Thyroid nodules are a very common problem. Since malignant thyroid nodules should be treated surgically, preoperative diagnosis of thyroid cancer is very crucial. Cytopathologic analysis of percutaneous fine-needle aspiration (FNA) specimens is the current gold standard for diagnosing thyroid nodules. However, this method has led to high rates of inconclusive results. Metabolomics has emerged as a useful tool in medical fields and shown great potential in diagnosing various cancers. Here, we evaluated the potential of nuclear magnetic resonance (NMR) analysis of percutaneous FNA specimens for preoperative diagnosis of thyroid cancer. We analyzed metabolome of FNA samples of papillary thyroid carcinoma (n = 35) and benign follicular nodule (n = 69) using a proton NMR spectrometer. The metabolomic profiles showed a considerable discrimination between benign and malignant nodules. Receiver operating characteristic (ROC) curve analysis indicated that seven metabolites could serve as discriminators . These findings demonstrated that NMR analysis of percutaneous FNA specimens of thyroid nodules can be potentially useful in the accurate and rapid preoperative diagnosis of thyroid cancer. Thyroid nodules are a very common problem, and they have been detected in up to 67% of the general population with ultrasonography (US) evaluations12However, this procedure has led to high rates of inconclusive results, including nondiagnostic and atypia of undetermined significance or follicular lesion of undetermined significance564567Due to the limitations of conventional cytopathologic analysis, several molecular analyses have been assessed and reported to have some potential as adjuncts to conventional cytopathologic analysis111213in vivo proton magnetic resonance spectroscopy (1H MRS), which might have limited clinical use, all of the previous studies were based on postoperative surgical specimens. The previous studies did not test the metabolomic approach as a preoperative diagnostic tool for thyroid nodules212223242526Metabolomics has emerged as a useful tool in multiple medical fields. It has shown great potential in diagnosing various cancers, such as breast, prostate, and colorectal cancers1516171819212223242526Therefore, the purpose of this study was to evaluate the potential of nuclear magnetic resonance (NMR) analysis of percutaneous FNA specimens for preoperative diagnosis of thyroid cancer.Among a total of 230 percutaneous FNA samples, 85 were benign  and 53 were malignant  based on cytopathologic analyses according to the Bethesda systemAmong them, 34 could not be analyzed due to the following reasons:Thirty-four thyroid nodules  were not analyzed because of insufficient FNA sampling amount (n = 29), failed freezing storage of the FNA sample (n = 1), sample loss (n = 1), and insufficient water suppression or shimming (n = 3).Finally, percutaneous FNA samples of 104 nodules from 100 patients  were analyzed using NMR . The sizOf 35 malignant nodules, 25 nodules of 24 patients underwent surgery. All of these were confirmed to be malignant (papillary thyroid carcinoma).V600E mutation were available in 22 cancers. There were 18 BRAFV600Emutation-positive nodules and four mutation-negative nodules.Of 25 surgically confirmed papillary thyroid carcinomas, the results of BRAFOnly one of 69 benign nodules underwent surgery because of concomitant thyroid cancer. This case was diagnosed as nodular hyperplasia.2 value of 0.33 (R2Y = 0.59). In the prediction validation study with three-fold cross-validation, the accuracy, sensitivity, and specificity for diagnosing papillary thyroid carcinoma were 88.6%, 75%, and 95.6%, respectively.We performed multivariate statistical analysis to discriminate benign and malignant nodules. The orthogonal projections to latent structures-discriminant analysis (OPLS-DA) has been applied because it can discriminate groups in the presence of high structured noise or confounding factors. As indicated in t test , choline (3.2 ppm), O-phosphocholine (3.2 ppm), and glycine (3.6 ppm) were greater in malignant nodules than in benign nodules. The relative amounts of citrate (2.6 ppm), glutamine (2.1 ppm), and glutamate (2.0 ppm) were greater in benign nodules than in malignant nodules . These ft test .For evaluating the discriminating model, we performed the receiver operating characteristic (ROC) curve analysis. The grades predicted by the PLS-DA model showed that area under the curves (AUCs) ranged from 0.64  to 0.85 (citrate). Citrate exhibited the sensitivity of 90% and specificity of 80% . To evalV600Emutation-positive papillary thyroid carcinomas and mutation-negative papillary thyroid carcinomas, there