features such as REM (Rapid eye movement) sleep disturbances, autonomic dysfunction, depression, anxiety, cognitive impairment or falling are associated with PD, and are moreover debilitating and unresponsive to dopamine-related treatments. Thus, PD is a complex systemic disorder with non-motor symptoms often preceding motor symptoms and worsening with disease progression  gene coding for α-synuclein protein ; presence of proteinaceous inclusions called Lewy bodies (LBs) and chronic inflammation. However, the initial causes and underlying mechanisms pertaining to these neuropathological features in the majority of patients, classified as sporadic PD, remain unknown. Recent studies indicate that combined genetic, environmental factors and aging confer risk for developing sporadic PD rather than genetic or environmental factor individually. Approximately 5–10% of PD patients present the familial form of the disease with either autosomal dominant or recessive mode of inheritance. Epidemiological analysis confirm that up to 40% of PD patients with age at onset of less than 30 years and 17% of those with age at onset of less than 50 years will probably present the familial form of the disease. At least 18 loci as well as 12 genes with Mendelian inheritance and highly penetrant mutations causing rare monogenic forms have been identified. The genetic discovery of point mutations, duplication or triplication of SNCA and LRRK2 (Leucine rich repeat kinase 2) loci consistently observed are also genes that have been identified in the monogenic, autosomal dominant familial PD patients. Occurrence of somatic mosaicism has been also hypothesized in the etiology of some cases of PD  causing spastic paraplegia  resulting in Rett syndrome  studies together with meta-analyses have led to significant and rapid advances in the genetic basis of sporadic PD, with realization both for its wide implication and complexity , and SNCA) cause rare monogenic forms of the disease; (d) somatic mosaicism could shed light on the heterogeneity of PD; and (e) additive mechanisms is suggested in risk for PD, increasing with the number of risk alleles carried by a single subject e.g., in HLA (human leucocyte antigen) region  although at present there is a rapid progress and evolution in technology to unravel genetic basis of PD, most PD risk is not understood; (b) the pathogenicity arising from several of identified gene mutations remains to be determined; (c) highly penetrant gene mutations , or BST-1  , DJ-1 and SNCA genes mutated in both familial and idiopathic PD are also known to function in microglia and astrocytes  showing their accumulation in both cerebrospinal fluid and post-mortem brain  positive antigen presenting cells, and the presence of barriers as the tanycytic barrier around the circumventricular organs or the neurovascular unit of the BBB. The latter is composed of endothelial cells, pericytes and astrocytes and associated strong, tight junctions prevent the entry of immune cells into the brain parenchyma. BBB is a metabolic and physical barrier that separates the CNS from the peripheral circulation, actively allowing the transports of nutrients to the brain but limiting passive diffusion of blood-borne solutes. However, in aging and in PD, a BBB disruption has been described with loss of the barrier permeability leading to secondary leukocyte migration within the brain parenchyma, reactive gliosis and damage to neurons  and the complement regulatory proteins, complement components C1q and C3 in order to eliminate cellular debris and damaged neurons by gliapses  are one of the most potent and effective anti-inflammatory agents in clinical use ever since the isolation of cortisone and its clinical application in the early 1950s by the Nobel Prize winners Hench, Kendall and Reichstein  are endogenous steroid hormones synthesized in adrenal glands and secreted into systemic circulation. The GC secretion occurs in ultradian pulsatile manner  as well as mineralocorticoid receptors (MR) that have restricted expression in neurons. However, it should be noted that MR is also expressed in glia  and immunophilins such as FKBP51 (FK506 binding protein51), FKBP52, CP44 and PP5  activation and intracellular signaling cascade is thus far best characterized  present in the promoter regions of its target genes. This transcriptional activity of GR requires the presence of chromatin modifiers , basal transcriptional machinery and co-factors  , Jun  as well as ATF (Activating transcription factor) families of transcription factors, which controls expression of many cytokines. AP1 activity is stimulated by MAPK cascade resulting in activation of c-Jun-N terminal kinase (JNK), which phosphorylates c-Jun. GR regulates AP1 activity by associating with Jun-Fos complex at AP1 DNA elements in promoter regions of genes, inducing a conformational change in the complex that is not functional , Liver X receptor (LXR), Estrogen receptor ER-β, Nuclear receptor NR4A family  are expressed in microglia, thus they can also control microglial activation. In this regard, Nurr1 inhibition was found to increase NF-κB activity in glia resulting in exaggerated expression of pro-inflammatory mediators and increased loss of dopamine neurons following LPS injection in substantia nigra (Saijo et al., Regarding GCs, it is possible that in PD, GR functions in immune cells are compromised because of chronically elevated levels of cortisol. The putative scenario of dysfunction of GR signaling in PD is illustrated in Figure 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.


Ndufs4 knockout mouse model of Leigh Syndrome. Although rapamycin is a widely used pharmaceutical agent dosage has not been rigorously examined and no dose-response profile has been established. Given these observations we sought to determine if increased doses of oral rapamycin would result in more robust impact on mTOR driven parameters. To test this hypothesis, we compared the effects of dietary rapamycin at doses ranging from 14 to 378 PPM on developmental weight in control and Ndufs4 knockout mice and on health and survival in the Ndufs4 knockout model. High dose rapamycin was well tolerated, dramatically reduced weight gain during development, and overcame gender differences. The highest oral dose, approximately 27-times the dose shown to extend murine lifespan, increased survival in Ndufs4 knockout mice similarly to daily rapamycin injection without observable adverse effects. These findings have broad implications for the effective use of rapamycin in murine studies and for the translational potential of rapamycin in the treatment of mitochondrial disease. This data, further supported by a comparison of available literature, suggests that 14 PPM dietary rapamycin is a sub-optimal dose for targeting mTOR systemically in mice. Our findings suggest that the role of mTOR in mammalian biology may be broadly underestimated when determined through treatment with rapamycin at commonly used doses.Rapamycin extends lifespan and attenuates age-related pathologies in mice when administered through diet at 14 parts per million (PPM). Recently, we reported that daily intraperitoneal injection of rapamycin at 8 mg/kg attenuates mitochondrial disease symptoms and progression in the  Rapamycin, a specific inhibitor of the mechanistic Target Of Rapamycin (mTOR), was the first pharmacological agent reproducibly demonstrated to extend lifespan in multiple organisms including budding yeast, nematodes, flies, and mice  mouse is a murine model of LS  is a severe mitochondrial disease that occurs in about 1:40,000 newborns and is associated with retarded growth, muscular deficits including myopathy and dyspnea, lactic acidosis, and a characteristic progressive necrotizing encephalopathy of the vestibular nuclei, cerebellum, and olfactory bulb  and are pooled as controls for the experiments described. All mice used in these studies were C57Bl/6NIA animals.Heterozygous Encapsulated rapamycin was obtained from the Barshop Institute at the University of Texas Health Science Center at San Antonio and Rapamycin Holdings, Inc. Standard mouse chow was ground to a powder and mixed with encapsulated rapamycin at 14, 42, 126, or 378 PPM. 300 mL of 1% agar melted in sterile water was added per kilogram of powdered chow and the mixture was pelleted and baked at 55°C for 2–3 h to harden. Pellets were then vacuum-sealed for short-term (4°C) or long-term (−20°C) storage. Control food contained either no drug or encapsulation material (eudragit) alone at a concentration matching that in the rapamycin chow, as indicated.Mice began receiving assigned diet treatments upon weaning, at 20–21 days of age. Mice assigned to rapamycin (8 mg/kg) or vehicle injection groups began treatments at post-natal day 10 (P10) as previously described . Circulating levels of rapamycin were measured after at least 7 days on the rapamycin diet , to a robust and highly significant reduction in body size at 378 PPM that is similar in magnitude to what we previously reported for daily injection of 8 mg/kg Figure . Rate ofNdufs4 KO animals fed a diet containing 42, 126, or 378 PPM rapamycin beginning at weaning. As previously shown, control treated Ndufs4 KO mice show a slightly reduced developmental weight gain compared to control animals until 35 days of age where disease symptoms onset. This is followed by a progressive decline in body weight concomitant with the presentation of neurological symptoms and a steady loss of fat mass  is 27 times the dose used by the ITP, while IP injection requires only 8 ug un-encapsulated rapamycin per gram-mouse weight to achieve comparable circulating levels. Alternative methods for delivery of oral rapamycin in mouse chow or the development of stable rapalogs will greatly accelerate efforts to study the effects of high-dose rapamycin in long-term treatment paradigms.We recognize that the use of high-dose rapamycin raises additional concerns regarding potential side effects. It is important to note that we observed beneficial effects of rapamycin in the mitochondrial disease model when initiated after developmental weight gain—this indicates that the effects of rapamycin treatment on disease in this setting can be at least partially uncoupled from the off-target effects on weight. Conversely, in the setting of a disease with 100% mortality early in life this off-target effect may be seen as tolerable. This will be an important consideration if rapamycin is brought to clinical trials for Leigh Syndrome.Even at the ITP dosage of 14 PPM, sustained periods of treatment may result in abnormal insulin and glucose responses reminiscent of diabetes . While more work is needed to define the physiological effects of high-dose rapamycin and the true relevance of observed metabolic shifts, our data clearly demonstrate that chronic, very high dose rapamycin has no overt toxicity in mice, and any abnormal physiological findings must be considered in the context of the known benefits of mTOR inhibition in aging or disease paradigms. Furthermore, while it was demonstrated that a modest extension of lifespan in female mice can be achieved in the context of mTORC1 reduction alone, the importance of mTORC2 in the beneficial effects of rapamyicn remains to be determined. The relative roles of mTORC1 and mTORC2 may partially explain the dosage-requirements demonstrated by our work, in addition to the pharmacokinetic properties and tissue specific effects of rapamycin, and will require further study.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.


