Hybrid positron emission tomography–magnetic resonance (PET-MR) imaging is a novel imaging modality with emerging applications for cardiovascular disease. PET-MR aims to combine the high-spatial resolution morphological and functional assessment afforded by magnetic resonance imaging (MRI) with the ability of positron emission tomography (PET) for quantification of metabolism, perfusion, and inflammation. The fusion of these two modalities into a single imaging platform not only represents an opportunity to acquire complementary information from a single scan, but also allows motion correction for PET with reduction in ionising radiation. This article presents a brief overview of PET-MR technology followed by a review of the published literature on the clinical cardio-vascular applications of PET and MRI performed separately and with hybrid PET-MR. Positron emission tomography (PET) and magnetic resonance imaging (MRI) are well established modalities for the investigation of cardiovascular disease. MRI provides high-resolution information on anatomy, morphology, function, and tissue characteristics. Parametric mapping with MRI allows visualization of quantitative changes in the myocardium based on changes in T1, T2, T2* and allows detection of myocardial fibrosis, infiltration, and inflammation and iron overload.This article presents a brief overview of positron emission tomography–magnetic resonance (PET-MR) technology followed by a review of the published literature on the clinical cardio-vascular applications of PET and MRI performed separately and with hybrid PET-MR.Simultaneous PET-MR was first demonstrated in preclinical phantom models in 1997.The integration of PET and MRI systems in a single hardware platform presented several technical challenges.Quantitative PET data requires an accurate method for photon attenuation, which occurs when photons that undergo annihilation are absorbed by the body. As such, PET pixel values need to be scaled to radioactivity concentrations units.18F-fluorodeoxyglucose (18F-FDG).Published comparisons between PET-CT and PET-MR quantitative data have demonstrated comparable standardized uptake values (SUV) of cardiac ,However, there are several challenges to AC with PET-MR, in particular, cortical bone (the tissue that contributes the greatest to attenuation) and air have low MRI signal, yet both are at the extremes of photon attenuation. Standard Dixon based MRI-based AC maps do not consider bone and solutions have been proposed with a Dixon based model-based bone segmentation algorithmHybrid PET-MR has potential to provide high-resolution MRI data with excellent soft-tissue contrast to track respiratory movement to motion-correct PET data.There are many potential clinical applications for PET-MR in cardiovascular imaging, but to date, the development has focussed on a small number of applications, which are discussed below.MRI and PET perfusion imaging are both in routine clinical use for ischaemia testing with similar levels of evidence in European Society of Cardiology guidelines for diagnosis of stable angina.Myocardial perfusion MRI tracks the first myocardial passage of a gadolinium-based contrast agent (GBCA) following intravenous injection. GBCAs shorten T1-relaxation time and increase signal intensity on T1-weighted MRI pulse sequences, which are used to acquire a dynamic series of images. Typically combined with vasodilator stress, myocardial perfusion MRI for ischaemia assessment is recommended in patients with intermediate risk of coronary artery disease (CAD).,In meta-analyses, myocardial perfusion MRI has a sensitivity of 87% and 89% and specificity of 91% and 87% at the vessel-level and patient-level respectively when compared with invasive fractional flow reserve as the reference standard.Table 15O-Water, 13N-Ammonia, 18F-Flurpiridaz) or generator (Rubidium-82) and allow visual or fully quantitative assessment of myocardial perfusion at rest and stress. A meta-analysis demonstrated sensitivity 92% and specificity 85% for PET to detect angiographically defined CAD.,Perfusion PET tracers . The distribution volume of the clinically used extracellular GBCAs is increased in infarcted tissue due to its larger extracellular volume (ECV) and slower clearance of GBCA. LGE imaging exploits these differences in contrast concentration and generates images of myocardial scar with high-tissue contrast and high-spatial resolution. On LGE, segments with <25% transmural extent of infarction have a high likelihood of functional recovery whereas segments with >75% transmural infarction are unlikely to recover after revascularization.18F-FDG is a radiolabelled glucose analogue that becomes trapped within cardiac myocytes following intracellular uptake and provides strong signal for PET imaging.,18F-FDG uptake.18F-FDG uptake), ‘hibernating’ , or ‘scarred’ (impaired perfusion and 18F-FDG uptake).18F-FDG to predict functional recovery following revascularization.Viability assessment by PET is based on metabolic assessment. As shown, MRI and PET use different biological approaches to assess myocardial viability. To date, most studies have focussed on cross-validation of techniques rather than exploitation of synergy.18F-FDG uptake with PET-MR in viability assessment.,,18F-FDG.18F-FDG was a better predictor for functional recovery than baseline LGE  with cardiac sarcoidosis  were found to have greater T1, T2, and ECV values compared with volunteers.Most commonly reported MRI findings in cardiac sarcoidosis include LGE of the mid-wall/epicardium of basal septum and lateral wall, and subendocardial and transmural enhancement.18F-FDG lends itself well to identification of inflammatory lesions in active sarcoidosis as there is increased glucose utilization from macrophage activity. Careful patient preparation with low-carbohydrate diet and fasting prior to imaging is essential to avoid false positive results.18F-FDG uptake with three patterns recognized: normal perfusion and no 18F-FDG uptake, abnormal perfusion or 18F-FDG uptake, and abnormal perfusion and metabolism.18F-FDG uptake) was associated with increased risk of cardiac death or ventricular arrhythmia.18F-FDG to detect cardiac sarcoidosis compared with Japanese Ministry of Health and Welfare (JMHW) criteria.A meta-analysis demonstrated sensitivity of 89% and specificity of 78% for Several studies have utilized PET-MR in patients with suspected cardiac sarcoidosis.18F-FDG uptake and LGE, although this is not an unexpected finding as the two modalities measure different entities of disease activity, and this study highlights the complementary information acquired from both modalities. At 2-year follow up, cardiac right ventricular PET involvement and presence of LGE were independent predictors of adverse events.In a study of 51 patients, the sensitivities for cardiac sarcoidosis detection of PET and MRI alone were 85% and 82%, respectively, which improved to 94% with hybrid PET-MR using the JMHW criteria as reference standard in patients with suspected cardiac sarcoidosis.18F-FDG uptake  for detection of active sarcoidosis.In another study, PET-MR was used in 25 patients with suspected active sarcoidosis, defined as positive LGE and 18F-FDG PET delineates acute myocardial inflammation from macrophage activity, whilst MRI reveals regional motion wall abnormalities from granuloma formation, fibrosis from LGE, oedema from T2, and changes in myocardial structure with parametric mapping. The combination of precisely coregistered data in a single scan may be used to devise and validate much needed accurate diagnostic criteria for cardiac sarcoidosis. Furthermore, hybrid PET-MR may provide unique insights into the pathophysiology of cardiac sarcoidosis and serve as an imaging biomarker to guide anti-inflammatory therapy. Hybrid PET-MR may also derive perfusion-metabolism information from MRI perfusion data and 18F-FDG activity at reduced radiation burden.Hybrid PET-MR combines two powerful imaging modalities to obtain complementary information on distinct pathological processes in cardiac sarcoidosis: Myocarditis is an inflammatory condition that causes chest pain, acute or chronic heart failure, life threatening arrhythmias, or cardiogenic shock.MRI provides important information in patients with myocarditis: increased signal intensity with T2-weighted imaging in acute myocarditis results from oedema, early gadolinium enhancement (EGE) demonstrates capillary leakage and LGE identifies infiltration and scar, typically in the inferolateral walls and less frequently in the anteroseptum.The ‘Lake Louise’ Criteria (which are currently being revised) requires two of three positive MRI features—EGE, LGE, and T2 imaging.18F-FDG is highly sensitive to metabolically active inflammation.18F-FDG had sensitivity and specificity of 100% for detection of endomyocardial biopsy proven active myocarditis in patients when performed within 14 days of onset.68Ga-DOTA-TOC in patients when PET-CT performed within 3–10 days of symptom onset.n = 55) with suspected myocarditis, there was good agreement between 18F-FDG and T2 and/or LGE  or wild type/familial-related amyloid (ATTR) is important as treatment and prognosis differ.Typical MRI findings in amyloidosis include symmetric or asymmetric left ventricular thickening, biatrial enlargement, and pleural effusions. LGE shows global transmural or subendocardial LGE in non-coronary artery territory distribution11C-Pittsburgh B, a radiotracer for detection of beta-amyloid deposits in Alzheimer’s patients, was detected in 13/15 patients with biopsy confirmed cardiac amyloid, whereas none was detected in patients without amyloid.18F-Florbetapir also detects beta-amyloid plaques and a pilot study demonstrated greater uptake in patients with cardiac amyloid compared with controls.,18F-Fluoride activity in areas of myocardial amyloid deposition depicted by LGE. ATTR amyloid patients had higher tissue-to-background ratios of 18F-Fluoride uptake compared with AL amyloid patients or controls.Only one published study to date has explored PET-MR in cardiac amyloid. Seven patients with AL or ATTR amyloid and 7 controls underwent PET-MR imaging.Hybrid PET-MR may have a role in the future to discriminate AL and ATTR amyloid heart disease, although larger clinical studies are required prior to consideration as a routine clinical application.Anderson-Fabry disease (AFD), an X-linked lysosomal storage disorder characterized by deficiency of alpha-galactosidase, results in glycosphingolipids accumulation and may cause left ventricular hypertrophy (LVH), myocardial fibrosis, systolic and diastolic dysfunction, arrhythmia, conduction disease, and sudden death.,Typical MRI findings in AFD include concentric, asymmetric, or apical LVH and mid-wall fibrosis of the basal inferolateral wall.15O-Water compared with controls.,Two studies reported that AFD patients had lower MPR with 18F-FDG uptake.18F-FDG uptake. The authors suggested PET-MR differentiated mature fibrosis or scar from fibrosis associated to active inflammation. However, this was a small sample size and several patients had homogenous 18F-FDG uptake, possibly indicating insufficient myocardial suppression.In a study of 13 patients with AFD who underwent PET-MR, 5 patients had LVH and focal LGE fibrosis of which 3 had positive STIR and focal PET-MR may aid diagnosis of AFD, provide insights into immunopathology of different disease stages and have potential as an imaging biomarker to guide EZT.In clinical practice, cardiac masses are primarily imaged with echocardiography and CT, while MRI and PET are reserved for further characterization.MRI can interrogate cardiac masses with T1-weighted black-blood imaging for mass localization, cine imaging for morphology, mobility and functional consequence, and tagging for assessment of attachments to other structures.18F-FDG uptake for staging, monitoring therapy, and prognosis. Malignant tumours have greater 18F-FDG uptake compared with benign tumours.max cut-off of 3.5–4.0.PET allows whole body evaluation of masses with 18F-FDG SUVmax was higher in malignant lesions and SUVmax cut-off of 5.2 provided 100% sensitivity and 92% specificity for malignancy detection.max cut-off of 5.2 increased sensitivity and specificity to 100%. These are interesting findings, although first-pass perfusion, EGE or LGE imaging was not reported as part of the ‘MRI only’ classification, which may have otherwise increased diagnostic accuracy for MRI alone.In a study of 20 patients who underwent PET-MR for cardiac mass assessment, 18F-FDG uptake in one sitting that may improve diagnosis, staging and provide further clarity on disease activity.Hybrid PET-MR can offer additional information though precise co-registration and localization of masses to be undertaken, with detailed interrogation with high resolution T1 and T2-weighted images, parametric mapping, perfusion, LGE imaging and PET-MR is an emerging modality gaining widespread interest for application to cardiovascular disease.The ability to obtain multiple radiation-free MRI data to motion-correct PET data is highly attractive, and future research will focus on improved efficiency and reduction in acquisition time of MRI sequences to simultaneously enhance PET images, quantitative PET data as well as MRI data, particularly for coronary imaging and the application of emerging radiotracers.AC has long been a challenge for PET-MR and novel methods to develop highly accurate AC maps such as with the inclusion of bone segmentation may be applied in future studies.PET-MR is an expensive modality that requires proximity to a cyclotron, specialist staff trained in PET and MRI for patient preparation, image acquisition, AC, post-processing, analysis and reporting. Simultaneous PET-MR imaging may improve patient workflow, with improved patient experience of a single scan. However, clear clinical indications for a hybrid PET-MR scan require evidence of incremental benefit compared with sequential imaging with separate MRI and PET scans. There is vast potential for application to research for cardiac PET-MR, particularly for coronary imaging which may pave the way for multicentre studies for identification of high-risk vulnerable plaques prior to rupture. With the current evidence available, clinical PET-MR may be well suited to conditions such as cardiac masses and suspected cardiac sarcoidosis where hybrid imaging offers precise co-registration and fusion of complementary information that may provide incremental value.Once clear clinical indications and pathways are developed, these will need to be recognized as cost-effective for healthcare systems with a defined route for reimbursement.Hybrid PET-MR combines two powerful imaging modalities to fuse and co-register anatomical detail, tissue characterization, and metabolic data. Technical challenges with AC remain and there is large potential for motion correction of PET data from MRI data. Several studies thus far have focussed on cross-validation of techniques and emerging studies indicate incremental benefit beyond sequential MRI and PET imaging. Future large studies will determine incremental utility of combined rather than sequential imaging and whether PET-MR will be restricted to the research domain or cement itself in the clinical arena.


Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) might contribute to bone loss and arthralgia before the onset of joint inflammation. We aimed to dissect additional mechanisms by which ACPAs might contribute to development of joint pathology.Fibroblast-like synoviocytes (FLS) were isolated from the synovial membrane of patients with RA. The FLS cultures were stimulated with polyclonal ACPAs (anti-CCP-2 antibodies) purified from the peripheral blood of patients with RA or with monoclonal ACPAs derived from single synovial fluid B cells. We analysed how ACPAs modulate FLS by measuring cell adhesion and mobility as well as cytokine production. Expression of protein arginine deiminase (PAD) enzymes and protein citrullination were analysed by immunofluorescence, and signal transduction was studied using immunoblotting.Challenge of FLS by starvation-induced stress or by exposure to the chemokine interleukin-8 was essential to sensitise the cells to ACPAs. These challenges led to an increased PAD expression and protein citrullination and an ACPA-mediated induction of FLS migration through a mechanism involving phosphoinositide 3-kinase activation. Inhibition of the PAD enzymes or competition with soluble citrullinated proteins or peptides completely abolished the ACPA-induced FLS migration. Different monoclonal ACPAs triggered distinct cellular effects in either fibroblasts or osteoclasts, suggesting unique roles for individual ACPA clones in disease pathogenesis.We propose that transient synovial insults in the presence of a certain pre-existing ACPA repertoire might result in an ACPA-mediated increase of FLS migration. Anticitrullinated protein/peptide antibodies (ACPAs) exist prior to the onset of rheumatoid arthritis (RA), however, it is unclear how autoimmunity in some but not all cases translate into manifest joint inflammation.Cellular stress and pro-inflammatory mediators (interleukin-8) can sensitise synovial fibroblasts to ACPAs by enhancing protein arginine deiminase enzyme expression and cellular citrullination.ACPAs promote synovial fibroblast migration through a phosphoinositide 3-kinase-mediated mechanism.Different monoclonal ACPAs have distinct cellular effects with three clones increasing migration of challenged fibroblasts, with no effect on osteoclasts and another clone increasing osteoclast differentiation with no effect on fibroblasts.Our results suggest that unique ACPAs may be responsible for specific pathological features in ACPA+RA.Inducible protein citrullination could be a key event in the transition of a systemic humoral autoimmunity towards the inflammation of the joints.Anti-citrullinated protein antibodies (ACPAs) are present in a majority of patients with rheumatoid arthritis (RA) and are specific for this disease.Previous studies have shown that in the presence of pre-existing joint inflammation in mice, transfer of a monoclonal ACPA may enhance synovial tissue injury,In the present report, we investigated ACPA effects on FLS. We demonstrate that stimuli such as cellular stress and/or exposure to pro-inflammatory mediators are needed for the sensitisation of FLS to ACPAs. Furthermore, some but not all ACPAs are able to affect FLS. Our findings thereby suggest that transient synovial insults in the presence of certain pre-existing ACPA clones might act in concert to promote ACPA-mediated FLS migration.Additional detailed information is provided in online 10.1136/annrheumdis-2018-214967.supp1Supplementary dataPolyclonal ACPA IgGs (anti-CCP2 antibodies) and non-ACPA control IgGs were purified from a pooled serum of patients with RAFibroblast migration was assessed by IncuCyte Zoom live cell imaging system or by scratch assay. Adhesion assays were performed using 16-well E-plates and the xCELLigence System Real-Time Cell Analyzer . When indicated, the cells were washed with phosphate buffered saline and starved for 2 hours in medium without fetal bovine serum. Citrullinated or native human purified fibrinogen, recombinant vimentin, α-enolase and histone H4 were added as pretreatment to ACPA and control IgGs at 4°C overnight. Phosphoinositide 3-kinase (PI3K) was inhibited by wortmanin and phosphatase and tensin homolog (PTEN) by SF-1670 . PAD activity was inhibited by Cl-amidine  or GSK199  for 72 hours prior to scratching. Histone acetyltransferase or deacetylase enzymes were inhibited by anacardic acid or trichostatin A , respectively. Fibroblasts were cultured in the presence or absence of IL-8  or tumour necrosis factor (TNF)-α . Cytokine levels were analysed using the Cytometric Bead Array  and matrix metalloproteinases were measured by ELISA .FLS were stained with indicated primary antibodies followed by horseradish peroxidase (HRP)-conjugated antibodies and developed by Vectastain Elite ABC and DAB  and then counterstained with Mayer’s haematoxylin. For confocal microscopy, cells and tissues incubated with primary and secondary antibodies and then counterstained with 4,6-diamidino-2-phenylindole.Starved and non-starved FLS whole cell lysate were analysed by mass spectrometry for detection of citrullination.Cells lysates were loaded an SDS-PAGE gel , transferred to nitrocellulose membranes and incubated with primary antibodies and peroxidase-conjugated secondary antibodies.Cells were collected using non-enzymatic detachment reagent , stained by fluorochrome-labelled antibodies and analysed by FACSVerse flow cytometer .The data were analysed using either Kurskal-Wallis test followed by Dunn’s multiple comparation test or two-way analysis of variance, followed by Tukey’s post hoc test by using GraphPad Prism 6 software . P values <0.05 were considered to be statistically significant.To test if FLS are or might be rendered sensitive to ACPAs, we compared the effects of polyclonal ACPA and non-ACPA control IgGs on FLS migration in both basal conditions and after challenging by a 2-hour serum starvation. Neither polyclonal ACPA IgGs nor non-ACPA control IgGs had any effect on the migration of non-starved FLS . In cont10.1136/annrheumdis-2018-214967.supp2Supplementary data10.1136/annrheumdis-2018-214967.supp3Supplementary data10.1136/annrheumdis-2018-214967.supp4Supplementary dataBesides migration, ACPA IgGs were able to increase adhesiveness of starved FLS , with an10.1136/annrheumdis-2018-214967.supp5Supplementary dataTo investigate the mechanisms that render FLS sensitive to ACPAs, we analysed the expression of PAD-2 and PAD-4, two PAD enzymes known to be present in RA synovium,10.1136/annrheumdis-2018-214967.supp6Supplementary data10.1136/annrheumdis-2018-214967.supp7Supplementary data10.1136/annrheumdis-2018-214967.supp8Supplementary dataPI3K activationis required forACPA-induced FLS migrationTo gain insight into the intracellular signals activated by ACPAs, we investigated two major regulators of FLS migration: PI3K and mitogen-activated protein kinases (MAPK). Polyclonal ACPAs induced a transient but robust peak in Akt phosphorylation at the Thr308 residue linked to PI3K activation , whereasTo identify additional factors that may render FLS sensitive to ACPAs, we investigated the potential contribution of pro-inflammatory stimuli, such as IL-8 and TNF-α. FLS express IL-8 receptors CXCR1 and CXCR2  and prodSince polyclonal ACPA IgGs contain a wide spectrum of antibodies, we investigated whether monoclonal ACPAs differ in their capacity to affect distinct cellular functions such as FLS mobility and OC differentiation. Among eight monoclonal ACPAs, each with different patterns of antigen recognition, three of them , enhanced FLS migration significantly , while n10.1136/annrheumdis-2018-214967.supp9Supplementary dataWe further investigated cellular binding patterns in synovial biopsies obtained from both healthy volunteers and patients with RA. None of the ACPA clones labelled the uninflamed synovial tissues . In contIn this report, we describe that steady-state FLS are unresponsive to ACPAs but can be rendered sensitive to these autoantibodies in the presence of additional stimuli. A key event in priming synovial fibroblasts to ACPA is an inducible PAD-2 and PAD-4 expression that is accompanied by increased protein citrullination. Importantly, individual monoclonal ACPAs exhibited different capacities to induce migration of challenged FLS and activate OC. We provide novel insights into how different ACPA clones in concert might contribute to distinct pathogenic events in the RA development. Furthermore, PAD inhibition may be beneficial in targeting FLS during the development of ACPA-positive arthritis.We demonstrated that polyclonal ACPA IgGs are able to induce migration and adhesiveness of challenged fibroblasts, independent of their tissue origin. This poses challenges in understanding the specific targeting of the synovial tissue in RA. While tissue-specific antibody-induced vascular changesIn contrast to OCs, no others tested RA-derived antibodies but ACPAs had an effect on FLS migration, suggesting a citrullination dependency of the observed effect. As such, the paucity of citrullinated targets observed on steady-state FLS might explain the lack of ACPA effects in unchallenged conditions. Indeed, FLS that were challenged by either cellular stress or certain pro-inflammatory signals increased their expression of both PAD enzymes and citrullinated proteins, which sensitised the cells to ACPA effects. Cellular citrullination appears to be important for the ACPA-mediated FLS mobility, as PAD inhibition completely abolished this effect. While other cellular effects of these inhibitors cannot be completely excluded, the blocking experiments with soluble citrullinated proteins and the reproducibility of the effects when using the F(ab’)2 ACPA fragments further strengthens this observation. Importantly, MDA modification, acetylation and homo-citrullination do not appear to influence FLS migration, as demonstrated by the lack of effect of anti-MDA antibodies, the lack of influence of increased or decreased acetylation on the ACPA effects and the migration promoting effects of both homo-citrullinated cross-reactive and non-cross-reactive monoclonal ACPAs.While citrullination appears to be central for the ACPA effects on FLS, precise identification of the exact cellular target(s) is still a challenge. Although citrullinated fibrinogen has been previously proposed as an interstitial tissue target able to generate immune complexes in the presence of ACPA,10.1136/annrheumdis-2018-214967.supp10Supplementary dataFLS motility is governed by complex mechanisms, involving a large array of intracellular signalling pathwaysA central finding of the current study resides in the selective cellular modulation by distinct monoclonal ACPAs. We show a selective increase of OC formation with the 1325:04C03 clone. This is in line with our previous reported increase of OC formation seen with the 1325:04C03 clone,In conclusion, we describe a novel potential pathogenic role for ACPAs in FLS migration. Our results also give further support for studies to investigate the early therapeutic and potentially preventive effect of drugs such as PAD enzymes inhibitors that affect the currently described mechanisms of FLS migration.