were no significant differences because two groups did not separated with negative Q2 value (−0.536) at OPLS-DA analysis.In the analysis of difference in metabolomic profiles between BRAFin vivo1H MRS differentiated between benign and malignant thyroid nodules, this technique cannot be easily applied to patients with thyroid nodules. This is because thyroid nodules are not usually large enough to place voxel for in vivo1H MRS and may be influenced by susceptibility artifact due to their anatomical adjacency to the airway21To our knowledge, the current study enrolled the largest number of thyroid nodules for NMR analysis. It was also the first study showing that NMR analysis of percutaneous FNA specimens of thyroid nodules could be applied for preoperative diagnosis of thyroid cancer. Although some previous studies reported that preoperative Although metabolic analysis usually uses biofluids such as urine, blood, cerebrospinal fluid, and bile juice15171922Although some specimens could not be used for metabolomic analysis in the present study, NMR analysis was performed in approximately 75% of all cases especially with only the remainder of the samples after allotment for conventional cytopathologic analysis. This indicates that the NMR approach is not an extra burden for patients in providing a better diagnosis.2 (R2Y) ranging from 0.37 (0.75) to 0.91 (0.82)25The present study had lower statistical power than previous studies using postsurgical specimens, which had Qin vivo1H MRS studies and studies using surgical specimens2123252627Higher relative concentrations of lactate and choline, and lower relative concentrations of citrate, glutamine, and glutamate in malignant thyroid nodules were found in the present study using preoperative percutaneous FNA specimens. These results are generally concordant with those of several previous Lactate concentrations are frequently increased in thyroid cancer and other malignant tumors2122232526293025262729303125de novo synthesis and catabolism3334Increased glycine in thyroid cancer may also result from increased glycolysis. Many recent studies reported that glycolytic intermediate metabolism plays an important role in tumorigenesis, and one of the main pathways is single carbon metabolism. Recently mitochondrial synthesis and consumption of glycine was proposed to be necessary for rapidly growing cancer cells. And glycine dehydrogenase (GLDC), which cleavages glycine and mediates folate cycle charging, are highly expressed on tumor promoting cells and its enhanced activity is associated with tumorigenesis. Therefore glycine could be one of important metabolites at tumorigenesis with its Unlike the results of previous studies using postoperative surgical specimens, this study presented that choline was increased in thyroid cancer25212223Citrate was the most powerful discriminator in the present study. Since proliferating cells exhibit aerobic glycolysis and convert glucose to lactate  at high levels, pyruvate-derived citrate synthesis in mitochondria may be reduced in these cells263638de novo lipogenesis36Recent publications on cell metabolism emphasize that proliferating cells exposed to hypoxic conditions rely almost exclusively on reductive carboxylation of glutamine-glutamate-derived α-ketoglutarate for This study has several limitations. First, since the reference used for comparison was based on Bethesda categories II and VI, which show typical benign and malignant cytologic features, the real metabolic spectra of benign and malignant thyroid nodules might be different from our results. Further study with a larger number of nodules should be performed to validate the diagnostic reference.Second, about 25% of samples (34/138) in the present study could not be used for metabolic analysis. The most important reason was insufficient remaining sample volume after allotment for conventional cytologic analysis. A more delicate preparation method is needed to ensure a sufficient sample amount for both conventional cytologic analysis and NMR analysis. However, in this study, as approximately one third of the thyroid nodules (n = 50) were less than 1 cm which are not usually indicated for FNA, this could also make sample volumes insufficient.Third, all malignant samples were papillary thyroid carcinomas. Further studies are necessary to evaluate the metabolic profiles of different thyroid neoplasms, such as follicular adenoma, follicular thyroid carcinoma, medullary thyroid carcinoma, and anaplastic thyroid carcinoma.V600E mutation, a useful prognostic marker for papillary thyroid carcinoma. Further studies using a large number of thyroid nodules should be planned to