The concept of a “local” renin angiotensin system (RAS) can mean different things to different people. Its main purpose is to differentiate the “local” RAS operating in tissues from the classical “circulating” RAS, but it is difficult to differentiate between the two systems because of their extensive overlap. The circulating RAS comprises kidney-derived renin acting on liver-derived angiotensinogen to generate angiotensin (Ang) I that is converted to Ang II by angiotensin converting enzyme (ACE). However, tissues are the main site of production of angiotensin peptides by the circulating RAS, whereby plasma-derived renin acts on plasma-derived angiotensinogen to generate Ang I, which is converted to Ang II by endothelial ACE –4.Local RAS refers to tissue-based mechanisms of Ang peptide formation that operate separately from the circulating RAS. Although many different concepts of local RAS have been described, a key feature is the local synthesis of RAS components including angiotensinogen and enzymes such as renin that cleave angiotensinogen to produce Ang peptides independently of the circulating RAS. ACE and Ang II type 1 (AT1) and type 2 (AT2) receptors are invariably locally synthesized, but these are also components of the circulating RAS. Many other potential components of local RAS have been described that may contribute to tissue-specific mechanisms of Ang peptide formation, and that may either participate in disease processes or contribute to mechanisms that protect from tissue injury. These include the (pro)renin receptor , renin-iThis short opinion piece discusses the potential clinical relevance of local RAS. The challenge in demonstrating the independence of local from circulating RAS, and the potential interaction of ACE inhibitor and AT1 receptor blocker (ARB) therapies with local RAS are discussed. Attempts to define local RAS that are independent of the circulating RAS have been primarily based on animal models and the clinical relevance of local RAS is uncertain. However, this area of research continues to evolve, and today’s opinions may change as we gain better understanding of how these novel components and mechanisms impact on clinical medicine.As reviewed elsewhere –12, manyBoth clinical experiences with ACE inhibitor and ARB therapies during pregnancy, and ACE, renin, angiotensinogen, and AT1 receptor gene mutation and knockout models demonstrate a critical role for the RAS in renal development and function in animals and humans –23. MoreSimilar to the case for angiotensinogen, mice with reduced renal expression of ACE had normal histology and urine concentrating ability , suggestEvidence for a pathogenic role of renal ACE is the demonstration that genetic deletion of renal ACE expression prevented hypertension produced by subcutaneous administration of Ang II , suggestAn alternative approach to defining a local tissue RAS was to use recombinant technology to express ACE as a reporter gene on the cardiomyocyte membrane . In thisin vivo levels of Ang peptides in tissues. For example, initial reports of substantial amounts of Ang II and Ang-(1-7) in the brain renin receptor, renin-independent mechanisms of Ang peptide generation from Ang-1-12), AT2 receptors, the ACE2/Ang-(1-7)/Mas receptor and Ang IV/insulin regulated aminopeptidase pathways, an intracellular (intracrine) RAS, and novel Ang peptides , 11, 12., AT2 recThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nature by Momo Bentires-Alj’s laboratory demonstrate that withdrawal of anti-CCL2 treatment accelerates lung metastasis and death in mice. The study highlights the need to consider longer term consequences of therapeutic intervention of metastatic disease, especially with regard to transient interference with the tumour microenvironment.Neutralisation of macrophage chemoattractant C-C chemokine ligand 2 (CCL2) has shown reduced metastasis and enhanced survival in numerous experimental models of tumorigenesis. However, important new findings reported in  The association between macrophage accumulation in primary tumours and poor prognosis is well established, and it is widely acknowledged that macrophages can contribute to metastatic onset . In breaBonapace and colleagues’ article demonstrates mammary tumour secretion of CCL2, enabling recruitment of inflammatory monocytes to the primary tumour and the metastatic site . The autBonapace and colleagues’ study identifies the surprising consequences of termination of an immunotherapy that has previously shown promise both in the laboratory and in clinical application ,6-8. TheThe immune system must have the ability to detect foreign antigen and respond in an appropriate and timely manner, but must also be adequately desensitized to avoid unnecessary response to self. The majority of studies into CCL2 signalling describe its tumorigenic features, but CCL2 is also known to provide protection, for example, by recruiting anti-tumorigenic T cell subsets to the tumour microenvironment . TherefoTo determine this hypothesis, further clarification is required to understand the extent to which monocytes are sequestered in the bone marrow during CCL2 neutralisation. It is difficult to interpret the distribution of these cells without appropriate data outlining their localisation and abundance in a resting mouse. This is exacerbated by differing patterns in monocyte distribution between experimental cell lines. Future studies must address bone marrow output during treatment to fully decipher whether monocytes are sequestered here. It is acknowledged that cancer patients often have augmented production of immature cells ; therefoCCL2 neutralisation has shown positive outcomes in several murine models of mammary, prostate and lung cancers ,12-14. D


Hepatocellular carcinoma is a major health issue, ranked the fifth most common tumor and currently being responsible for a third of the cancer-related deaths globally, with an ever-increasing number of fatalities. Current advances in contrast-enhanced imaging techniques such as contrast-enhanced ultrasonography, multi-detector computed tomography and diffusion-weighted magnetic resonance imaging are improving the rate of hepatocellular carcinoma diagnosis. Contrast-enhanced ultrasonography has widely become the first choice in liver tumor assessment, as it is faster, simpler and safer than other forms of diagnostic imaging. On the other hand, cross sectional computed tomography is frequently employed when a hepatic formation is suspected of malignancy and allows a more accurate characterization of lesions through multiphasic multi-detector computed tomography technology. Diffusion weighted magnetic resonance imaging represents another addition to the wide range of diagnostic and prognostic techniques available for patients with hepatocellular carcinoma and is currently regarded as one of the best tools for the characterization of these lesions. Furthermore, groundbreaking biomarkers for hepatocellular carcinoma are being discovered, although alpha-fetoprotein remains one of the most frequently used serum test in the early stages. Nonetheless, further advances are required for the detection of small liver carcinomas.Abbreviations : AASLD = American Association for the Study of Liver Diseases, AFP = Alpha-fetoprotein, AFP-L3 = Alpha-fetoprotein isoform 3, CEUS = Contrast-enhanced ultrasonography, DCP = Des-gamma-carboxy-prothrombin, DW-MRI = Diffusion weighted magnetic resonance imaging, FNA = Fine neddle aspiration, Gd-EOB-DTPA = Gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid, GGT-II = Gamma-glutamyl transpeptidase II, GP73 = Golgi protein 73, HCC = Hepatocellular carcinoma, HCCR-1 = Human cervical cancer proto-oncogene 1, IL-18 = Interleukin 18, MDCT = Multi-detector computed tomography, PET-CT = Positron emission tomography – computed tomography, SUV = Standardized Uptake Value Alternatively, advances in various imaging techniques (contrast-enhanced ultrasonography (CEUS), multi-detector computer tomography (MDCT), diffusion weighted magnetic resonance imaging (DW-MRI) have significantly improved the detection of hepatocellular carcinoma, with limitations of less than 2 cm in diameter in the case of liver tumors, where further studies are required [2]. An accurate screening method or a combination of methods for early HCC detection are often evaluated based on sensitivity and specificity rates, as these statistical indicators are not dependent on disease prevalence and offer the possibility of comparing studies performed in populations with varying characteristics [3].Hepatocellular carcinoma (HCC) is a major health issue, as recognized by the World Health Organization (WHO), being ranked the fifth most common tumor, currently responsible for a third of cancer-related deaths on a global scale, with an ever-increasing number of fatalities. As a result, there have been numerous attempts to establish a set of guidelines for an early liver cancer diagnosis and long-term prognosis based on different serum biomarkers such as alpha-fetoprotein (AFP), des-gamma-carboxy-prothrombin (DCP) or other novel markers that are currently being studied . The echographic contrast agent approved for use in Europe (SonoVue) is composed of small micro-bubbles, which can flow inside capillaries and, as a result, can generate a map of the intratumoral vascularization with a high degree of accuracy [5]. The evaluation of hepatic nodular lesions undergoes three vascular phases: the arterial and portal phases, which occur during the first 2 minutes after the contrast agent is injected, and a late phase that lasts until the contrast is eliminated. The typical appearance of HCC in contrast enhanced ultrasound is usually hyperenhancement in the arterial phase, with a chaotic vascular pattern . In the portal venous and late phases, HCC usually shows hypoenhancement apart from well-differentiated HCC that may be iso-enhancing  [6]Contrast-enhanced ultrasonography, a non-invasive diagnostic method for various types of hepatic tumors such as hemangioma, focal nodular hyperplasia or adenoma, has recently been introduced as an imaging technique useful for differentiating HCC from cirrhosis nodules based on the different vascularization patterns of the two pathologic entities . Hepatic carcinogenesis is a multistage process, which frequently has a cirrhosis regenerative nodule as a starting point. During CEUS, this type of lesion does not differ from the surrounding parenchyma and, consequently, is not visible in any vascular phase. For this reason, any change in behavior during the arterial phase, such as hypervascular images, can be suggestive for a dysplastic nodule, which can be very difficult to differentiate from early HCC Fig. 4). This method allows the acquisition of multiple cross sections simultaneously and at rapid speeds, which in combination with an intravenous contrast agent allows a multiphasic analysis of liver parenchyma: a pre-contrast phase, an arterial phase and a portal phase of approximately 30 seconds each and a delayed phase up to 3 minutes [12].Cross sectional computer tomography is frequently the first diagnostic imaging technique employed when a malignant hepatic lesion is suspected and, currently, a more accurate characterization of this type of lesions can be obtained by using multiphasic multi-detector CT technology , whereas a significant number of well differentiated together with poorly differentiated HCC showed hypoattenuation in the arterial phase. A possible explanation for these findings may be related to the gradual changes in tumor vascularization that occur during hepatic carcinogenesis, ranging from reduced blood flow during early angiogenesis in well differentiated tumors to increased vascularization for those moderately differentiated and continuing with a decrease in blood supply as the tumor becomes less differentiated. Furthermore, the correlation between the length of time necessary for contrast washout during portal phase and differentiation grade (faster washout as tumor grading increases) allows a better tumor characterization and is a valuable tool for prognosis and a