Salmonellosis. Previous studies have revealed important roles of statins in regulating innate immunity. Therefore, we investigated the effects of statins on innate immunity in Salmonella-infected intestinal epithelial cells (IECs), which are involved in mucosal innate immunity. SW480 cells and Akt siRNA- or vitamin D receptor (VDR) siRNA-transfected SW480 cells were infected by wild-type S. Typhimurium strain SL1344 in the presence or absence of statins. The mRNA or protein expression was analyzed by real-time quantitative PCR or western blot analysis, respectively. Simvastatin or fluvastatin caused IL-8 (interleukin-8) suppression, but increased hBD-2 mRNA expression in Salmonella-infected SW480 cells. Both statins enhanced phosphorylated Akt and VDR expressions. Akt or VDR knockdown by siRNA counteracted the suppressive effect of simvastatin on IL-8 expression, whereas VDR knockdown diminished the enhanced hBD-2 expression in Salmonella-infected SW480 cells. Therefore, we observed differential regulation of statins on inflammatory IL-8 and anti-microbial hBD-2 expressions in Salmonella-infected IECs via PI3K/Akt signaling and VDR protein expression, respectively. The enhanced activity of antimicrobial peptides by statins in Salmonella-infected IECs could protect the host against infection, and modulation of pro-inflammatory responses could prevent the detrimental effects of overwhelming inflammation in the host.Alternative therapies are needed to reduce the use of antibiotics and incidence of drug-resistant  Salmonella enterica serotype enteritidis infections has increased substantially. Antibiotic resistance among some serotypes of clinical Salmonella isolates has been increasing, even though multi-drug resistance (resistance to three or more classes of antibiotics) in Salmonella remained steady in the United States [The incidence of food-borne human d States , with sid States .Salmonella. Antimicrobial peptides, such as human β–defensins-2 (hBD-2), are secreted to kill the microorganisms at mucosal surfaces, while chemokines, such as interleukin-8 (IL-8), promote the transmigration of basolateral neutrophils to the intestinal lumen and induction of diarrhea, and give rise to the characteristic pathology of colitis [Intestinal epithelial cells (IECs) act not only as a barrier to gut pathogens, but as an essential element of the mucosal innate immune system by the secretion of inflammatory cytokines, chemokines, and antimicrobial peptides following infection with  colitis .Antimicrobial therapies, including microbicidal and microbiostatic therapies, are powerful tools for the treatment of several infectious diseases. Antibiotics are a type of antimicrobial drugs used in the treatment and prevention of bacterial infections. However, the increased resistance of microorganisms to currently used antibiotics has generated the need for alternative agents with potential antimicrobial activity.Clostridium difficile-associated diarrhea [Statins are cholesterol-lowering drugs targeting HMG-CoA reductase, thereby reducing the risk of coronary disorders and hypercholesterolemia. The beneficial effects of statins beyond cholesterol lowering have been explored. A growing body of evidence from both clinical trials and basic science studies suggest that statins have anti-inflammatory properties, which may additionally lead to clinical efficacy. Statin therapy might be associated with a lower incidence of infection-related morbidity and mortality. Patients receiving statin therapy may experience reduced infection-associated mortality due to pneumonia , bloodstdiarrhea  as suggediarrhea . Statinsdiarrhea . HoweverSalmonella-infected IECs [Salmonella-infected IECs as well as the signaling pathways and proteins that are involved.We have previously demonstrated that phosphoinositide-3 kinases (PI3Ks)/Akt have an anti-inflammatory effect on interleukin-8 (IL-8) production in ted IECs , and NODted IECs . Vitaminted IECs . TherefoSalmonella-infected IECs. Intestinal epithelial SW480 cells were either uninfected or infected with wild-type S. Typhimurium strain SL1344 for one hour in the presence or absence of different simvastatin concentrations. Real-time quantitative PCR (RT-PCR) was performed to measure IL-8 and hBD-2 mRNA expressions. In Salmonella-infected SW480 cells when its concentration was increased to 10 and 20 μM, whereas it enhanced hBD-2 mRNA expression. In contrast, Salmonella-induced IL-8 mRNA expression was enhanced by a high concentration (50 μM) of simvastatin.We examined the effect of statins on IL-8 and hBD-2 mRNA expressions in Salmonella-induced activation of PI3K/Akt signaling resulted in the inhibition of extracellular signal–regulated kinase (ERK) and consequent suppression of IL-8 expression, [Salmonella-infected SW480 cells by statins. SW480 cells were uninfected or infected with S. Typhimurium wild-type strain SL1344, and activation of Akt and ERK was analyzed by Western blotting. The results showed that simvastatin significantly upregulated Akt (p-Akt) activation in Salmonella-infected SW480 cells, and ERK was downregulated  [Salmonella invasion [Salmonella colitis and its invasion.A retrospective case-control study conducted in three hospitals including all hospitalized patients diagnosed with a (CDAD)  revealedinvasion . These rSalmonella infection, IECs represent the first defense barrier against the bacteria invading the intestinal tissues. IL-8 released from the infected IECs recruits neutrophils from the circulation into the sub-epithelial region to help contain and eliminate the infection. However, accumulation of neutrophils results in colitis [Salmonella invasion. We observed that statins upregulate anti-microbial peptide hBD-2 but downregulate pro-inflammatory IL-8 expression in Salmonella-infected IECs. This suggests that statins may be used as an alternative therapy for Salmonella colitis by defending against and suppressing the pro-inflammatory responses to the luminal bacterial infection. To our knowledge, there have been no reports on how statins affect innate immunity to control Salmonella infection. Further in vivo studies to clarify the effect of statins on Salmonella colitis and invasiveness are required.Following oral  colitis . In contSalmonella-infected IECs. Clinically, acute gastroenteritis with pathogens such as Salmonella is followed by increased risk of inflammatory bowel disease (IBD) [Salmonella-induced IL-8 expression, which could be hazardous for patients with IBD complicated by intracellular bacterial infection, such as Salmonellosis. Although colitis is a rare complication of simvastatin treatment, development of ulcerative colitis has been reported to be an adverse reaction to simvastatin [Simvastatin negatively regulates inflammation in chemical-induced experimental colitis . Our stuse (IBD) . These ose (IBD) . Our stuvastatin , which, vastatin , though Antibiotics, such as clarithromycin or erythromycin, may inhibit the metabolism of statins and increase statin concentration in the blood, which can cause muscle or kidney damage, and even death. However, these adverse reactions are rare. In contrast, an increasing number of studies are emerging which detail the attenuation of bacterial growth as well as the in vitro and in vivo virulence by statin treatment . AlthougSalmonella-infected IECs. This suggests the involvement of VDR in statin-mediated suppression of inflammation during Salmonella colitis. Another possibility is the bactericidal effect of statins on Salmonella extracellularly. However, in the review by Hennessy et al. [Salmonella enterica serovar Typhimurium, as used in our study. This reinsures the justification of our experiments because the cellular innate immunity we investigated seems not to be influenced by the direct antimicrobial effect. Additionally, the differences in the production of IL-8 could be independent of the bacterial load.Accumulating evidence has also shown that VDR is critical for the control of innate immunity in the gastrointestinal tract and in experimental IBD ,26. VDR y et al. , they haSalmonella-infected IECs [Salmonella-infected IECs via the VDR-PI3K-AKT pathways. However, high-dose statins on the other hand may deplete the membrane cholesterol in Salmonella-infected IECs and lead to upregulation of IL-8 expression via inhibition of Akt activation [The beneficial effects of the statin therapy were mediated via VDR activation by this group of drugs because a striking similarity between the benefits of vitamin D and statin therapy was observed . Each ofted IECs . This sutivation . This efAdditionally, PI3K/Akt mediates anti-apoptotic effects in IECs to enhance their resistance to injury . The intS. enterica [Chlamydia pneumoniae [Salmonella-infected IECs, and may lead to the direct elimination of mucosal bacteria, which can explain the protective effects of statins against the invasiveness of Salmonella infections [Salmonella-infected IECs via VDR. We observed that statins enhanced VDR mRNA and protein expressions and subsequent NOD2 expression, resulting in enhancement of hBD-2 expression. However, the mechanisms how statins upregulate VDR expression remain unknown. Two reports have indicated that statins increased gene expression through the inhibition of histone deacetylase activity [Statin treatment can lower bacterial burden during enterica  and Chlaeumoniae  infectiofections . The obsfections  and thatfections  suggestsactivity , and thaactivity , suggestactivity  or a senSalmonella enterica serovar Typhimurium (S. Typhimurium) strain SL1344 was used in this study, and the Salmonella inoculum was prepared as described previously [The wild-type eviously ,43.8 bacteria cells) were prepared as previous described [Caco-2 (passage 20–23) and SW480 cells (passage 52–63) were purchased from the American Type Culture Collection  and were grown as previously reported to 50−75% confluency ,43. Aliqescribed ,43 and uStandard laboratory reagents were purchased from Sigma-Aldrich  or Fisher Scientific . The standard stock solution of simvastatin and fluvastatin  was prepared in dimethyl sulfoxide (DMSO) and water, respectively, stored at −20 °C, and further adjusted to the appropriate concentration with cell culture medium immediately before use.IL-8 protein secreted in the culture supernatants were measured by ELISA , according to the manufacturer’s instructions and modified as described previously .Extracted protein fractions from untreated and treated cultured cells were prepared by Nuclear/cytosolic Fractionation or Membrane Protein Extraction Kit  as recommended by the manufacturer or modified as in previous studies ,43. The Equal amounts of extracted protein from the fractionation of cultured cells were separated by SDS-PAGE and then transferred to nitrocellulose membranes by semi-dry blotting as previously described ,43. AfteTotal RNA was extracted from cultured cells using Trizol reagent , following the manufacturer’s directions. The cDNA generated by reverse transcription was amplified using PCR as described previously ,43.Ct) method of relative quantitation.Real-time reverse transcription-PCR analyses were performed in a fluorescence temperature cycler  as described previously ,43 to deRNAi experiments in cultured cells were performed as previously reported. Cultured cells were transfected according to the manufacturer’s protocol, which was modified in our laboratory ,44,45. BRepresentative cell populations from each condition were examined by light microscopy. No significant morphological change was observed under experimental condition. Cell viability was also confirmed by trypan blue exclusion.t-test for comparison of two means and ANOVA for three or more means . p values < 0.05 were considered significant.All above experiments were carried out at least in triplicate with similar results. Statistical analysis was performed using the paired Student’s Salmonella-infected IECs via the expression of different proteins and signaling pathways. Downregulation of the pro-inflammatory response by statins prevents the detrimental effects of overwhelming inflammation in the host, whereas enhanced activity of antimicrobial peptides in the IECs protects the host against infection. Thus, the effects of statins on innate immunity in IECs may provide an alternative or combination therapy for Salmonella colitis and IBD. However, well-controlled clinical trials are needed for the administration of statins as an adjunctive treatment in Salmonella infection or IBD.In conclusion, we observed the differential regulation of statins on pro-inflammatory chemokine IL-8 and antimicrobial peptide hBD-2 in