correlate between various molecular markers and metabolic profiles.Fourth, this study failed to reveal the correlation between metabolic spectra and BRAFLastly, studies with a large number of patients should be undertaken to verify the usefulness of NMR analysis in various clinical applications including the nodules with insufficient or indeterminate results and how to use NMR analysis in conjunction with the current cytopapthologic analysis.In summary, NMR analysis of preoperative percutaneous FNA specimens of thyroid nodules presented different metabolomic profiles for benign and malignant thyroid nodules in this study. NMR analysis of FNA specimens of thyroid nodules may be useful in the accurate and rapid preoperative diagnosis of thyroid cancer.This prospective study was approved by the institutional review board of Seoul National University Hospital. All the methods used in this study were carried out in accordance with the approved guidelines. Informed consent was obtained from all patients.From November 2012 to June 2013, 230 nodules of 214 consecutive patients underwent FNA for diagnosis of thyroid nodules and subsequent sampling for NMR analysis by 9 board-certified radiologists specializing in head and neck imaging . Patients who had a history of previous FNA, thyroid surgery, or thyroid hormonal treatment were excluded from the study. For patients with multiple thyroid nodules, only the nodules with different ultrasonographic features from one another and located in different thyroid lobes were included.Under the guidance of high-resolution US machines  with 10–12MHz linear transducers, FNA was performed with up to 4 needle passes using capillary or aspiration FNA techniques according to the characteristics of thyroid nodules. Immediately after FNA procedures, aspiration specimens were smeared on a slide, fixed with alcohol, and stained with Papanicolaou for conventional cytologic analysis. The remainders of aspiration specimens were collected in Eppendorf tubes (20–40 μL). The tubes were kept in a dry-ice box and stored in a liquid nitrogen tank until metabolomic analysis was performed.V600E mutation status was analyzed by direct DNA sequencing and metabolomic profiles were analyzed between mutation positive and negative groups.Cytopathologic analyses were done according to the Bethesda system2O to a final volume of 35 μL. The one-dimensional spectra of thyroid samples were measured using an NMR spectrometer  equipped with a 1.7-mm PATXI probe operating at a proton NMR frequency of 700.193 MHz.Samples from thyroid nodules were slowly thawed in an icebox, after which they were centrifuged at 13,000 rpm. The supernatant was collected with a pipette and placed in 1.7-mm SJ tubes with 0.25% trimethylsilane propionic acid (TSP) buffer in Dδ = 1.342 ppm) was used as a reference value.The acquisition parameters were: pulse, CPMG; time domain size, 32,768; relaxation delay, 2 s; number of scans, 128; spectral width, 14,097 Hz; mixing time, 76 ms; and temperature, 25 °C. The lactate signal  against total integration values and TSP buffer to exclude the effects of different volumes and NMR measurement variations. Data was then converted into an ASCII text file. The regions corresponding to water (4.71–5.1 ppm) were removed from all spectra. Binning, normalization, and conversion were performed using an in-house Perl program.P value < 0.05) between benign and malignant groups in terms of area normalization values were determined using the unpaired Student t test. Thereafter, metabolites were identified using Chenomx  by fitting the experimental spectra  to those in the database.The signals in specific bins that showed significant differences , and mean centering was performed with Pareto scaling for multivariate statistical analysis. Furthermore, OPLS-DA was performed with one predictive component and two orthogonal components for benign and malignant thyroid nodules and also for BRAFa priori cut-off value of 0.5 was used to evaluate the prediction results2 and R2). Eventually, the specific signals were compared to metabolites identified using the Chenomx data base.Class discrimination models were created while ensuring that the cross-validated predictability value did not significantly increase to avoid over-fitting of the statistical model. Diagnostic performance was obtained by predicting three-fold cross-validation samples on the basis of a distinction model constructed using the rest of the samples. An www. roccet.ca).The performance of the prediction derived from OPLS-DA modeling of NMR spectra was evaluated by computing the AUC using an open source ROC curve analysis tool for metabolomics data .