correct treatment scheme [13]. Other studies suggest that further optimization can be achieved by assessing tumor washout both in the portal as well as in the delayed phase because certain tumors, especially the smaller ones, present late hypoattenuation and can remain unnoticed  .A 2011 retrospective study conducted over the course of 7 years on 243 patients with histologically confirmed HCC analyzed contrast enhancement patterns on MDCT and their correlation with tumor differentiation based on attenuation, washout and various other case specific factors such as necrosis, aneurysms or dysmorphic intratumoral vessels. According to the results, the typical pattern of hypervascularization normally seen in HCC was most frequent in moderately differentiated tumors and only partly present in well differentiated carcinomas  present a higher uptake in malignant cells, yet due to the normally increased glucose metabolism of hepatocytes, there are some challenges in successfully applying PET-CT as a diagnostic tool for liver carcinomas [17]. It has been proven that PET-CT is essential in establishing the extent of metastasis  in patients already diagnosed with HCC, therefore offering a reliable long-term prognosis.Currently, there is a growing interest in determining the feasibility and effectiveness of PET-CT in HCC detection and also in establishing which metabolic tracer offers the most accurate results. Due to the fact that this method focuses mainly on metabolism intensity, quantified through the standardized uptake value (SUV), and less on anatomical morphology, it is commonly combined with MDCT in order to optimally pinpoint the location of areas with atypical metabolic rates [18]. Numerous studies have been conducted on various techniques of enhancing PET-CT detection rate of primary HCC and secondary metastatic sites. One of them focuses on a dual-phase whole body scan. Phase one consists of a full body scan 90 minutes after the tracer is injected and is followed by phase two, a second scan is performed only in the abdominal region, in order to allow an increased ratio between the tumor and the normal parenchyma SUV and a better contrast. Although analyzed only in a limited number of studies, this approach appears to be promising, as it may improve primary HCC detection by up to 20%, and allow a more exact delimitation of tumors in relation to unaffected surrounding liver tissue .However, a recent study conducted on 138 patients with confirmed liver carcinomas revealed that, when compared to other ultrasonic or tomographic diagnostic methods, PET-CT is significantly less sensitive as a detection tool [21]. Despite the increase in the sensitivity of up to approximately 80%, a major drawback is that both FDG and 11C-Acetate PET-CT reach this sensitivity only for tumors of over 5 cm, whereas small tumor detection rate (1-2 cm) is less than 40% [22]. An alternative option is the association of dual tracers such as FDG and 11C-acetate, a substance involved in lipid metabolism, which increases PET-CT sensitivity. Certain tumors that remain undetected with one tracer may become visible when using another [Magnetic Resonance Imaging (MRI)23]. DW-MRI has proven itself a more accurate tool than the conventional T1 or T2–weighted MRI [24]. The use of a 3T (Tesla) MRI in conjunction with subsequent Dynamic Contrast Enhanced and Diffusion are regarded as the best diagnostic tool for HCC , although some limitations still exist for tumors under 10 mm [25].Diffusion weighted MRI (DW-MRI) represents another addition to the wide range of diagnostic and prognostic techniques available for patients with HCC. The functioning principle of this method is based on the diffusion capacity of water molecules, which varies depending on the density of cells in different areas of the studied tissue. As a result of the increased cellularity characteristic to tumors, liquid particles are displaced over shorter distances under the action of the magnetic field in comparison to normal cellular architecture [26]. From a different perspective, two studies that assessed 162 tumor nodules and 83 respectively showed that, although DW-MRI is more susceptible to artifacts than MDCT, its greater contrast offers comparable or even superior sensibility in liver carcinoma diagnosis. Furthermore, for small HCC (<10 mm), this novel MRI detection instrument is twice as accurate as the 64-row multidetector CT [27]. With the development of hepatocyte-specific magnetic resonance imaging contrast agents such as gadoxetic acid disodium (Gd-EOB-DTPA), there is an interest in combining multiple MRI techniques in order to obtain the best possible sensibility without affecting specificity, especially in hipovascular tumor .A 2011 study revealed that contrast-enhanced imaging such as Dynamic Contrast Enhanced MRI offers reduced sensibility for small liver tumors due to the difficulty in distinguishing venous washout phase on images less than 1 cm. Although for larger tumors, DW-MRI and Dynamic Contrast Enhanced MRI offer similar results, for lesions of this size sensibility increases from 72% to 92%, thus providing a viable option for early HCC detection [Fine Needle Aspiration Liver Biopsy28]. This is opposed to the Asian Pacific Association for the Study of the Liver and EFSUMB (European Federation of Societies for Ultrasound in Medicine and Biology) guidelines  which consider CEUS comparable to MDCT as previously discussed studies have shown [30]. For tumors between 1 and 2 cm, there are various approaches ranging from noninvasive use of one or two imaging techniques in conjunction with invasive liver biopsy, which remains the option of choice in case of uncertainty. Lesions under 1 cm remain under periodic surveillance (every 6 months) until changes in size are observed, although the Korean Liver Cancer Study Group guidelines currently consider that combining MDCT with Gadoxetic Acid Enhanced MRI is successful in detecting even infracentimetric HCCs [31].Current diagnostic guidelines of the American Association for the Study of Liver Diseases (AASLD) refer to liver biopsy only after other less invasive methods have been exhausted. According to a 2010 update, these methods now solely include imaging methods, as the use of AFP as a diagnostic criterion has been abandoned due to its reduced specificity and no other serum markers have yet been proven a viable replacement. There is a consensus that for hepatic tumors larger than 2 cm, one contrast enhanced imaging diagnostic tool, either MDCT or Dynamic Contrast Enhanced MRI, presenting a typical vascularization pattern is sufficient for diagnosis. The AASLD and European Association for the Study of the Liver guidelines have excluded CEUS from this list due to false positive results in cases with cholangiocarcinoma [32].As previously stated, fine needle aspiration (FNA) liver biopsy is reserved only for atypical HCC due to the inherent risks involved in any invasive procedure such as intraperitoneal or needle tract-seeding and possible bleeding. Percutaneous FNA accuracy has been reported to be up to 100%, granted that the biopsy has been taken from the suspected tissue and not from normal liver parenchyma. These errors can be avoided by performing a CT or ultrasound guided FNA. The development of endoscopic ultrasound guided FNA has largely increased the area of the liver available for biopsy and has offered a fast and relatively safe method for the histopathological diagnosis of HCC [Prognostic Markers33]. A recent study that proposed the associating AFP with human cervical cancer proto-oncogene 1 (HCCR-1) determined that the latter is actually more accurate to some extent and that a large number of subjects were positive for at least one of the two markers, which lead to a significant increase in early stage HCC diagnosis [34]. Furthermore, another diagnostic option for patients with AFP < 20ng/ ml is the alpha-fetoprotein isoform 3 (AFP-L3) fraction, which presents a higher specificity (80%-97%) and has proven to be useful in clinical practice [35]. Regarding the prognostic value of serum markers, Survivin, an apoptosis inhibitor and a promising therapeutic target, although significantly correlated with the clinical stage, portal thrombi and lymph node metastasis, has not proven to be more reliable than AFP [36].Among the earliest discovered oncofetal proteins, AFP is currently one of the most frequently used serum tests for early stage HCC and, although groundbreaking biomarkers are being discovered, to this date, it still plays an important role in terms of HCC detection. The introduction of new serum markers in order to improve or replace AFP is encouraged by its poor specificity and sensibility coupled with the fact that recent studies have yet to provide sufficient unbiased data necessary for establishing it alone as an efficient surveillance, diagnostic and prognostic tool [37], des-gamma-carboxy-prothrombin  has been suggested as a measurement for cancer progression. Tumor size, as well as vascular invasiveness and metastasis frequency, have been reported to correlate with serum DCP levels [38]. Moreover, increased DCP values are an indicator for the reoccurrence and low survival probability. The applicability of this marker is limited by the fact that it has a low positivity rate for small HCC with a current sensitivity of around 50%, despite the introduction of more accurate detection techniques [39]. A case-control study conducted on 1031 patients concluded that the sensitivity of DCP and AFP increases with HCC progression and proposed further studies regarding the use of a combination of the two markers in early diagnosis and surveillance of HCC’s response to treatment [40].Despite its modest sensitivity in preclinical tumor detection at its current cut-off values . A novel study has indicated that the use of GP73 in conjunction with gamma-glutamyl transpeptidase II (GGT-II) alone did not lead to any conclusive results; however, a triple marker assay including AFP yielded promising results [43]. A 2007 study adopted a new approach by establishing a prognostic correlation between HCC tumor stage and aggressiveness and interleukin 18 (IL-18), which is speculated to be part of an antitumor response by adjacent healthy hepatocytes [44]. According to another recent study, one option would be an extensive serum and urine metabolite profiling technique, which would take into consideration various alterations of metabolic pathways determined by HCC evolution, which unfortunately may also occur in hepatitis and cirrhosis. Although a promising screening tool, this approach, as well as most of the before mentioned markers, requires further research and refining in order to be useful in clinical settings [45].Numerous other biomarkers have been also studied and compared to AFP. One of these is Golgi protein 73 (GP73), a transmembrane protein, which has yielded contradictory data in several studies and thus research is required into whether it is more accurate than the traditional serum markers [In conclusion, the multitude of current imaging techniques available for HCC assessment allows a higher rate of detection and significantly contributes to a more accurate long-term prognosis. Contrast-enhanced ultrasonography has proven to be a cheaper, faster, simpler, non-invasive and safer method for the patient, which offers high accuracy for the characterization of focal hepatic lesions. In order to obtain a precise characterization of HCC, MRI and CT capabilities are continuously evolving and offering better results from both a morphological point of view through DW-MRI and MDCT, as well as from a functional perspective through PET-CT.Nevertheless, there is hope that future constant research for a rapid and specific screening serum marker would lead to an early detection of small liver tumors and thus to a better chance of a successful treatment.AcknowledgmentsThis paper was published under the frame of European Social Fund, Human Resources Development Operational Program 2007-2013, project no. POSDRU/159/1.5/136893 and UEFISCDI Partnership project VIP SYSTEM, ID: 2011–3,2-0503.