Neuromuscular impairment and reduced musculoskeletal integrity are hallmarks of spinal cord injury (SCI) that hinder locomotor recovery. These impairments are precipitated by the neurological insult and resulting disuse, which has stimulated interest in activity-based physical rehabilitation therapies (ABTs) that promote neuromuscular plasticity after SCI. However, ABT efficacy declines as SCI severity increases. Additionally, many men with SCI exhibit low testosterone, which may exacerbate neuromusculoskeletal impairment. Incorporating testosterone adjuvant to ABTs may improve musculoskeletal recovery and neuroplasticity because androgens attenuate muscle loss and the slow-to-fast muscle fiber-type transition after SCI, in a manner independent from mechanical strain, and promote motoneuron survival. These neuromusculoskeletal benefits are promising, although testosterone alone produces only limited functional improvement in rodent SCI models. In this review, we discuss the (1) molecular deficits underlying muscle loss after SCI; (2) independent influences of testosterone and locomotor training on neuromuscular function and musculoskeletal integrity post-SCI; (3) hormonal and molecular mechanisms underlying the therapeutic efficacy of these strategies; and (4) evidence supporting a multimodal strategy involving ABT with adjuvant testosterone, as a potential means to promote more comprehensive neuromusculoskeletal recovery than either strategy alone. Spinal cord injury (SCI) results in profound sensorimotor impairment below the lesion site, which is precipitated by the insult to the spinal cord and exacerbated by a cascade of secondary cellular and molecular processes occurring in the acute and chronic post-injury phases ,2,3. NeuLow testosterone (T) is also a secondary consequence of SCI, with hypogonadism being ~four times more prevalent in men after SCI than in non-injured populations . Non-neuMotoneuron innervation of skeletal muscle fibers influences muscle mass and function, and fiber-type distribution . After SThe muscular adaptations to SCI have been detailed previously , which w3 ubiquitin ligases that stimulate atrophy in response to disuse and other catabolic states . How. How5]. an males , which inal cord .In rodent SCI models, BWSTT produces relatively consistent improvements in muscle integrity, even following complete transection. As evidence, after spinal transection, the incorporation of BWSTT completely preserved the muscle mass:body mass ratio ,196 and The structural improvements mentioned above appear to be influenced, in-part, by reduced catabolic signaling resulting from BWSTT. As evidence, in a rodent SCI model, three sessions of BWSTT suppressed >10 protein ubiquitination pathway genes, an effect accompanied by the full preservation of muscle mass . SimilarMhyl, Mybph, and Myh4 gene expression (fast-twitch fiber markers) was apparent in rodent muscle within five-days of initiating BWSTT [In addition to muscle preservation, BWSTT is reported to reduce soleus fatigue and attenuate the slow-to-fast fiber transition occurring in rodent moderate-contusion SCI models . Similarng BWSTT , indicatng BWSTT  and a ~tng BWSTT  to six-yng BWSTT , suggestIn rodent SCI models, ABTs limit injury severity, evidenced by the preservation of myelin, axons, and collagen morphology and decreased lesion volume , and proIn individuals with incomplete SCI, beneficial adaptations to spinal neuronal pathways have been demonstrated in response to BWSTT. For example, the soleus H-reflex phase-dependent modulation during walking normalizes homosynaptic facilitation, reverses homosynaptic depression, and improves the presynaptic inhibition of Ia afferents ,215,216.While the molecular signals regulating BWSTT-induced motoneuron recovery after SCI require further elucidation, there is growing evidence that several inducible neurotrophic factors influence neuroplasticity . For exa2 treatment increases NT-3 levels in several CNS regions [2-mediated mechanisms. BWSTT is also reported to upregulate genes involved in neuroplasticity  and angiogenesis , which may be important given that improved neurovascular remodeling has the potential to improve locomotor function after SCI [Neurotrophin-3 (NT-3) is another endogenous neurotrophin that regulates synaptic transmission, promotes neuromuscular junction maturation, and improves the survival and function of sensory neurons via the activation of TrkC (receptor), both of which are expressed in the spinal cord ,272,273. regions , althougfter SCI .We are unaware of published results from any randomized study evaluating T treatment as an adjuvant to BWSTT. However, at least one ongoing study is evaluating TRT as an adjuvant to electric stimulation-based resistance training in men with motor-complete SCI . A singlTRT is approved for the treatment of hypogonadism in adult men and has been shown to increase muscle protein synthesis in women . Howevern = 83,010 men with low T) that indicated TRT reduced all-cause mortality by 56% and MI and stroke risk by 24–36% in comparison with untreated hypogonadal men [To-date, meta-analyses have confirmed that TRT produces three adverse events (AEs) in non-neurologically impaired adult men: (1) polycythemia, which occurs in ~6% of men treated with TRT; (2) a higher number of cumulative prostate-related events (discussed below); and (3) a slight decrease in HDL cholesterol, which is of unknown clinical significance . The devadal men . Of noteadal men .The risk for increased cumulative prostate-related events also merits mention because prostate growth and worsened urinary symptoms resulting from TRT have the potential to worsen bladder dysfunction, which is already common in the SCI population . In thisSeveral less common risks are also associated with TRT, which we have previously reviewed , includi2. Regardless, these improvements appear to result in only minor improvement in locomotor recovery in rodents after moderate-severe contusion SCI, likely because only limited descending corticospinal input remains. In comparison, BWSTT improves descending corticospinal drive and/or facilitates reorganization of spinal cord neural circuitry below the lesion, which produces neuromuscular improvement and some restoration of musculoskeletal integrity. However, findings from well-controlled randomized clinical trials indicate that BWSTT only promotes limited clinically meaningful improvement in voluntary locomotor recovery in humans after SCI. Interestingly, activity-mediated neuromuscular plasticity appears to rely, in-part, upon the upregulation of BDNF/TrkB in the spinal cord. Androgen/AR signaling is required for BDNF/TrkB-mediated dendritic restoration in androgen sensitive motoneurons in the lumbosacral spinal cord, suggesting that T treatment may enhance activity-mediated neuromuscular plasticity after SCI. Indeed, our preliminary data supports this contention, given that adult male rodents receiving TE adjuvant to BWSTT exhibited profound musculoskeletal and neuromuscular improvement and recovery of voluntary weight-supported overground plantar stepping after moderate-severe contusion SCI. Elucidation of the molecular mechanisms underlying these neuromusculoskeletal and locomotor improvements will assist in validating our hypothesis and will improve the likelihood for the translation of this promising multimodal therapeutic strategy.Upregulation of FOXO signaling occurs in the immediate days to weeks after SCI, an effect that likely influences the rapid muscle loss in this population. Similarly, muscle PGC-1α and -1β are reduced in response to SCI, underlying the slow-to-fast fiber change and impaired mitochondrial function subsequent to SCI. In comparison, downregulation of IGF-1 and PI3K/Akt signaling occurs more chronically after SCI. These molecular changes are precipitated by the spinal injury and subsequent disuse, but may also be exacerbated by low T that occurs in many men with SCI. T promotes muscle anabolism via direct AR-mediated genomic signaling and/or via cross-talk with IGF-1 and the PI3k/Akt signaling pathway. T may also promote anticatabolic effects in muscle by directly or indirectly suppressing FOXO signaling. Further, androgen treatment increases PGC-1α in muscle and attenuates the slow-to-fast muscle fiber-type transition occurring in response to spinal transection. In addition, T improves the preservation of motoneuron dendritic length after SCI directly via AR-mediated mechanisms and/or indirectly, following aromatization to E


The temperature responsive behavior investigated by transmittance and dynamic light scattering showed that some of the obtained mPEG-PAAHPs exhibited obvious temperature responsiveness and could be used to prepare nanoparticles by quickly heating. Drug paclitaxel can be encapsulated into mPEG-PAAHP based nanoparticles with a high encapsulation efficiency up to 99% (corresponding to a drug loading content of around 9.9%). Dynamic light scattering results showed that the PTX-loaded nanoparticles had a mean size around 80 nm (PDI<0.2) and good stability in PBS with 150 mM ionic strength. In vitro cytotoxicity results showed that mPEG-PAAHP did not show any toxicity to HeLa cells, but the PTX-loaded nanoparticles based on mPEG-PAAHP exhibited obvious anti-cancer activity. Thus, the temperature responsive PEGylated polyaspartamide derivative mPEG-PAAHP may be a promising drug delivery system.The temperature responsive PEGylated polyaspartamide derivative, denoted as mPEG-PAAHP, was synthesized by the click reaction. FTIR and  Polymeric drug carriers with various stimuli responsiveness have attracted great attention in the past two decades ,2. TheseL-aspartic acid (NK105  were synthesized via the click reaction successfully. The structures of obtained mPEG-PAAHPs were confirmed by FTIR and