Migration, proliferation and stem cell-like activity are all key cellular characteristics which aid the formation and progression of breast cancer, in addition to involvement in treatment resistance. Many current therapies aim to target tumour proliferation, and although successful, mortality rates in breast cancer remain significant. Our main objectives were to investigate the relationship between proliferation, migration and stem cell-like activity in breast cancer.We used a panel of cell lines and primary human breast cancer samples to assess the relationship between migration, proliferation and stem cells. We performed live cell sorting according to cell cycle (Hoechst-33324) and in combination with stem-cell markers (CD44/CD24/ESA) followed by assessment of migration and stem cell activity (mammosphere formation).We identified an inverse relationship between proliferation and migration/stem cell-like activity. G0/1 cells showed increased migration and mammosphere formation. Furthermore we identified a subpopulation of low proliferative stem-like cells (CD44+/24lo/ESA+) with increased migration and mammosphere formation that are specifically inhibited by Dickkopf 1 (DKK1) and Dibenzazepine (DBZ) known stem-cell inhibitors.These data show the co-ordination of migration, proliferation and stem cell activity in breast cancer, and has identified a sub-population of stem-like cells, greatly adding to our understanding of the complex nature of stem cell biology. Breast cancer is one of the most common diseases in women in the Western world, but despite the introduction of anti-cancer treatments such as radiotherapy and targeted drugs such as the anti-oestrogen Tamoxifen, a significant proportion of patients are either resistant to treatment or show disease recurrence. Given that breast cancer currently accounts for approximately 200 000 deaths each year and that the incidence of breast cancer is increasing worldwide, it is essential that we have a better understating of tumour characteristics in order to develop more effective targeted therapies -3.Recurrences at metastatic sites, in particular lung and bone represent the major cause of mortality in breast cancer patients , 5. MigrA number of general tumour characteristics have been described with loss of control of proliferation considered a hallmark of many cancer types including breast cancer. Normal cellular proliferation is a highly regulated process however when the signals that control proliferation are deregulated, cancer may develop. This deregulation of proliferation may occur due to epithelial mutations or altered regulation of genes that control growth and proliferation, with numerous tumour suppressor genes having been identified. Furthermore, surrounding cells within the tumour stroma may secrete growth factors which in turn allow the uncontrolled proliferation of the cancer cell .+/24−, or by mammosphere formation and self-renewal ). Cells were grown for 5 days and maintained in a humidified incubator at 37°C at an atmospheric pressure in 5% (v/v) carbon dioxide/air. Spheres >50μm were counted using an eye piece graticule, and the percentage of cells plated which formed spheres was calculated and is referred to as the percentage mammosphere formation (%MSF).: Live cell imaging of mammospheres was performed using an Olympus CKX41 inverted light microscope at x40 magnification, scale bar represents 50μm. Secondary Mammospheres: After 5days of culture, mammospheres were collected by centrifugation at 800rpm for 2 minutes, dissociated by manual disaggregation into a single cell suspension at re-plated at 500 cells/cm2 in mammosphere medium for a further 5 days. Culture of cells in these conditions allows the survival of stem-like cells and subsequent spheroid growth from a single cell using both cell lines and primary tissue samples. Mammospheres grow at a similar rate when plated as single cells or at higher densities indicating that mammospheres are truly clonal structures and not formed through aggregation [A single cell suspension was prepared using enzymatic , and manual disaggregation (25 gauge needle). Patient-derived breast cells did not require the addition of trypsin, a single cell suspension was created using manual disaggregation. Cells were plated at a density of 500 cells/cmregation , 22.Transwell chambers with a diameter of 6.5 mm and a pore size of 8 μm were used to assess migration . Migration chambers were incubated with 150 μl serum-free media for an initial equilibrium period of 1 hour. Cells (transfected the day before) were resuspended in serum-free media and 50 000 cells were added to each migration chamber. Next, chambers were placed into wells containing 600 μl 10% FCS medium, and cells were allowed to migrate for 5 hours  or overnight . Cells that had not migrated were removed with a cotton swab, whereas the migrated cells situated on the lower side of membranes were fixed in 4% paraformaldehyde for 25 minutes. Membranes with the migrated cells were mounted on glass slides for DAPI staining and counted using a fluorescent microscope (cells in six X10 magnification fields were counted). Experiments were repeated at least three times.Wnt pathway inhibition. MDA-MB-231, MDA-MB-468, MCF7, and T47D cells and primary human breast cancer cells were plated into MS culture following FACS cell sorting and treated with a single dose of human recombinant Dickkopf 1( DKK1) (R and D systems) (50ng/ml).Notch pathway inhibition. MCF7, T47D cells and primary human breast cancer cells were plated into MS culture following FACS cell sorting and treated with a single dose of Dibenzazepine .Throughout the paper data is represented as mean ±SEM taken over a minimum of three independent experiments. Statistical significance was measured using parametric testing, assuming equal variance, with standard t-Tests for two paired samples used to assess difference between test and control samples.