DELAY OF GERMINATION1. While new mechanisms are emerging from genetic studies of seed dormancy, novel hypotheses are also generated from seed germination studies with high throughput gene expression analysis. Recent studies on tissue-specific gene expression in tomato and Arabidopsis seeds, which suggested possible “mechanosensing” in the regulatory mechanisms, advanced our understanding of embryo-endosperm interaction and have potential to re-draw the traditional hypotheses or integrate them into a comprehensive scheme. The progress in basic seed science will enable knowledge translation, another frontier of research to be expanded for food and fuel production.Seed dormancy has played a significant role in adaptation and evolution of seed plants. While its biological significance is clear, molecular mechanisms underlying seed dormancy induction, maintenance and alleviation still remain elusive. Intensive efforts have been made to investigate gibberellin and abscisic acid metabolism in seeds, which greatly contributed to the current understanding of seed dormancy mechanisms. Other mechanisms, which might be independent of hormones, or specific to the seed dormancy pathway, are also emerging from genetic analysis of “seed dormancy mutants.” These studies suggest that chromatin remodeling through histone ubiquitination, methylation and acetylation, which could lead to transcription elongation or gene silencing, may play a significant role in seed dormancy regulation. Small interfering RNA and/or long non-coding RNA might be a trigger of epigenetic changes at the seed dormancy or germination loci, such as  The ultimate role of seeds is to produce offspring and maintain species. Therefore, plants have evolved diverse strategies to ensure successful germination of this genetic delivery system. Proper distribution of seed germination, in both temporal and spatial manners, is critical for survival and proliferation of seed plants. Spatial distribution of germination is generally controlled through seed and fruit morphology, which enhances dispersal of the offspring from the maternal habitat. In contrast, temporal distribution of germination is controlled mainly by the physiological status of seeds. A variation among individual seeds in a population, in terms of physiological status, allows each seed to germinate at a different timing, which is an important strategy for seeds to avoid competition with their siblings or extinction of all individuals due to a disastrous condition. Plants have evolved seed dormancy, temporal suppression of germination under the conditions favorable to germination. Induction of seed dormancy during the maturation stage and its release at a dry state after a certain period of time, which is called “after-ripening,” are widespread phenomena observed in diverse species of seed plants , an ABA biosynthesis gene and GA2ox, a GA deactivation gene, and intensive analysis of their regulatory mechanisms in the last decade, have provided a comprehensive picture of ABA and GA involvement in the seed dormancy mechanisms  and dormancy release by gibberellin (GA) are observed in many species. The molecular mechanism of antagonistic function of these two hormones was unclear for many years. However, identification of the rate-limiting hormone metabolism genes, such as nine-Previously unknown seed dormancy-associated factors are emerging from on-going research, some of which enhance seed dormancy while others negatively affect it. The positive and negative regulators of seed dormancy, which will be discussed in this section, are summarized in Table DELAY OF GERMINATION (DOG) genes  analysis using natural variation in Arabidopsis has identified the “seed dormancy-specific” loci, including the DOG1 is still a mystery. DOG1 encodes an unknown protein, for which only limited information is available. The DOG1 cDNA shows highest similarity with a Brassica napus EST from an embryo library, however this gene also is not annotated. The protein with a known function that shows the highest similarity with DOG1 is the wheat transcription factor Histone gene Binding Protein-1b (HBP-1b)  regulated genes  or dormancy down-(Ddown) regulated genes  in the DOG1 near isogenic line (NIL) have been identified  , which activates ABI5, was induced in imbibed seeds, ABI5 was able to suppress seed germination  has not been experimentally examined. Investigation of molecular consequences upon DOG1 induction at the right timing, including gene expression, protein phosphorylation and epigenetic changes (discussed below), will provide useful information. It should be noted that there are other dormancy(-specific) genes recently discovered, such as Seed dormancy 4 (Sdr4) in rice  mutants  to the DNA template and the rate of transcription elongation after its binding to DNA. The efficiency of transcription elongation is influenced by an arrest of Pol II and its recovery from the arrest   gene, an Arabidopsis ortholog of Bre1  also causes reduced dormancy in seeds  monoubiquitination  seeds exhibit reduced dormancy, genes down-regulated in the hub1 mutant are good candidates for seed dormancy-imposing genes, the expression of which is promoted through transcriptional elongation. ABA INSENSITIIVE4 (ABI4), DOG1, NINE-CIS-EPOXYCAROTENOID DIOXYGENASE9 (NCED9) and other genes have been identified as possible targets of HUB1/RDO4  and RDO4 (HUB1), two positive regulators of transcription are induced during the same stages of seed maturation (~18–19 DAP). There is a significant overlap between rdo2 and rdo4, in terms of differentially expressed genes in the mutants. These results suggest that RDO2 and RDO4 might share common targets. Intriguingly, DOG1 is one of the genes commonly down-regulated in the two mutants  interacts with SWI-INDEPENDENT3 (SIN3), an amphipathic helix repeat protein, removes acetyl groups from lysine in the histone tails, and creates a transcriptionally inactive state of chromatin , ACO4, and ACO5 and ethylene response genes, such as ETHYLENE RESPONSE FACTOR9 (ERF9), ERF105, and ERF112, were up-regulated in the mutant  and sensitivity (negative) in the regulatory mechanisms of seed dormancy is not known. It is possible that the seemingly counterintuitive effects are associated with negative feedback regulation.In contrast, the same study suggests that SNL-HDA19 increases ABA levels and thereby enhances seed dormancy. HISTONE DEACETYLASE 2B (HD2B), another HDAC gene, is also involved in seed dormancy. In this case, it negatively affects seed dormancy , mature seeds of ColHD2B/Cvi exhibited reduced dormancy, which was not evident immediately after harvest without cold stratification but became clear when seeds were stratified or partially after-ripened  associated with seed dormancy are regulated by histone deacetylation. These results demonstrate that epigenetic regulation through chromatin remodeling is a robust mechanism to alter hormone levels in seeds.Cold stratification releases seed dormancy through an increase in GA levels. SUPERMAN (SUP) locus identified the KRYPTONITE (KYP) methyltransferase, which causes H3K9me2  or negatively (HD2B), depending on the target genes. Histone methylation also affects seed dormancy in both ways. While H3K4 and H3K79 methylation activates gene expression and causes seed dormancy as mentioned above (Set1 or ATXR7), dimethylation of H3K9 (H3K9me2), a repressive mark, occurs on the chromatin associated with seed dormancy genes. Analysis of gene silencing at the Arabidopsis 2 Figure  4_9 gene in barley, is expressed differentially in the embryos of dormant and non-dormant grains and is thought to function as a negative regulator of seed dormancy through RdDM . AGO802B expression is significantly lower in preharvest sprouting (PHS)-resistant  varieties than in susceptible ones , which is triggered by small interfering RNAs (siRNAs) produced by DICER-LIKE3 (DCL3) and their loading onto ARGONAUTE4 (AGO4) , is confusing. It is even more confusing when the description is combined with different terminology of histone modification, such as histone (de)acetylation, monoubiquitination or (de)methylation, because they could be either repressive or active marks depending on the position of residues in the histone tail. To avoid the confusion, the positive and negative regulators of seed dormancy, their roles in chromatin and DNA modification, and possible consequences in gene expression downstream are summarized in Figure A quiescent state of the embryo is changed when molecular repression on seed germination genes is removed, which is probably orchestrated with silencing of dormancy genes. However, an active embryo is still unable to complete germination when the suppressive force, or mechanical resistance, of the covering tissues, such as the testa and endosperm, exceeds embryo growth potential. When the embryo is not dormant, it is the mechanical resistance of the covering tissues that mainly determines whether the embryo emerges from imbibed seeds. In fact, the embryos in dormant seeds in many species are able to grow when they are excised from seeds, which is called coat-imposed dormancy , an experimentally validated upstream regulator of the PR genes, was also one of the ME-enriched genes in tomato seeds , peripheral endosperm , radicle (RAD), and cotyledons (COT)  gene also isolated PHS-resistant wheat lines  gene, which is a recently identified member of the ABA and GA signaling pathways in Arabidopsis , was minimized in the discussion above, because it is well summarized elsewhere (Finkelstein et al., NCED with RNA interference can promote germination in lettuce seeds (Huo et al., NCED, a single gene, was sufficient to suppress precocious germination in Arabidopsis, which can also be applied to PHS prevention in cereal crops (Martinez-Andujar et al., NECD enhancement, which does not require chemical application, has been established recently, using a positive feedback mechanism. In this system, a chimeric NCED gene, which is designed to trigger positive feedback regulation, amplifies ABA biosynthesis and signaling in seeds and causes hyperdormancy in a spontaneous manner (Nonogaki et al., NCED expression in seeds (Nonogaki et al., More progressive efforts are being made to translate seed hormone biology. It has been demonstrated that direct manipulation of the rate-limiting enzymes in the hormone metabolism pathways can successfully be used to alter seed performance. Silencing A patent application has been filed for a technology described in this article. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Primary immunodeficiencies are rare inherited disorders that may lead to frequent and often severe acute respiratory infections. Respiratory syncytial virus (RSV) is one of the most frequent pathogens during early infancy and the infection is more severe in immunocompromised infants than in healthy infants, as a result of impaired T- and B-cell immune response unable to efficaciously neutralize viral replication, with subsequent increased viral shedding and potentially lethal lower respiratory tract infection. Several authors have reported a severe clinical course after RSV infections in infants and children with primary and acquired immunodeficiencies. Environmental prophylaxis is essential in order to reduce the infection during the epidemic season in hospitalized immunocompromised infants. Prophylaxis with palivizumab, a humanized monoclonal antibody against the RSV F protein, is currently recommended in high-risk infants born prematurely, with chronic lung disease or congenital heart disease. Currently however the prophylaxis is not routinely recommended in infants with primary immunodeficiency, although some authors propose the extension of prophylaxis to this high risk population. Pneumovirus genus and Paramyxoviridae family responsible for frequent acute respiratory infections especially in young infants. Specific antibodies are detectable in 87% of infants younger than 18 months [RSV is a ubiquitous RNA virus of theIt was estimated that over 33 million episodes of RSV-related lower respiratory tract infections (LRTI) occurred worldwide in 2005 in children younger than 5 years of age [RSV epidemics in infants younger than 5 years old have a strong impact on pediatric healthcare. In 2000 in the USA, RSV infections lead to 86,000 hospitalizations, 402,000 emergency room visits, 1.7 million office visits, and 236,000 hospital outpatient visits. The estimated cost of these visits and hospitalization was near $258 million [Although RSV-associated mortality is ninefold higher than influenza-associated mortality during the first year of life , mortaliTwo different subtypes (A and B) of RSV were identified depending on variations in G protein and the two subtypes coexist during every RSV season. Subtype A is probably associated to more severe infections , 15. TheSimilar to other respiratory viruses, RSV infection does not induce an effective immunological memory because of the high heterogeneity of protein A, B, and G  and for Especially in younger infants, a primary RSV infection evokes a poor immune response, and it has limited effect on subsequent reinfection , 23. OncThe response to RSV infection induces both serum and secretory antibody production, even in the first years of life when the antibody titers are lower , 28. In T-cell mediated immune response is also involved in the protection against RSV associated illness through the induction of viral clearance, even if the mechanisms of the immune response has not been extensively clarified , 26.The role of CD4 T cells is crucial to maintain the balance between the effector and the regulatory immune response in the lungs infected by RSV. An abnormal CD4 T-cell response was observed to lead to a more severe disease through the activation of Th2-, Th9-, and Th17-related cytokines as observed in murine models or in some vaccination trials . MoreoveRSV infection leads to a CD4 Th2-mediated immune response resulting in mucus production and airway hyperactivity that determine the symptoms of bronchiolitis and LRTI; young infants are more predisposed toward this response than adults .Newborn and infants, particularly those born prematurely, are susceptible to severe LRTI because of the small size of the airways where the airway edema and sloughing of epithelial cells cause atelectasis and subsequent mismatching of ventilation and perfusion and decreased oxygenation.CD4 Th2 cells also play a key role in the regulation of the immune response aimed to protect the lungs to an immunomediated toxic effect; this homeostasis is mediated by regulatory T cells (Tregs) and IL-10 –36. AlteSeveral hypotheses on the mechanisms modulating the immune response against RSV infections may be inferred from an unfortunate experience with a potential RSV vaccine that occurred in the 1960s. In the 1960s, an experimental vaccine against RSV inactivated with formalin (FI-RSV) was administered in a randomized controlled trial to a group of four-month-old infants and their siblings younger than 10 years (219 subjects received FI-RSV vaccine) and to a group of healthy infants between the ages of 6 and 24 months . As a reThe lack of protective effect of FI-RSV vaccine and its correlation to a more severe clinical course of the infection was probably due to several factors, including the inadequate development of poorly neutralizing antibodies against RSV-encoded epitopes, incomplete affinity maturation of the antibodies, and lack of an anti-RSV cytotoxic T lymphocyte response . The resChildren with T-cell deficiencies are unable to efficiently clear RSV, confirming that T cells indeed play a role in virus neutralization and subsequently they are at a higher risk for severe and potentially lethal viral infections, including RSV LRTI , 43. MorThe real incidence of primary immunodeficiency is probably underestimated , since tClinical studies considering the incidence of RSV infections in immunocompromised infants often consider both primary and acquired immunodeficiencies, probably because RSV causes the same clinical manifestations in infants with any severe immunodeficiency including hematopoietic stem cell transplantation and oncologic disorders , 48, 49, in vivo proliferative tests showed an increased susceptibility to infectious pathogens. These data were confirmed by a recent cohort study [The first study considering immunocompromised infants with RSV infections was conducted over ten consecutive winter seasons,  and analrt study  that enrEnvironmental prevention is extremely important to reduce both nosocomial and community-acquired respiratory infections, in particular in high-risk patients as immunocompromised infants.The diffusion of the virus can be controlled by protective measures such as hand washing, isolation of positive hospitalized cases (cohorting) and with the use of gloves, masks, and protective clothing by dedicated staff. Moreover, in epidemic seasons immunocompromised infants and children should be hospitalized in special designated areas where other patients, caregivers, and visitors should be checked for respiratory infections and where the admission of visitors is limited .Pharmacological prophylaxis in immunocompromised infants is based on the use of palivizumab. Palivizumab is a humanized IgG1 monoclonal antibody obtained through recombining DNA technique specific for an epitope of antigen A of F (fusion) protein of RSV. The mAbs have the same properties of a human IgG1, with a long half-life (28 days). It is free from the risk of transmission of blood-borne pathogens, which is particularly important in immunocompromised infants. Palivizumab was approved by the Food and Drug Administration in the United States in 1998; it is currently the only approved monoclonal antibody used for RSV prophylaxis and it was observed to determine a 55% decrease in RSV-related hospitalizations . It is cThe severity of RSV infections in infants with primary immunodeficiency and the lack of recommendations for prophylaxis in current guidelines have led several authors to consider the “off-label” extension of the use of palivizumab to these uncommon and severe diseases. A Delphi study was performed by Gaboli et al.  to obtaiMortality and morbidity due to RSV LRTI infections are higher in infants with primary immunological disorders than in healthy infants. The risk is similar to other rare and severe disorders such as malformations of the airways, cystic fibrosis, and neuromuscular diseases, which are also not included in current recommendations for prophylaxis. The experience of clinicians treating these rare and severe disorders is essential to assess that prophylaxis should be extended to all infants at increased risk for severe and potentially lethal RSV infections.The use of palivizumab in addition to environmental prophylaxis in selected immunocompromised patients was proved to be effective and free from adverse events. Current guidelines for this prophylaxis do not contain recommendations for these high risk infants, although some authors endorse the extension for treatment to immunodeficient subjects. The inclusion of such very high risk patients in guidelines will prevent unnecessary hospitalizations and deaths.At present, randomized clinical trials (RCT) on the efficacy of palivizumab prophylaxis in immunocompromised infants have not been conducted, probably because of the low incidence of these disorders and the ethical controversies surrounding them.RCT conducted in patients with immunological disorders could confirm the efficacy and the cost-effectiveness of prophylaxis with palivizumab in these infants, allowing the inclusion of infants and children with primary immunodeficiency in international recommendations and guidelines.


Neisseria meningitidis is an important cause of invasive bacterial infection in children worldwide, and is a rare example of a bacterium that has evolved to become an obligate human commensal which commonly colonizes the oropharyngeal mucosa. Carriage is age-dependent and appears to be very common in young adults. The relationships between carriage and invasive disease are not completely understood .Each year approximately 1.2 million cases of invasive meningococcal disease (sepsis or meningitis) with 135,000 deaths are estimated to occur worldwide [Epidemiology and serogroup distribution differs geographically, as the example in Figure ©, Novartis Vaccines, Italy). This vaccine is protein-based and, therefore, compared to meningococcal conjugate vaccines , has a different mechanism of action, along with different safety, reactogenicity and immunogenicity profiles in the various age groups.In early 2013, a new vaccine developed specifically to prevent disease caused by group B meningococci (MenB) was licensed in Europe  and outer membrane vesicles derived from the NZ98/254 strain and has the potential to reduce mortality and morbidity associated with serogroup B meningococci infections, but uncertainty remains about the breadth of protection the vaccine might induce against the diverse serogroup B meningococci strains that cause disease: Meningococcal Antigen Typing System predicted that 78% of all MenB strains would be killed by postvaccination .In Italy (updated April 2014) the Basilicata Region .For parents and clinicians, the predicted benefits of 4-component meningococcal group B vaccine (4CMenB) outweigh existing uncertainties about the potential impact of the MenB vaccine against invasive disease, but future introduction of the vaccine must be followed by rigorous post-implementation surveillance to assess its value to health systems.