Physical inactivity measured during late-life is a modifiable risk factor for dementia, but many studies use concurrent assessments with limited longitudinal follow-up. Less is known regarding life course exposure to physical inactivity. Physical activity patterns at different ages may make independent contributions to dementia risk, which would point to multiple critical periods for intervention. Using Health and Retirement Study Life History Mail Survey data , latent growth curves tested whether retrospectively-reported activity in early (18-29 years) and middle (40-49 years) adulthood predicted later-life memory trajectories over 18 years . Total metabolic equivalents were computed from reports of moderate and vigorous physical activity. Biennial memory performance was modeled from study entry (between 1996 and 2014) to 2014. Self-reported physical and mental health at study entry were modeled as independent mediators. Models were adjusted for age at study entry, sex, education, race, ethnicity, childhood socio-economic status, year of study entry, and year of mail survey enrollment. More physical activity at ages 18-29 and 40-49 were independently associated with better initial memory, but not subsequent memory change. The association between physical activity at ages 40-49 and initial memory was partially mediated by better mental and physical health. These observational results support the possibility that physical activity interventions during multiple stages of the adult life course might be effective at lowering dementia risk. In particular, mid-life physical activity may have broad effects on later mental, physical, and cognitive health.


In this work, we propose ReStoCNet, a residual stochastic multilayer convolutional Spiking Neural Network (SNN) composed of binary kernels, to reduce the synaptic memory footprint and enhance the computational efficiency of SNNs for complex pattern recognition tasks. ReStoCNet consists of an input layer followed by stacked convolutional layers for hierarchical input feature extraction, pooling layers for dimensionality reduction, and fully-connected layer for inference. In addition, we introduce residual connections between the stacked convolutional layers to improve the hierarchical feature learning capability of deep SNNs. We propose Spike Timing Dependent Plasticity (STDP) based probabilistic learning algorithm, referred to as Hybrid-STDP (HB-STDP), incorporating Hebbian and anti-Hebbian learning mechanisms, to train the binary kernels forming ReStoCNet in a layer-wise unsupervised manner. We demonstrate the efficacy of ReStoCNet and the presented HB-STDP based unsupervised training methodology on the MNIST and CIFAR-10 datasets. We show that residual connections enable the deeper convolutional layers to self-learn useful high-level input features and mitigate the accuracy loss observed in deep SNNs devoid of residual connections. The proposed ReStoCNet offers >20 × kernel memory compression compared to full-precision (32-bit) SNN while yielding high enough classification accuracy on the chosen pattern recognition tasks. The proliferation in real-time content generated by the ubiquitous battery-powered edge devices necessitates a paradigm shift in neural architectures to enable energy-efficient neuromorphic computing. Spiking Neural Networks (SNNs) offer a promising alternative toward realizing intelligent neuromorphic systems that require lower computational effort than the artificial neural networks. SNNs encode and communicate information in the form of sparse spiking events. The intrinsic sparse event-driven processing capability, which entails neuronal computations and synaptic weight updates only in the event of a spike fired by the constituting neurons, leads to improved energy efficiency in neuromorphic hardware implementations  spiking non-linearity  and +1 (whigh), to produce the convolutional output maps. The convolutional outputs, referred to as post-synaptic currents, are fed to non-linear layer of Leaky-Integrate-and-Fire (LIF) spiking neurons  after which the membrane potential is reset. The IF neurons, in effect, fire based on the average spiking activity of the spatially pooled convolutional spike maps. We low-pass filter the spike trains of the pooled maps by integrating the spikes at every time instant and decaying the resultant sum between successive spike timing instants to estimate their spiking activations over the time period for which the input is presented. The spiking activations of the pooled maps pertaining to all the convolutional layers are fed to the fully-connected layer composed of ReLU neurons  while retaining the salient features. Spatial pooling also renders the network invariant to slight translations in the input features , integrating Hebbian and anti-Hebbian learning mechanisms to train the binary synaptic weights constituting an SNN. We present two versions of the HB-STDP learning rule, namely, excitatory HB-STDP (eHB-STDP) and inhibitory HB-STDP (iHB-STDP) to train the binary synaptic weights connecting excitatory and inhibitory pre-neurons, respectively, to excitatory post-neurons. An excitatory neuron is modeled as a neuron firing unit positive spikes while an inhibitory neuron fires unit negative spikes. Input image pixels with intensities ranging from 0 to 255 are mapped to excitatory pre-neurons firing unit positive spikes at a rate proportional to the respective pixel intensities. On the contrary, input images when pre-processed by normalizing the raw pixel intensities to zero mean and unit variance result in normalized images with positive and negative pixel intensities. The normalized pixels with negative intensities are mapped to inhibitory pre-neurons firing unit negative spikes. The normalized input maps containing excitatory and inhibitory pre-neurons offer richer spike-encoding of the image patterns, resulting in efficient STDP-based feature learning. We find that input normalization is critical for natural images like those from the CIFAR-10 dataset  triggers the post-neuron to fire  and the difference between the respective spike times (Δt = tpost−tpre) is smaller than a pre-specified time period (tHebb_pot), we switch the synapse from low to high (‘L’ → ‘H’) state with a constant probability, pHebb_pot, as illustrated in Binary synapses require a probabilistic learning rule to prevent rapid switching of the weights between the allowed levels, which could otherwise render the synapses memoryless. Both the proposed eHB-STDP and iHB-STDP learning rules map the time difference between a pair of pre- and post-spikes to the switching probability of the interconnecting binary synapse. We first detail the eHB-STDP learning rule for excitatory pre-neurons and subsequently discuss how the learning dynamics are adapted for inhibitory pre-neurons. According to eHB-STDP, if an excitatory pre-spike  window. It is important to note that eHB-STDP contains a dead zone in the STDP timing window, where the switching probability is zero, between the Hebbian potentiation and anti-Hebbian depression windows as depicted in where t (tpost−tpre) axis. As a result, the erstwhile potentiation windows are converted to depression windows, and vice versa. According to iHB-STDP, if an inhibitory pre-spike causes the post-neuron to fire and the spike timing difference is smaller than a pre-specified time period, we probabilistically depress the binary synaptic weight. This ensures that the strongly correlated inhibitory (negative) pre-spike modulated by the depressed synaptic weight causes an effective increase in the post-neuronal membrane potential, thereby improving the chances of a post-spike at subsequent time instants. Probabilistic synaptic depression enables a post-neuron to integrate the small positive time difference between an inhibitory pre-spike and the ensuing post-spike, which conforms to the Hebbian learning theory. Probabilistic synaptic potentiation, on the other hand, causes an inhibitory pre-spike modulated by the synaptic weight to lower the post-neuronal membrane potential, thus reducing the chances of a post-spike at subsequent time instants. Hence, it is carried out for large positive time difference (anti-Hebbian in nature) or small negative time difference (Hebbian in nature) between the pre- and post-spikes. The iHB-STDP learning rule for inhibitory pre-neurons effectively incorporates the learning dynamics of eHB-STDP for excitatory pre-neurons by mirroring the potentiation and depression windows about the Δt axis.Next, we discuss how the eHB-STDP dynamics are adapted for binary synapses connecting inhibitory pre-neurons firing negative spikes. The iHB-STDP dynamics shown in  are obtapre) that is reset to unity at the time instant of a pre-spike, and sampling it in the event of a post-spike. Smaller the time difference between the pre- and post-spikes, larger is the sampled pre-trace, and vice versa. Every pre-neuron has a pre-trace that is sampled upon a post-spike to obtain the positive spike timing difference. Likewise, every post-neuron has a post-trace (with time constant τpost) that is sampled upon a pre-spike to obtain the negative spike timing difference. As a result, the eHB-STDP (iHB-STDP) hyperparameters, namely, tHebb_pot (tHebb_dep), tantiHebb_dep (tantiHebb_pot), and tHebb_dep (tHebb_pot) are mapped to preHebb_pot (preHebb_dep), preantiHebb_dep (preantiHebb_pot), and postHebb_dep (postHebb_pot), respectively.In this work, we use trace-based technique to estimate spike timing differences as it is commonly adopted for efficient implementation of STDP learning rules  and jth output map in layer “l” (output mini-batch). We first average the spike timing differences between the spiking post-neurons and the respective pre-neurons, estimated using fixed STDPstride, over each output map in the mini-batch to obtain the resultant spike timing difference per output map in the mini-batch. We subsequently average the resultant spike timing differences of the output maps across the mini-batch and probabilistically update TSTDP, for which the training patterns are presented.STDP-based learning is typically performed in an online manner by feeding the input patterns sequentially. STDP-based online learning has been shown to work well particularly for two-layer fully-connected SNNs, where each output or excitatory neuron learns to spike exclusively for a unique class of input patterns by encoding a general input representation in the input to excitatory synaptic weights  in layer “l” at any given time t, τlpf is the low-pass filter time constant, Δtsim is the simulation time-step, Tsim is the simulation period for which the input patterns are presented, and l” over the simulation period. The spiking activation thus obtained accounts for the highly non-linear leaky-integrate-and-fire and membrane potential reset dynamics of the spiking neurons in the convolutional