Background: Recent studies suggested that cardiac conduction in murine hearts with narrow perinexi and 50% reduced connexin43 (Cx43) expression is more sensitive to relatively physiological changes of extracellular potassium ([K+]o) and sodium ([Na+]o).Purpose: Determine whether similar [K+]o and [Na+]o changes alter conduction velocity (CV) sensitivity to pharmacologic gap junction (GJ) uncoupling in guinea pigs.Methods: [K+]o and [Na+]o were varied in Langendorff perfused guinea pig ventricles (Solution A: [K+]o = 4.56 and [Na+]o = 153.3 mM. Solution B: [K+]o = 6.95 and [Na+]o = 145.5 mM). Gap junctions were inhibited with carbenoxolone (CBX) (15 and 30 μM). Epicardial CV was quantified by optical mapping. Perinexal width was measured with transmission electron microscopy. Total and phosphorylated Cx43 were evaluated by western blotting.Results: Solution composition did not alter CV under control conditions or with 15μM CBX. Decreasing the basic cycle length (BCL) of pacing from 300 to 160 ms decreased CV uniformly with both solutions. At 30 μM CBX, a change in solution did not alter CV either longitudinally or transversely at BCL = 300 ms. However, reducing BCL to 160 ms caused CV to decrease more in hearts perfused with Solution B than A. Solution composition did not alter perinexal width, nor did it change total or phosphorylated serine 368 Cx43 expression. These data suggest that the solution dependent CV changes were independent of altered perinexal width or GJ coupling. Action potential duration was always shorter in hearts perfused with Solution B than A, independent of pacing rate and/or CBX concentration.Conclusions: Increased heart rate and GJ uncoupling can unmask small CV differences caused by changing [K+]o and [Na+]o. These data suggest that modulating extracellular ionic composition may be a novel anti-arrhythmic target in diseases with abnormal GJ coupling, particularly when heart rate cannot be controlled. Normal cardiac conduction is critical for maintaining efficient pumping of the heart, and abnormal conduction can lead to arrhythmias and sudden cardiac death. Cardiac conduction is dependent on propagation of electrical signals from cell-to-cell. Cell membrane depolarization can occur by raising the intracellular potential via direct axial current through high resistance gap junctions , or by decreasing the extracellular potential between closely adjacent cells (ephaptic coupling)  and potassium ([K+]o) are complex  would unmask conduction sensitivity to [K+]o and [Na+]o in guinea pig hearts with narrow perinexi.The purpose of this study was twofold. First, we wanted to determine if the observed response of cardiac conduction to . The heart was extracted, retrogradely perfused in a Langendorff perfusion apparatus and the atria excised to reduce competitive stimulation. The hearts were perfused with constant flow to maintain pressure between 40 and 55 mm Hg. Tyrode's solutions were altered as described in Table Male retired breeder Hartley albino guinea pigs  and [Na+]o by 5.4% (145.5 to 153.3mM) from historic guinea pig Tyrode's solutions  in Solution A or B.All conduction measurements were taken 10 min after the new perfusate reached the heart to control for the amount of time each heart was exposed to the solution, and because we previously demonstrated CBX slowed conduction to near steady-state values within 10 min  was sectioned into 1 mmThe voltage sensitive dye di-4-ANEPPS (15 μM) was perfused for 10 min before the start of the experimental protocol. Cardiac motion was reduced with 2,3-butanedione monoxime . Hearts were further stabilized by applying light pressure on the posterior surface of the heart.The dye was excited with a halogen light source  with an excitation filter of 510 nm (Brightline Fluorescence Filter). An emission filter of 610 nm  was used before the emitted light was recorded using a MiCam Ultima CMOS L-camera (SciMedia), sampling