Neuronal expression of major histocompatibility complex I (MHCI)-related molecules in adults and during CNS diseases is involved in the synaptic plasticity and axonal regeneration with mechanisms either dependent or independent of their immune functions. Motor neurons are highly responsive in triggering the expression of MHCI molecules during normal aging or following insults and diseases, and this has implications in the synaptic controls, axonal regeneration, and neuromuscular junction stability of these neurons. We recently reported that MHCI and immunoproteasome are strongly activated in spinal motor neurons and their peripheral motor axon in a mouse model of familial amyotrophic lateral sclerosis (ALS) during the course of the disease. This response was prominent in ALS mice with slower disease progression in which the axonal structure and function was better preserved than in fast-progressing mice. This review summarizes and discusses our observations in the light of knowledge about the possible role of MHCI in motor neurons providing additional insight into the pathophysiology of ALS. G93A), developed more than 20 years ago, has been widely used to unravel the cascade of molecular mechanisms underlying the pathogenesis and progression of ALS  is an irreversible, fatal adult onset neurodegenerative disorder causing degeneration of motor neurons in the brain stem, spinal cord, and cerebral cortex. The muscle denervation leads to impairment of the neuromuscular system, resulting in paralysis and death for respiratory failure within 2–3 years after symptom onset. The majority of ALS cases are sporadic, while about 10% are inherited; however, the two forms are clinically and pathologically indistinguishable. A limited number of genes, including SOD1, TARDBP, FUS, C9orf72, and TBK1, are responsible for a significant proportion of familial and sporadic ALS cases .An intriguing hypothesis suggests that the evolution of the adaptive immune system may have coincided with elaboration of the neural crest and tissues derived from it, such as the jaws, at the time of increasing complexity of the nervous system , 15. In One of the main factors in the adaptive immune system is the MHCI, classically involved in the presentation of peptides derived from proteolysis mediated by the immunoproteasome (IP) to cytotoxic lymphocytes . MHCI beG93A markedly activated the expression of several molecules associated with MHCI at the onset and during the progression of the disease (+ T cells) or whether alternative mechanisms can influence motor neuron viability in an immune system-independent manner. In this review, we will comment on both these possibilities trying to define a potential role of MHCI in ALS.Motor neurons seem to be the most responsive in triggering the MHCI during normal aging , 35, aft disease –41. The 2m) chains that can bind a large set of antigenic peptide fragments (8–11 amino acids) produced in the cytosol by the IP and translocated into the ER through the transporter associated with antigen presentation (TAP) (+ T lymphocytes. These cells recognize MHCI through the CD8 receptor and the antigenic peptide trough the T cell receptor (TCR).Major histocompatibility complex class I molecules are trimeric complexes assembled in the endoplasmic reticulum (ER). It consists of two non-covalently linked polypeptide chains, known as α-chain  and beta 2 microglobulin (βon (TAP) . The MHC+ T lymphocytes recognize the peptides as non-self or neo-self-antigens, they activate the IP and kill the presenting cells directly through the Fas or perforin pathways and/or indirectly by the release of cytokines (2m is located on a different chromosome (chr 15 in humans and chr 2 in mice)   .The MHC region is the most polymorphic of the entire human genome . Each HLGene alterations in the MHC class I region have been linked to various disorders with neurological symptoms, including spinocerebellar ataxia, Huntington’s disease, Parkinson’s disease, multiple sclerosis, narcolepsy, dyslexia, schizophrenia, and autism –47. ThisA high frequency of the HLA-A3 and HLA-B12 antigens was initially found to be associated with ALS patients with rapid and slow disease progression, respectively. However, this was not validated in a subsequent study comparing the HLA antigenic frequency in ALS patients and control groups , 48. BotTap1 and Tap2) lie in the MHC class II region close to the IP genes (Lmp2 and Lmp7), whereas the gene for tapasin (Tapbp), a chaperon protein essential for peptide loading, lies at the edge of the MHC locus nearest the centromere  , 43. In entation . No corrloidosis , whereasloidosis .2m are strongly expressed by cortical and hippocampal neurons in Rasmussen’s encephalitis, a childhood disease causing chronic inflammation of the brain with infiltration of T cells in the parenchyma  maintained by tight junctions between brain endothelial cells, the basal lamina of these cells, and astrocyte end-feet processes. In addition, cells in the CNS display very little or no expression of immune molecules, such as the MHCI, thus avoiding attack by immunocompetent cells and this contributes to the immunoprivileged status . In pathrenchyma . The samrenchyma , all dis tissues .in vitro and in vivo models under inflammatory stimuli such as cytokines or viral infection  cells in vivo   . We also+ T cells leading to damage to the neurons. Studies in vitro on primary mouse neurons infected by neurotropic Borna disease virus (BDV) or pulsed by lympho-choriomeningitis virus envelope glycoprotein (GP33) confirmed this indicating that activated CD8+ T cells establish stable contacts with axons in an antigen- and MHCI-dependent manner (+ T cells) contacts rapidly cause segmental damage of neurites in the form of spheroids or beads, independent of detectable morphological changes to the somata  in favor of a long terminal depression (LTD) between CNS neurons. The pleiotropic role MHCI was further investigated in adult mice after a mechanical insult or trauma. These studies produced controversial results about the specific role of MHCI in CNS.In the past 10 years, the use of animal models has enables us to identify an additional non-immunological function for MHCI in CNS neurons, strictly related to synaptic remodeling and plasticity. More specifically, the lack of a functional MHCI in mice profoundly affects synaptic density and transmission of neuronal cells in hippocampus, visual cortex, and cerebellum during normal development . From thEscande-Beillard et al.  showed t2mβ−/− mice subjected to axonal transection strongly influenced synapse density and axonal regeneration of spinal motor neurons. In 2mβ−/− mice, the synaptic detachments of the soma of lumbar motor neurons 1 week after peripheral sciatic nerve transection were more extensive than in wild-type animals. This surplus removal selectively involved the clusters of inhibitory synapses without affecting the loss of excitatory synapses, which remained the same in normal and 2mβ−/− mice. Thus, after axon lesion motor neurons preferentially remove excitatory inputs, maintaining a subset of inhibitory inputs on the cell and this last effect is strictly dependent on the presence of MHCI. In the same study, Oliveira et al.  had an abnormal pattern of innervation of the hind limb muscles and diaphragm compared with wild-type mice. There was a higher density of acetyl choline receptor (AChR) clusters, disruption of synaptic band structure, and fewer terminal Schwann cells (SCs). These events, which profoundly affect axonal regeneration and muscle reinnervation, are probably responsible for the delayed improvement in grip strength of Kb−/− Db−/− versus wild-type mice after sciatic nerve crush  for 48 h triggers a death pathway in cultured mSOD1 motor neurons  is more complex and plastic than previously anticipated. Its composition varies from one cell type to another and can be perturbed by cell-intrinsic and -extrinsic factors including dysregulation of cellular metabolism and infection  exacerbate cell death and damage by releasing toxic pro-inflammatory mediators over a long period of time. Successful axonal regeneration depends on the coordinated effort of non-neuronal cells that, while removing motor axon debris, release extracellular matrix molecules, cytokines, and growth factors that support axon regrowth  and international laws and policies .GN wrote the manuscript and edited figures. MT wrote part of the manuscript. CB wrote the manuscript and supervised the final version.The authors declare that the research was conducted with no commercial or financial conflict of interest.


Rac1 is a major player of the Rho family of small GTPases that controls multiple cell signaling pathways, such as the organization of cytoskeleton (including adhesion and motility), cell proliferation, apoptosis and activation of immune cells. In the nervous system, in particular, Rac1 GTPase plays a key regulatory function of both actin and microtubule cytoskeletal dynamics and thus it is central to axonal growth and stability, as well as dendrite and spine structural plasticity. Rac1 is also a crucial regulator of NADPH-dependent membrane oxidase (NOX), a prominent source of reactive oxygen species (ROS), thus having a central role in the inflammatory response and neurotoxicity mediated by microglia cells in the nervous system. As such, alterations in Rac1 activity might well be involved in the processes that give rise to Amyotrophic Lateral Sclerosis (ALS), a complex syndrome where cytoskeletal disturbances in motor neurons and redox alterations in the inflammatory compartment play pivotal and synergic roles in the final disease outcomes. Here we will discuss the genetic and mechanistic evidence indicating the relevance of Rac1 dysregulation in the pathogenesis of ALS. Sod1, many fundamental achievements in clinical, genetic, and basic biology processes related to ALS have profoundly improved our knowledge of the disease . Since the discovery of the first gene associated to familial forms of ALS, TARDBP , the core modules of the splicing machinery , namely Spinal Muscular Atrophy (SMA), sustain the idea that post-transcriptional gene regulation is profoundly affected in MND, including ALS. Indeed, SMA is caused by a decreased availability of the Survival Motor Neuron protein (SMN), due to gene mutations or deletions in the The overlap between ALS and SMA is not however restricted to RNA control. Indeed, ALS and SMA share actin dynamics disturbances as a central route for motor neuron degeneration. In particular, the regulation of actin dynamics at the synapse, which is accomplished by a plethora of actin-binding/-regulating proteins is crucial for the proper function and maturation of neuromuscular junctions (NMJs), that are supposed to be a primary, early target of both ALS and SMA  gene have been identified in late 2012 to cause ALS in two large families  for Rho family of small GTPases, including Rac1, and mutation in alsin are expected to affect GEF activity of this protein : when mitochondrial damage is prevented by p66Shc functional inhibition, Rac1 activity is rescued, and cell survival recovered. Thus, damage to mitochondria, which represent a well recognized, early sign of motor neuron degeneration in ALS, might represent the trigger of a pathological cascade involving Rac1 dysregulation, eventually leading to both actin cytoskeleton disturbances and loss of cell viability. In addition, these data point to mitochondria-derived reactive oxygen species (ROS) as mediators of Rac1 inhibition. Many types of hints are indeed accumulating, which indicate that Rho family GTPases are a redox sensitive family of proteins , the cytosolic subunits p40Phox, p47Phox, p67Phox and Rac1. Upon activation, the cytosolic factors translocate to membranes to interact with cytb558, through which electrons from NADPH are transported to reduce molecular oxygen to superoxide. Active, GTP-bound Rac1 acts both as an adaptor, to ensure correct positioning of p67Phox toward cytb558, and as a player in the electron-transfer reaction  is a major source of superoxide by phagocytic cells  ATP, enhances the neurotoxic potential of SOD1-G93A microglia and increases the production of ROS through the recruitment of Rac1 and NOX2. Conversely, different P2X7 antagonists restore ROS production to basal levels decreasing the amount of Rac1-GTP and inhibiting NOX2 activity (Apolloni et al., The overactivation of Rac1/NOX2 pathway A role for Rac1 in ALS neuroinflammation is also suggested by the interplay between alsin and SOD1-G93A in influencing the dynamics of ROS production in glial cells. Alsin is known to bind three components of the redoxosome: it possesses Rho-GEF domains for Rac1 and Rab5 and it also interacts with SOD1. When coexpressed with SOD1-G93A, alsin was shown to attenuate SOD1-dependent Rac1 activation, ROS generation by NOX2, NFκB induction, TNFα secretion and to protect neurons from toxicity in co-culture studies (Li et al., In this review, we have shown how cytoskeletal dynamics alterations in motor neurons as well as neuroinflammatory events in microglia underlie the pathogenesis of ALS and other MNDs, and how Rac1 GTPase might be involved in both processes. Although many gaps exist in our knowledge about the mechanistic aspects of these processes, Rac1 should be considered as a valuable target to interfere with the disease development.Like other Rho GTPases, Rac1 represents a hub that responds to different signaling networks and in turn results in different, even contrasting outcomes by interacting with many diverse downstream effectors, that might well be different depending on the cell type as well as the stimulus involved. Thus, the apparently contradictory evidence about the activity of Rac1 in ALS motor neurons and microglia should not be regarded as surprising. Yet, the antagonistic effects that Rac1 targeting might have on the motor neurons as opposed to the inflammatory compartments poses some questions about how to devise an effective strategy, that will need to be answered in the near future. Nonetheless, a significant body of studies performed in contexts where aberrant Rac1 signaling is associated to alterations in neuronal structural plasticity and actin cytoskeleton dynamics indicates that the modulation of Rac1-dependent signaling pathways could be effective to protect neuronal degeneration (Lorenzetto et al., 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.