layers. The spiking activations of the pooled maps of all the convolutional layers are concatenated and fed to the fully-connected layer, trained using error backpropagation  SNN under iso-accuracy conditions.The binary fully-connected SNN  SNN, which is specified byWe first train a binary fully-connected SNN of 400 excitatory neurons using the three different eHB-STDP configurations on 3500 MNIST digit patterns. #input neurons is 784 for the MNIST dataset. where whigh) with a probability, phigh, which is specified byThe MNIST dataset contains 60,000 training patterns and 10,000 test patterns of handwritten digits that are stored as 28 × 28 Grayscale images. In this work, we developed a custom simulation framework using Pytorch . The firing threshold of the LIF neurons in every convolutional layer are initialized to zero.where αWe first simulated a 16C3-2P-10FC ReStoCNet, composed of single convolutional layer with 16 maps and 3 × 3 binary kernels followed by pooling layer whose spiking activations are directly fed to the final softmax layer. The input image pixels are mapped to excitatory pre-neurons firing at a rate constrained between 0 and 200 Hz depending on the corresponding pixel intensities. The eHB-STDP model parameters are provided in The CIFAR-10 dataset contains 50,000 training images and 10,000 test images, 32 × 32 × 3 in dimension, spanning 10 output classes. We pre-processed the CIFAR-10 images using global contrast normalization followed by ZCA whitening  and kernel memory compression as the evaluation metrics. The memory compression offered by ReStoCNet as a result of using binary kernels in the convolutional layers, referred to as kernel memory compression, is computed as specified byNReStoCNet (Nbaseline) and ksizeReStoCNet (ksizebaseline) are the number of kernels and kernel size, respectively, in ReStoCNet , and nbitsbinary and nbitsfull_precision are the hardware bit-precision required for storing the binary and full-precision kernels, which are set to 2-bits and 32-bits, respectively. Note that the binary kernels in ReStoCNet require storage capacity of 2-bits per synaptic weight since they are constrained to binary states −1 and +1. where TrueNorth  , parallel efforts are underway to develop neuromorphic hardware implementations with on-chip intelligence, which can exploit the inherent computational efficiency offered by the SNN algorithms. IBM Stochastic device technologies such as Conductive-Bridge Random Access Memory (CBRAM)  learning rule, integrating Hebbian and anti-Hebbian learning mechanisms, for training the binary kernels constituting ReStoCNet in a layer-wise unsupervised manner. We demonstrated up to 3-layer deep ReStoCNet and showed that residual connections are critical to enabling the deeper convolutional layers to self-learn useful high-level input features and improving the scalability of deep SNNs. ReStoCNet offered 98.54% accuracy and 39.5 × kernel memory compression compared to full-precision (32-bit) convolutional SNN under iso-accuracy conditions for MNIST digit recognition. On the CIFAR-10 dataset, ReStoCNet provided 66.23% accuracy and 21.7 × kernel memory compression, albeit with 5% accuracy degradation compared to full-precision convolutional SNN. We believe that ReStoCNet, with event-driven computing capability and memory-efficient probabilistic learning with binary kernels, is ideally suited for neuromorphic hardware implementations based on CMOS and stochastic emerging device technologies like Resistive Random Access Memory, Phase-Change Memory, and Magnetic Tunnel Junctions that can potentially lead to much improved energy efficiency in battery-powered IoT edge devices.https://www.cs.toronto.edu/~kriz/cifar.html.Publicly available datasets were analyzed in this study. This data can be found here: GS wrote the paper and performed the simulations. All authors helped with developing the concepts, conceiving the experiments, and writing the paper.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.


Opisthorchis viverrini-related cholangiocarcinoma, Schistosoma haematobium-associated bladder cancer, Helicobacter pylori-infected gastric cancer, human papillomavirus-related cervical cancer, Epstein-Barr virus-infected nasopharyngeal carcinoma) and pro-inflammatory factors . Interestingly, several of our studies suggested that inflammation-associated DNA damage in cancer stem-like cells leads to cancer development with aggressive clinical features. Reactive oxygen/nitrogen species from inflammation damage not only DNA but also other biomacromolecules, such as proteins and lipids, resulting in their dysfunction. We identified oxidatively damaged proteins in cancer tissues by 2D Oxyblot followed by MALDI-TOF/TOF. As an example, oxidatively damaged transferrin released iron ion, which may mediate Fenton reactions and generate additional reactive oxygen species. Dysfunction of anti-oxidative proteins due to this damage might increase oxidative stress. Such damage in biomacromolecules may form a vicious cycle of oxidative stress, leading to cancer development. Epigenetic alterations such as DNA methylation and microRNA dysregulation play vital roles in carcinogenesis, especially in inflammation-related cancers. We examined epigenetic alterations, DNA methylation and microRNA dysregulation, in Epstein-Barr virus-related nasopharyngeal carcinoma in the endemic area of Southern China and found several differentially methylated tumor suppressor gene candidates by using a next-generation sequencer. Among these candidates, we revealed higher methylation rates of RAS-like estrogen-regulated growth inhibitor (RERG) in biopsy specimens of nasopharyngeal carcinoma more conveniently by using restriction enzyme-based real-time PCR. This result may help to improve cancer screening strategies. We profiled microRNAs of nasopharyngeal carcinoma tissues using microarrays. Quantitative RT-PCR analysis confirmed the concordant downregulation of miR-497 in cancer tissues and plasma, suggesting that plasma miR-497 could be used as a diagnostic biomarker for nasopharyngeal carcinoma. Chronic inflammation promotes genetic and epigenetic aberrations, with various pathogeneses. These changes may be useful biomarkers in liquid biopsy for early detection and prevention of cancer.Infection and inflammation account for approximately 25% of cancer-causing factors. Inflammation-related cancers are characterized by mutagenic DNA lesions, such as 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-nitroguanine. Our previous studies demonstrated the formation of 8-oxodG and 8-nitroguanine in the tissues of cancer and precancerous lesions due to infection (e.g.,  It is estimated that infectious diseases and chronic inflammation account for approximately 25% of cancer-causing factors . Under cOpisthorchis viverrini; OV) infection and its related cholangiocarcinoma. OV infection is considered “carcinogenic to humans” (group 1) by the International Agency for Research on Cancer (IARC). In a collaboration with Khon Kaen University, Thailand, we firstly reported 8-nitroguanine and 8-oxodG formation in the livers of hamsters treated orally with the liver fluke in an animal model for OV-related cholangiocarcinoma [Schistosoma haematobium-associated bladder cancer [Helicobacter pylori-infected gastric cancer [The Khon Kaen region, in the northeastern part of Thailand, is a local endemic area for liver fluke , and prc cancer , 13, nanc cancer –17, and c cancer , 19, andc cancer ). In addIn our early studies on inflammation-related carcinogenesis, we detected the formation of 8-nitroguanine and 8-oxodG in inflammatory and cancer tissues by immunohistochemistry (IHC) and an electrochemical detector coupled to HPLC (HPLC-ECD). HPLC-ECD analyses also demonstrated that urinary 8-oxodG levels were significantly higher in OV-related cholangiocarcinoma patients than in OV-infested patients and healthy subjects and higher in OV-infested patients than in healthy subjects . The 8-oVarious death signals including ROS and DNA damage induce apoptosis via the mitochondrial pathway. Melatonin is a candidate chemopreventive agent for inhibiting cholangiocarcinoma development. The mechanisms of anti-cancer activity by melatonin remain unclear. We examined the induction of apoptosis in human cholangiocarcinoma cell lines treated with melatonin . FlowcytChronic inflammation causes various types of damage to nucleic acids, proteins, and lipids via ROS/RNS generation, resulting in tissue damage. The tissue injury may activate progenitor/stem cells for tissue regeneration. Stem cells are damaged by ROS/RNS from inflammation, and the resulted mutations can accumulate, which could generate cancer stem cells . In OV-r2O2) every day for more than 2 months, to be H2O2-resistant cells . The resistant cells grew more rapidly and had higher expressions of anti-oxidant enzymes such as catalase than the original parental cells. Recently, we found downregulation of early B cell factor 1 (EBF1), a tumor suppressor gene, in cholangiocarcinoma-cultured cells and ox-MMNK1-L cells compared to normal cholangiocyte cells (MMNK1) [We established a hydrogen peroxide-resistant cholangiocyte cell line, as a novel model of oxidative stress-related cholangiocarcinoma genesis . Parenta (MMNK1) . InteresEpstein-Barr (EB) virus infection is assessed as group 1 (carcinogenic to humans) by IARC. Nasopharyngeal carcinoma is a human epithelial tumor with a high prevalence in Southeast Asia and Southern China and has a strong association with EB virus infection , 26. We Inflammation-derived ROS/RNS damage not only DNA but also proteins. Carbonylation is an irreversible and irreparable protein modification induced by oxidative stress. Carbonylated proteins can be detected by treatment with 2,4-dinitrophenylhydrazine (DNPH), leading to a 2,4-dinitrophenyl (DNP) hydrazone adduct , which i2•−) and NO. NO reacts with O2•− to form highly reactive peroxynitrite (ONOO−), causing 8-oxodG and 8-nitroguanine. H2O2 generated from dismutation of O2•− reacts with Fe(II) to produce hydroxyl radical (•OH) in the Fenton reaction. Highly reactive •OH can attack DNA, proteins, and lipids. In addition to the above mentioned DNA and protein oxidation, inflammation also contributes to lipid peroxidation, for example, higher plasma levels of isoprostanes and malonaldehyde in cholangiocarcinoma patients [In the inflammatory microenvironment, ROS/RNS damage biomacromolecules, including DNA, proteins, and lipids, as shown in Fig. patients . Lipid ppatients .Fig. 1BiEpigenetics is the phenomenon of heritable changes in gene expression that occur without a change in DNA sequence , throughHelicobacter pylori infection-related gastric cancer [Human malignant tumors are characterized by changes in the patterns of DNA methylation, which include a globally hypomethylated tumor cell genome and the focal hypermethylation of numerous CpG islands; many of them are associated with gene promoters . Promotec cancer , 42 and c cancer , indicatProfiling of DNA methylation across the genome is extremely important to understanding the influence of epigenetics. There has been a revolution in DNA methylation analysis technology over the past decade, and analyses that previously were restricted to specific loci can now be performed on a genome scale . The maiMicroRNAs are a broad class of non-coding RNAs, 18–25 nucleotides in length in mature form, which control gene expression post-transcriptionally through binding to the 3′-untranslated region (3′-UTR) of mRNA transcripts, stimulating translational suppression or breakage of the target gene. MicroRNAs are involved in a wide range of biological processes including cell proliferation, differentiation, and apoptosis. Various microRNAs are frequently dysregulated in human cancers. We profiled microRNAs of nasopharyngeal carcinomas using microarrays and confirmed the results by quantitative RT-PCR. Among the dysregulated microRNAs in the tissues, plasma miR-497 was lower in cancer patients relative to non-cancerous control patients. Functional analyses revealed that miR-497 mimic-transfected cancer cells suppressed cell growth and migration and induced apoptosis, and showed slower tumor growth in subcutaneous xenografts. These results indicate that miR-497 has a potent tumor suppressor function and may be used as a diagnostic biomarker for nasopharyngeal carcinoma [Head and neck squamous cell carcinoma (HNSCC) is a prevalent malignancy worldwide, and its risk factors such as smoking, alcohol consumption, and HPV infection, are related to inflammation. In a collaborative study with the Department of Otolaryngology and Head and Neck Surgery, Mie University, we compared microRNA expression levels between HNSCC tissues and the adjacent non-cancerous tissues by microarray. Let-7c was one of the downregulated microRNAs in HNSCC tissues. To elucidate the molecular mechanisms underlying the progression of HNSCC, we investigated the function of let-7c as a tumor suppressor. In vitro and in vivo studies revealed that let-7c negatively regulated cancer cell proliferation, migration, and epithelial-mesenchymal transition (EMT) via dysregulation of its direct target genes, insulin-like growth factor 1 receptor (IGF1R), and the high mobility group AT-hook 2 (HMGA2) . FurtherLiquid biopsy is an approach to determine the genomic profile of patients with cancer for monitoring treatment responses and to assess the emergence of therapy resistance . Body flWe elucidated the molecular mechanisms of inflammation-related carcinogenesis from the aspects of DNA damage and epigenetic alteration and found several candidate biomarkers. Inflammation plays pivotal roles not only in cancer but also in neurodegenerative diseases , cardiov


Non-Alcoholic Fatty Liver Disease (NAFLD) is the growing epidemic which is rapidly increasing in USA . The earEpigenetic alterations play a critical role in the age-associated liver disorders. However, very little is known about age-dependent epigenetic signatures of liver disorders including hepatic steatosis. Epigenetic changes include methylation of CpG islands and changes of chromatin structure by modifications of histones. A recent report identified 152 age-dependent differentially methylated CpG islands between controls and NASH patients. These changes correlated with fibrosis . AnotherIn the first paper, Guillory et al investigated age-associated development of hepatic steatosis in mice with deletion of ghrelin, a hormone which promotes adiposity in animals and in humans . At younIn this regard, a recent paper by Nguyen et al examined the role of cdk4 in age-dependent hepatic steatosis using three settings: liver biopsies from old patients with NAFLD, cdk4-resistant C/EBPα-S193A mice and inhibition of cdk4 in old WT mice , heteroc


Epidermal growth factor receptor (EGFR) plays critical roles in cell proliferation, tumorigenesis, and anti-cancer drug resistance. Overexpression and somatic mutations of EGFR result in enhanced cancer cell survival. Therefore, EGFR can be a target for the development of anti-cancer therapy. Patients with cancers, including non-small cell lung cancers (NSCLC), have been shown to response to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) and anti-EGFR antibodies. However, resistance to these anti-EGFR treatments has developed. Autophagy has emerged as a potential mechanism involved in the acquired resistance to anti-EGFR treatments. Anti-EGFR treatments can induce autophagy and result in resistance to anti-EGFR treatments. Autophagy is a programmed catabolic process stimulated by various stimuli. It promotes cellular survival under these stress conditions. Under normal conditions, EGFR-activated phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase (AKT)/mammalian target of rapamycin (mTOR) signaling inhibits autophagy while EGFR/rat sarcoma viral oncogene homolog (RAS)/mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) signaling promotes autophagy. Thus, targeting autophagy may overcome resistance to anti-EGFR treatments. Inhibitors targeting autophagy and EGFR signaling have been under development. In this review, we discuss crosstalk between EGFR signaling and autophagy. We also assess whether autophagy inhibition, along with anti-EGFR treatments, might represent a promising approach to overcome resistance to anti-EGFR treatments in various cancers. In addition, we discuss new developments concerning anti-autophagy therapeutics for overcoming resistance to anti-EGFR treatments in various cancers. Constitutive signaling from the EGFR promotes cell survival, proliferation , and invAutophagy is a self-digesting cellular process that allows cells to sequester cytoplasmic contents, through the formation of double membrane vesicles (autophagosomes). Autophagy as a survival mechanism provides an alternative energy source and facilitates the disposal of unfolded proteins during metabolic stresses ,9. AllelEGFR signaling both suppresses and promotes the autophagic response. All EGFR downstream signaling pathways are involved in autophagy modulation. The PI3K/AKT1 axis downstream of EGFR can inhibit autophagy by activating mTOR, an inhibitor of autophagy . EGFR-meThis review focuses on the relationship between EGFR signaling and autophagy. We review recent reports concerning the emergence of autophagy as a mechanism of resistance to anti-EGFR treatments. We discuss the relevance of targeting both EGFR signaling and autophagy as a potential strategy for overcoming resistance to anti-EGFR treatments. We also review recent developments of therapeutics, such as chemicals, peptides, and microRNAs (miRNAs), that may overcome resistance to anti-EGFR treatments.EGFR plays critical roles in cell proliferation , differeThe extracellular ligand-binding domain of EGFR contains four distinct subdomains (I–IV)  Figure A. DomainEGFR mutations account for 10%–17% of NSCLC cases in North America and Europe and 30%–50% of NSCLCs in Asian countries. They are most common among patients with adenocarcinoma NSCLC and a light or non-smoking history ,39. Two Activating mutations in the tyrosine kinase domain of the EGFR among NSCLC cells can enhance responses to EGFR-tyrosine kinase inhibitors (EGFR-TKIs)  Figure . Point mEGFR D761Y mutation is associated with resistance to first-generation EGFR-TKIs such as gefitinib and erlotinib  Figure . EGFR G7There have been considerable advances in the treatment of NSCLC patients with EGFR mutations. Acquired resistance to EGFR-TKIs such as gefitinib and erlotinib is a critical obstacle in the treatment of EGFR mutant-positive NSCLCs.Signaling from EGF plays critical roles in growth, survival, proliferation and differentiation , anti-caAutophagy is a conserved catabolic process that involves self-digestion and the removal of dysfunctional proteins and organelles . HypoxiaDuring macroautophagy, cytoplasmic components are bordered by phagophores, which enlarge and seal to form autophagosomes . These aEGFR tyrosine kinase can induce the tyrosine phosphorylation of Beclin1 , forming Beclin1 homodimers, and inhibiting autophagy . The tyrAt present, EGFR-targeting anti-cancer drugs consist of two types: (1) EGFR monoclonal antibodies that inhibit the activation of the EGFR ligand-binding domain ; and (2)Cetuximab, a murine–human chimeric antibody, can bind to the extracellular domain of EGF receptor  Figure B. CombinDacomitinib A, a secoSTAT3 mediates EGFR signaling A. InhibiResistance to erlotinib and gefitinib occurs through EGFR T790M mutation. This mechanism  accounts for approximately 50% of the resistance to these EGFR-TKIs in NSCLC cells . EGFR T7Autophagy may provide cells with energy to survive a stress condition (such as under therapy with anti-cancer drugs) by recycling their own proteins and damaged organelles. Increased expression of Sequestosome 1 (SQSTM1/p62), a selective adaptor protein in autophagy, is associated with a poor response to cetuximab therapy . IncreasTumors treated with single agents usually develop resistance. In lung cancer cells, autophagy may be induced by EGFR-TKIs. Co-inhibition of EGFR signaling and autophagy has demonstrated promising results in vitro ,127. AutHydroxychloroquine (HCQ), chloroquine (CQ), and bafilomycin A1 can inhibit the fusion of autophagosome with lysosome  Figure B. LapatiMiRNAs (micro RNAs), 21–23 nucleotides in length, are endogenous non-coding RNAs that can regulate the expression of target genes either through the translational inhibition or destabilization of messenger RNA (mRNA) . MiRNAs Tumor suppressor miRNAs, such as let-7 and miR-34, can enhance the anti-cancer effects of erlotinib on NSCLC cells . MiR-217MiRNAs that show differential expression between anti-cancer drug-sensitive and anti-cancer drug-resistant NSCLC cells have been identified . These mExosomes are extracellular vesicles (EVs) produced in the endosomal compartment of eukaryotic cells. Multivesicular body (MVB) is an endosome that buds inward into the endosomal lumen. If MVB fuses with the plasma membrane, these vesicles are released as exosomes. Recent reports suggest a role of inactive EGFR in autophagy, which is stimulated by serum starvation and EGFR-TKIs . Endosom266AQTGTGKT273) corresponds to DEAD box domain of CAGE, binds to CAGE, inhibits the interaction between CAGE and Beclin1, decreases autophagic flux in PC-9/ER cells, and enhances the sensitivity of PC-9/ER cells to erlotinib and osimertinib [269GTGKT273) corresponds to DEAD box domain of CAGE, can inhibit the interaction between CAGE and glycogen synthase kinase 3 beta (GSK3β) and enhance the sensitivity of anti-cancer drug-resistant melanoma cells to anti-cancer drugs [CAGE, a cancer/testis antigen, can confer resistance to various anti-cancer drugs in melanoma cells. CAGE binds to EGFR and HER2. The downregulation of EGFR enhances the sensitivity of melanoma cells to anti-cancer drugs . CAGE bimertinib . CAGE-deer drugs . AQTGTGKer drugs . It willDistinct subgroup of the Ras family (DIRAS) can act as a tumor suppressor. DIRAS binds to Beclin1 and forms an autophagy initiation complex. DIRAS-derived peptides can bind to Beclin1 and inhibit DIRAS-mediated autophagy . These DEGFR has emerged as a valuable target for the development of anti-cancer drugs. EGFR-targeting antibodies (such as cetuximab and panitumumab) and EGFR-TKIs  have been developed. First generation EGFR-TKIs have shown promising effects on cancer patients with EGFR L858R mutation . HoweverResistance to these EGFR-TKIs can also occur through downstream signaling activation of Kirsten rat sarcoma viral oncogene homolog (KRAS), NRAS, BRAF, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), and tyrosine-protein kinase Met (MET) mutations  Figure A and pheUnderstanding the functional association of autophagy with anti-EGFR resistance may provide a promising therapeutic strategy to enhance the effects of EGFR-TKIs in cancer patients with EGFR mutations and/or overexpression. Autophagy induced by anti-EGFR treatments can promote the survival of tumors with high levels of basal autophagy, ROS, and DNA damage or under metabolic stress A. Report