The identification of ATM as a positive modulator of several signalling networks that sustain tumorigenesis, including oxidative stress, hypoxia, receptor tyrosine kinase and AKT serine-threonine kinase activation, raise the question of whether ATM function in cancer may be more complex. This review aims to give a complete overview on the work of several labs that links ATM to the control of the balance between cell survival, proliferation and death in cancer.Ataxia-telangiectasia mutated (ATM) kinase is a one of the main guardian of genome stability and plays a central role in the DNA damage response (DDR). The deregulation of these pathways is strongly linked to cancer initiation and progression as well as to the development of therapeutic approaches. These observations, along with reports that identify ATM loss of function as an event that may promote tumor initiation and progression, point to ATM as a  ATM has been identified as a major player of the DNA damage response (DDR) elicited by double strand breaks (DSBs). According to this observation, ATM kinase activity is induced by DSBs and participates in the different responses to this stress including cell cycle arrest, DNA repair apoptosis and senescence, in order to prevent genomic instability. The molecular mechanisms through which ATM exerts its canonical function as a guardian of genomic stability have been largely investigated and have been already reported in several excellent reviews [Ataxia-telangiectasia mutated (ATM) is a serine threonine kinase originally identified as the product of the gene mutated in ataxia telangiectasia (AT). This gene is located on human chromosome 11q22-23 and codes for a large protein of about 350 kDa characterized by the presence of a phosphatidylinositol 3-kinase (PI3K)-like serine/threonine kinase domain flanked by FAT (FRAP-ATM-TRRAP) and FATC J recombination, where DSBs physiologically occur and promote a DDR.Evidence for a role of ATM in tumor initiation and progression comes also from studies aimed at the generation of mouse models in which ATM activity has been genetically modulated. To date several models of enerated –16. In ain vivo has been provided [in vivo. Overall, the ATM knockout and the ATM knockin mouse models provide genetic support in vivo for a significant increase in the rate of lymphoid tumor development associated with ATM deficiency.More recently, evidence for the ability of ATM kinase dead protein to induce genomic instability provided ,18. Surpprovided ,18. For in vivo. ATM loss promotes intestinal tumor development in APC mouse models [The central role of ATM in the prevention of genomic instability, as well as the occurrence of the activation of the DDR at early stages of tumor initiation, prompted several groups to investigate the role of ATM expression in several tumor models e models . Loss ofe models ,21. In te models .3.The first clear link between ATM and cancer consists of the observation that AT patients, among other features, display an increased predisposition to the development of lymphoma and leukaemia ,24. ThisSeveral reports suggest that germline ATM mutations enhance cancer predisposition not only when both alleles are affected but also when a single copy is hit. Indeed, heterozygous ATM mutations are associated with up to a fivefold increased risk of breast cancer development, depending on the type of mutation ,29. In aMoreover, wide scale studies identified selective changes in ATM expression levels in human tumors. These changes are dependent on different causes among which are ATM somatic gene mutations or deletions. ATM point mutations or deletions are frequently found in chronic lymphocytic leukaemia and are associated with poor prognosis .Interestingly, the analysis of data from the Catalogue of Somatic Mutations in Cancer (COSMIC) identified ATM mutations or deletions in about 5% of about 9000 samples of all types of solid tumors included in this catalogue . In addi4.in vitro and in vivo experiments support the requirement of the DDR for senescence induction in response to replicative stress elicited by oncogenes [The DNA damage response (DDR) has been suggested to be activated and to act as a barrier at early stages of tumor initiation to prevent tumor progression beyond their early stages ,38. Thisncogenes –41. The ncogenes .cMyc, H-ras, cyclin E, Cdc25A, E2F1, cdc6, and ERBB2 [Several issues still deserve further investigation. For example neither the molecular mechanism that allows some, but not all oncogenes to trigger DDR, nor the significance of DDR activation in a subset of solid tumors, have been clearly elucidated so far. It has been shown that a large number of oncogenes may elicit the DDR, including nd ERBB2 ,40,43–45nd ERBB2 . Regardind ERBB2 .The hypothesis of DDR activation as a cancer barrier, fits well with the observation that DDR activation precedes genetic alterations and genomic instability, which are detected at later stages of cancer progression. In this light, the idea is that an activated DDR would act as a barrier to cancer progression, but at the same time would exert a sort of selective pressure for mutations or epigenetic silencing of checkpoint kinases that may occur at later stages and rescue proliferation of incipient cancer cells, counteract cell death and therefore ultimately promote cancer progression . This hyin vivo is indeed very limited [in vivo functional experiments [ATDL/ATDLMre11, DB/DBNbs1, Chk2−/−, DC/DCNbs1Chk2−/−, 515C/515Cp53, p53−/− and p53BP1−/−, each of which shows defects in checkpoints activation, apoptosis and/or DNA repair [ATDL/ATDLMre11 genetic background promotes NeuT-dependent tumor development and ATDL/ATDLMre11 mammary tumors display high-grade histopathology and are highly metastatic to lung compared to wild-type (wt) [The functional effect of DDR activation as a barrier to tumor progression deserves further investigation. So far it is mainly based on: (1) correlative evidence: (a) mutations affecting components of the DDR are frequently associated with predisposition to cancer; and (b) co-expression of DDR activation and senescent or apoptosis or cell growth arrest markers; (2) functional requirement of DDR for senescent phenotype induction. Despite these supportive genetic data, causal demonstration that oncogene-induced DDR may suppress tumorigenesis  limited –49. A ro limited . The auteriments . More reA repair . The autype (wt) .Recently the identification of an unexpected link between the DDR and the ARF (Alternate reading frame) tumor suppressor suggest a more complicated role of the DDR, and in particular of the ATM kinase, in restraining of tumor progression upon oncogene expression . The aut5.During the last 15 years, work of many laboratories identified a role for ATM in the modulation of numerous signalling pathways whose deregulation in cancer may promote tumorigenesis and tumor progression. These include death receptor-induced apoptosis oxidative stress, receptor tyrosine kinases, metabolic changes, hypoxia and angiogenesis, and the reactivation of pathways involved in stem cell maintenance. Support for this comes also from global proteome analysis, which identified about 1000 ATM targets in response to ionizing radiation (IR), among which are several proteins that exert important functions outside of the DNA damage response ,55. Impo5.1.Fas receptor (CD95/APO-1) and TRAIL receptor belong to the death receptor family which plays a crucial role in the transduction of the extrinsic apoptotic signal elicited by specific ligands that upon binding to their target receptors trigger their activation. These signaling pathways play an essential role in the control of the development and homeostasis of the immune system , and morOur laboratory identified an interplay between Fas and TRAIL death receptors and the ATM signaling pathway, showing that lymphoblastoid cells derived from AT patients which lack ATM expression are significantly resistant to Fas- and TRAIL- induced apoptosis ,27. LossCollectively, these data identify a molecular mechanism through which ATM kinase may contribute to the immune system homeostasis and to prevent lymphoma development ,64.Recently, we identified a molecular mechanism by which ATM kinase triggers FLIP degradation: ATM enhances ITCH E3-ubiquitin ligase activity, which in turn promotes FLIP ubiquitination and degradation . The obs5.2.Amplification or upregulation of growth factor receptor tyrosine kinases (RTKs) are responsible for human cell transformation, cancer progression and are considered signs of poor prognosis in human cancers .Early work showed a defect in the binding affinity of insulin receptors to insulin in AT cells that expressed low levels of insulin-like growth factor-I receptor (IGF-IR), which could be restored by expression of ATM cDNA .Following these observations, Yang and Kastan reported a cytosolic role for ATM in insulin signaling. They showed that insulin can activate ATM kinase activity and the absence of ATM negatively affected insulin signaling. ATM was shown to phosphorylate 4E-BP1 (eIF-4E-binding protein 1) on S111 in response to insulin stimulation, therefore modulating translation initiation . FurtherAKT and ATM display similarities in terms of phenotypic abnormalities such as growth retardation, defects in the maturation of the immune system, infertility, resistance to insulin and radiosensitivity [Importantly, ATM has been shown to be a major modulator of AKT activation. AKT plays a key role in pathways related to survival by inhibition of apoptotic signals and promotion of cell cycle progression, with a clear implication in cancer and other pathologies . Moreovesitivity ,73. Sevesitivity ,74–76. Msitivity ,78.The identification of ATM as a positive regulator of AKT activity strongly supports the idea that ATM may play a role downstream a panel of RTKs and more importantly that in certain contexts ATM may positively modulate cell survival and proliferation and tumor progression.Recently, evidence for a role of the ATM protein, not only downstream of RTKs, but also upstream as a modulator of RTK expression on the cell surface has been provided . The aut5.3.It is well accepted that oxidative stress may play a role in cancer, as several oncogenes induce oxidative stress, cancer cells generally display abnormalities in metabolism and mitochondrial functionality and are resistant to hypoxia .Atm−/− mice, the authors demonstrated that the absence of ATM increased the levels of ROS in vivo and caused signs of oxidative stress in the central nervous system, in particular