We have further observed that c-MET is also highly expressed in a peculiar type of cells of the CLL-microenvironment showing nurturing features for the leukemic clone (nurse-like cells: NLCs). Since HGF treatment drives monocytes toward the M2 phenotype and NLCs exhibit features of tumor associated macrophages of type 2 we suggested that HGF, released either by cells of the microenvironment or leukemic cells, exerts a double effect: (i) enhances CLL cells survival and (ii) drives differentiation of monocytes-macrophages to an oriented immune suppressive phenotype. We here discuss how paracrine, but also autocrine production of HGF by malignant cells, may favor leukemic clone expansion and resistance to conventional drug treatments in CLL, as well as in other hematological malignancies. Novel therapeutic approaches aimed to block HGF/c-MET interactions are further proposed.Chronic lymphocytic leukemia (CLL) is characterized by the progressive expansion of B lymphocytes CD5+/CD23+ in peripheral blood, lymph-nodes, and bone marrow. The pivotal role played by the microenvironment in disease pathogenesis has become increasingly clear. We demonstrated that bone marrow stromal cells and trabecular bone cells sustain survival of leukemic B cells through the production of hepatocyte growth factor (HGF). Indeed the trans-membrane kinase receptor for HGF, c-MET, is expressed on CLL cells and STAT3 TYR Chronic lymphocytic leukemia (CLL), the most common leukemia in the western world, is characterized by a progressive expansion of relative mature B lymphocytes, CD19+/CD5+/CD23+, in peripheral blood, secondary lymphoid tissues, and in bone marrow (BM) [Chronic lymphocytic leukemia cells in vivo show an apparent long half-life while they tend to rapidly undergo apoptosis when cultured in vitro, in medium supplemented with only fetal bovine serum. This observation suggests that prolonged in vivo survival may be due to cell-to-cell interactions or soluble factors, present in the microenvironment, protecting leukemic cells from apoptotic cell death ,6,7,8,9.The interaction between hepatocyte growth factor (HGF) and its receptor c-MET regulates various physiological processes such as embryogenesis, regeneration of damaged organs, hematopoiesis, and immune cell functions. However the aberrant expression of c-MET and of HGF is implicated in neoplastic cells spreading and tumor progression in many solid tumors ,11,12,13To date, only a few studies have addressed the role of HGF in lymphomas. Moreover data from these reports, on the supposed relationship between enhanced c-MET expression and disease pathogenesis, are sometimes conflicting, especially in diffuse large B cell lymphoma (DLBCL) or in Hodgkin disease (HD) ,15. NumeFrom our studies, first focused to clarify whether various cell types sharing the same embryonic origin  affect survival and expansion of the leukemic B cell clone, we identified HGF as a novel factor, released from cells of the CLL-microenvironment, capable of protecting leukemic cells from apoptosis . We haveWe here describe how the HGF/c-MET axis is involved in a complex scenario taking place in different hematological malignancies: we will deepen details relative to the CLL microenvironment to explicit how HGF, produced within the stroma by different mesenchymal cell types and by the CLL cells themselves, contributes to increase viability of the c-MET+ leukemic clone and further affects differentiation of surrounding cells, leading to suppression of normal immune responses. Moreover, we will summarize and explore therapies aimed to block survival advantages provided by c-MET/HGF interactions in leukemia.HGF first discovered as a potent stimulator of cultured hepatocytes  is produc-MET is largely expressed on epithelial cells but certain hematopoietic cells may also carry this receptor and gain sensitivity to HGF: c-MET is present in B cells of the germinal center or in terminally differentiated plasma-cells . In normThe HGF/c-MET pathway appears further largely activated in malignant cells from primary effusion lymphoma (PEL): here the expression of plexin B1 is required for c-MET-mediated survival of PEL cells. Interestingly, targeting of c-MET/HGF pathway successfully suppresses PEL diffusion in a pre-clinical mouse model ,31,32. cMultiple studies have established that the HGF/c-MET pathway may have a critical role in MM pathogenesis: elevated HGF and HGFA concentrations were in fact detected in sera from MM patients and were correlated with progressive disease ,43. FurtHGF and c-MET clearly contribute to MM pathogenesis, while data reported from various authors about a potential relationship between prognosis and c-MET expression in Hodgkin lymphomas are instead sometimes discordant ,52,53. TThis brief overview outlines how upregulated c-MET expression on leukemic cells, and paracrine but also autocrine release of HGF , may creThe peripheral blood represents only part of the CLL life cycle. The observation that most circulating CLL cells are out of the cell cycle but have undergone a significant number of divisions, as demonstrated by in vivo kinetic studies with deuterated water labeling , suggestHerishanu et al.  through TYR705 and AKT phosphorylation. ERK1/2 appeared instead phosphorylated only after the addition of exogenous CXCL12 to CLL cells. These findings may apparently suggest that two different signaling pathways should be alternatively activated via HGF or via CXCL12, after CLL cells cross-talk with osteoblasts or with fibroblasts, respectively [136. Although we could not detect significant amounts of HGF in media from fresh CLL B cells cultured for 72 h, we instead found higher HGF levels in sera from CLL patients than in sera from normal controls  as already reported [We first focused our attention to dissect whether different stromal components with the same mesenchymal origin may differentially sustain survival of CLL cells . We deteectively . The invectively . Observaectively . They dereported . These fTYR705 phosphorylation. More importantly, we further confirmed c-MET and IDO positivity, through immune-histochemical staining, in cells resembling NLCs morphology within BM and lymph-nodes biopsies from CLL patients. Moreover, in vivo, IDO appeared co-expressed with CD163, an antigen typically displayed by tumor associated macrophages [It has been described that HGF promotes monocyte differentiation toward the generation of cells producing IL-10 but not IL-12 and favors the expansion of T regulatory cells (Treg) ,63. Whenrophages . The prerophages ,68,69 throphages ,72. In arophages . IDO degrades trypthophan into kynurinine which, in turn, inhibits effector T cells and stimulates Treg expansion. It is of interest to note that kynurinine–tryptophan ratios, reflecting increased IDO activity, have been found higher than normal in sera from CLL patients . In addi−/− knockout mouse. Here the authors suggest that MIF sustained the expansion of the CLL clone through interaction with macrophages: the last appeared in fact reduced in the spleen and in BM of the Eµ-TCL1+/wt MIF−/− mice [Whether particular soluble factors may be involved in increasing HGF release from leukemic B cells is still unknown. It has been reported that in naïve splenic B cells from mice, HGF, produced after the interaction between CD74 and macrophage migration inhibitory factor (MIF), regulates the survival of these cells . MIF, am−/− mice .The presence of elevated MIF levels have been also observed in MM patients and higher concentrations of MIF appear implicated in homing of MM cells to bone marrow and in resistance to chemotherapy . In addi−/− and c-MET−/− exploitation in a whole living organism is limited unless targeting c-MET deletion in specific tissues through conditional KO mice [Co-culture systems are in part capable of recapitulating CLL cells/tumor microenvironment interactions. Co-cultures of CLL cells with stromal cells, T cells or transfected-fibroblasts expressing CD40L or CD31 ,83 have  KO mice .Finally, a better comprehension of the complex cross-talk among leukemic cells, stromal cells, and immune cells within a structured model of culture should further clarify the potential role of HGF/c-MET axis in pathogenesis and progression of hematological disease.The tumor microenvironment has been recognized as a key contributor in progression of neoplastic diseases. Inhibition of c-MET signaling may impair cancer cell growth and at the same time stimulate immune responses via relieving HGF/c-MET induced immunosuppressive effects on mono-macrophages and on T cells. We demonstrated that the c-MET tyrosine kinase inhibitor SU11274 or the neutralizing anti-HGF moAb L2G7  counteracted increased CLL cells viability induced by HGF treatment or after co-culturing leukemic cells with specific stromal cells (BMSC, trabecular bone cells, the osteoblasto-like cell line MG63  and FiguThe utilization of mesenchymal-derived exosomes further represents today a novel and emerging alternative chance of intervention to contrast cancer-supportive microenvironments . ExosomeThe tumor micro-environment has a pivotal role in promoting tumor development. Stromal cells, integral components of the tumor microenvironment, activate c-MET through HGF release. Therefore, high c-MET expression and involvement of HGF/c-MET pathway has been observed in a large part of cancer and has been correlated with poor patient survival. Abnormal activation of c-MET tyrosine kinase is further critical for resistance to targeted therapies. Although studies referring to activation of the HGF/c-MET axis in hematological diseases are relatively a few, it appears that this interaction may, directly or indirectly, favor the expansion of the leukemic clone. We in particular described a double effect of HGF leading to enhanced survival of CLL cells but also driving monocytes/macrophages toward an alternative M2 suppressor phenotype, thus facilitating tumor evasion from immune control. Our findings may thus indicate that targeting of HGF, a novel microenvironmental factor involved in CLL pathogenesis, could improve conventional therapies to cure this disease by inhibiting leukemic cell growth and by restoring specific immune responses against the tumor.