in cells of the cerebellum, the primary site of neurodegeneration in AT patients [Over the past two decades, evidence has accumulated that links ATM deficiency to increased oxidative stress. The first hint that ATM may modulate the response to oxidative stress came from the observation that ATM deficiency leads to increased levels of ROS which may play an active role in neurodegeneration linked to AT. Using patients . More repatients –84, RTK patients  and hypopatients ,87, thatThe molecular mechanisms that link the induction of ROS in the absence of ATM on one side and the activation of ATM by ROS on the other side have not been fully elucidated.Recently, a potential role for ATM in the control of an antioxidant response via the pentose phosphate pathway (PPP) has been reported and may explain the increase in oxidative stress shown in ATM null tissues . The autin vivo, loss of ATM results in mitochondrial dysfunction in thymocytes, including elevated mitochondrial number and increased mitochondrial ROS production [Since mitochondria are also the major source of intracellular ROS, several laboratories investigated whether ATM may modulate their functionality. Indeed, cells lacking ATM function exhibit alterations in both mitochondrial homeostasis  ,90, and oduction .A milestone in the elucidation of the connection between oxidative stress and ATM has been established by the identification of a direct activation of ATM in the cytoplasm in response to oxidative stress. The authors could demonstrate that in this context, ATM activity is induced via a molecular mechanism entirely different from that which ensures ATM activation by DNA damaging agents . ATM canAlthough oxidative stress and DNA damage may independently trigger ATM activation and may therefore target different substrates, caution should be taken as oxidative stress and ROS production usually induce DNA damage, and therefore ATM is often exposed to both DNA damage and oxidation simultaneously . This ob5.4.Hypoxia is a common phenomenon in cancers. Interestingly, low oxygen tension or hypoxia is a common feature of all solid tumors, and hypoxia is associated with tumor development, malignant progression, metastatic outgrowth, and resistance to therapy and is considered an independent prognostic indicator for poor patient prognosis in various tumor types . A serie2O2 [Evidence for a link between ATM and hypoxia has been provided by several laboratories ,87,95–982O2 . Consist2O2 . The con2O2 .The comprehension of the molecular mechanism through which hypoxia triggers ATM activation deserves further investigation, although it seems independent of the DDR . InteresAtm in mice is activated specifically in immature vessels in response to the accumulation of ROS. Moreover, they could show that Atm deficiency lowered tumor angiogenesis and enhanced the antiangiogenic action of agents that block VEGF, suggesting that ATM activation in response to ROS may positively impinge on tumor growth because of its ability to promote angiogenesis [One of the most important responses to hypoxia is angiogenesis. The expression of HIF-1 correlates with hypoxia-induced angiogenesis as a result of the induction of a major HIF-1 target gene, the vascular endothelial cell growth factor (VEGF), also in tumor development where novel vessels are required to sustain tumor growth . Okuno aogenesis .5.5.It has been largely demonstrated that several pathways that are involved in development and in stem cell proliferation are aberrantly upregulated in cancer. This observation has driven the hypothesis of cancer initiation and propagation by a limited number of cancer cells, termed cancer stem cells, or more correctly, cancer initiating cells . SeveralAtm−/− NSCs induced by oxidative stress [Regarding the first point, early evidence for a role of ATM in regulation of stem cell survival was first described in neuronal stem cells (NSCs) . In parte stress ,104. More stress .Interestingly, ATM function in the oxidative stress response is also important for homeostasis of normal haematopoietic stem cells (HSCs) . Normal In conclusion these papers suggest that the ability of ATM in the control of oxidative stress may contribute to the regulation of stem cell survival.in vitro and in vivo genetic evidence that ATM kinase enhances ITCH enzymatic activity and triggers ubiquitination/degradation of ITCH (itchy E3 ubiquitin protein ligase) substrates such as FLIP-L and JUN [Evidence for a role of the ATM protein in signalling pathways classically involved in stem cell maintenance has been provided. Recently we showed that ATM is a novel positive modulator of ITCH E3-ubiquitin ligase activity. A single residue on ITCH protein, S161, which is part of an ATM S/T-Q consensus motif, is required for ATM-dependent activation of ITCH. In fact, we provide  and JUN . ITCH is and JUN . We coul and JUN . Interes and JUN ,110 and  and JUN . A secon and JUN , is invo and JUN . It has  and JUN . Finally and JUN ,114. Fut6.in vivo xenograft tumor growth in mice by increasing their responsiveness to chemotherapeutic treatment with etoposide and irinotecan treatment [Ionizing radiation (IR) and many classical chemotherapeutic drugs trigger ATM kinase activation and their outcome is strongly dependent on the functionality of the DDR in general as well as on ATM expression and activity. The activation of DDR may promote genomic instability in those tumor contexts where its tumor-suppressive function has already been lost, for example following p53 mutation or loss of expression. Importantly, cells derived from AT patients clearly display a significantly augmented sensitivity to IR, pointing to ATM targeting as a valuable tool to modulate the sensitivity of tumors to IR or other chemotherapeutic agents ,32. The reatment , while Kreatment .Although various ATM inhibitors have been effective in some situations, evidence for off-target effects caused by the currently available inhibitors has also been provided. As an example, KU-55933 has been identified in a screening for compounds that may efficiently inhibit autophagy by targeting Vps34 independently of ATM kinase activity .Atm−/− models. In addition, mice expressing the kinase dead allele are embryonically lethal, in contrast to the viable KO mice. While further experiments will clarify this issue, it has been suggested that during development inactive Atm may impair the function of the DDR to a higher degree than Atm deficiency, particularly affecting DNA repair and resulting in higher levels of chromosome instability [One major problem behind the idea of targeting ATM activity for cancer therapy comes from the observation that the output of the inhibition of ATM activity does not fully mimic the effect of ATM loss of expression. It has been show that when ATM kinase activity is inhibited, but not when ATM expression is lost, Sister Chromatin Exchange (SCE) and subsequently HR and DSB repair, are impaired ,121. Contability .Overall, these findings provide support, but at the same time raise some concerns about the pharmacological targeting of ATM in cancer therapy. The support is linked to the possible utility of ATM kinase inhibitors in the treatment of cancer cells upon establishment of the genetic context. For example it has been well documented that, while ATM inhibition sensitizes p53 deficient/mutated tumor cells to chemotherapy, it induces chemoresistance in p53 wt backgrounds . The conAnother possible exploitation of ATM inhibition in cancer therapy is linked to the idea that ATM kinase inhibition may be synthetically lethal with the inhibition of other components of the DDR. For example, since ATM is involved in the homologous recombination, its inhibition may be convenient to sensitize cells to PARP-inhibitors ,125.In vitro and in vivo experiments showed higher expression of activating phosphorylation of ATM, RAD17, CHK1 and CHK2 checkpoint proteins in these cells after IR treatment [+ cells are characterized by preferential survival after irradiation, which may be overcome by treatment with CHK2 inhibitor. Consistent with these studies, two grade IV glioma cell lines displaying pronounced and poor stem phenotypes respectively, were pre-incubated with a non toxic concentration of the ATM inhibitors KU-55933 and KU-60019 and then irradiated, in order to improve the therapeutic efficacy of radiation on glioma stem cells (GSCs). GSCs were sensitized to radiation and radio-mimetic chemicals by ATM inhibitors, showing significantly reduced survival. No sensitization was observed after cell differentiation, indicating that ATM inhibitors specifically sensitize GSCs [+/CD24−/low cells, enriched for CSCs, of two breast cancer cell lines and the primary culture of patient breast cancer cells, demonstrated enhanced expression of phosphorylated ATM after radiation, which correlates with increased radioresistance. Using the ATM inhibitor KU-55933 they obtained significantly decreased radiation resistance of the CD44+/CD24−/low subset isolated both from cell lines and from primary culture [Interestingly, a role for the DDR and ATM activation in the regulation of cancer stem cell survival has been proposed. Cancer stem cells are a small fraction of cells in the tumor, with the abilities of self-renewal and multipotent differentiation that seems to have crucial roles in tumor initiation, metastasis and resistance to anti-cancer therapies . In partreatment . Indeed,ize GSCs . Very si culture . All togImportantly, according to the wide range of functions exerted by ATM kinase activity, ATM inhibition may be useful to sensitize tumor cells also to other therapeutic approaches that do not target the DDR. In particular a positive role of ATM inhibition has been described in combined approaches with MET inhibitors  and ATM 7.Several pathways link ATM kinase expression and activity to the modulation of the balance between cell proliferation and cell death. Although several lines of evidence support a tumor suppressor function of ATM, in agreement with its canonical function as safeguard of genomic stability, the identification of ATM as a modulator of several additional cellular responses aberrantly activated in cancer has provided evidence for new and unexpected roles of ATM as a promoter of tumorigenic signals in particular contexts. This duality might be explained by the plethora of functions that ATM may exert, and could also be dependent on the specific genetic background. Future experiments will clarify the underlying molecular basis and more importantly will uncover its significance for cancer therapy.