at a rate of 1 kHz. Images were captured on a 100 × 100 array with an inter-pixel resolution of 0.1 mm.L and CVT, respectively), with anisotropic ratio (AR) calculated as CVL/CVT. Conduction in each direction was calculated from a group of vectors that were within 5 pixels and with an angle of ±8° from direction of longitudinal (fastest) or transverse (slowest) propagation. The first vectors immediately adjacent to the site of pacing were excluded to reduce pacing artifacts in the CV analysis. CV was only quantified up to 32 pixels from the site of pacing in the longitudinal direction and 16 pixels in the transverse direction to reduce the contribution of conduction breakthrough. Longitudinal CV was only quantified if there were at least 10 vectors meeting the criteria above and transverse CV was quantified if at least 50 vectors met the criteria above.CV was calculated as previously described  and transverse (RTT) directions. Action potential duration (APD) was calculated as the difference between the activation time and 85% repolarization from peak action potential amplitude.Rise time (RT) was calculated by the time difference from 20 to 80% of the peak fluorescent action potential amplitude during depolarization. RT was calculated both in the longitudinal (RTn = 3), perfused with respective solutions for 30 min, were snap frozen. Tissue was homogenized in a RIPA lysis buffer  supplemented with HALT Protease and Phosphatase Inhibitor Cocktail (ThermoScientific). Following sonication and clarification by centrifugation, the BioRad DC protein assay was employed to determine and normalize protein concentrations prior to analysis. SDS-PAGE electrophoresis was performed as previously described using 4–20% NuPage Bis-Tris or 3–8% Tris-Acetate gels (Life Technologies) which were then transferred using the Trans-Blot Turbo system (BioRad) to low-fluorescence PVDF membrane and blocked with 5% BSA in TNT buffer  for 1 h at room temperature. Membranes were then incubated with rabbit anti-phospho-Cx43Ser368  overnight at 4°C. Following several washes in TNT, secondary detection was performed using goat anti-rabbit HRP antibody  for 1 h at room temperature. Bound antibody was detected post washing using Clarity Western ECL Substrate (BioRad) and imaged using the BioRad Chemidoc MP system. Membranes were then stripped with Re-Blot Plus Strong (Millipore) according to manufacturer's instructions. To detect total Cx43, stripped membranes were blocked for 1 h at room temperature in 5% milk in TNT buffer, and subsequently incubated overnight at 4°C with primary antibodies against Cx43 , and alpha-tubulin  diluted in 5% milk TNT. Membranes were then washed and incubated with the fluorescently distinct secondary antibodies goat anti-mouse AlexaFluor555 and goat anti-rabbit AlexaFlour647  for 1 h at room temperature. Following several washes in TNT, membranes were fixed in methanol, dried, and imaged using the Biorad Chemidoc MP System. Protein expression was quantified by densitometry using ImageLab software (BioRad), Cx43 expression levels were normalized to alpha-tubulin, and Cx43 phosphorylate Serine 368 (Cx43-p368S) to total Cx43.Hearts . On the other hand, changing BCL with Solution B caused a significant decrease in CVT. For all experiments, solution composition did not significantly change CVL or CVT under control conditions  in contrast to control.We previously demonstrated that GJ inhibition with 15μM CBX does not significantly alter CV . Since APD shortened approximately equally with Solutions A and B, and the magnitude of APD shortening was similar, these data suggest that APD restitution kinetics may not explain the changes in CV reported above. In fact, one might expect that Solution A, which produced the longer APD, would decrease CV more at a 160 ms BCL, and this was not the case.APD during perfusion of Solution B was shorter than in hearts perfused with Solution A for all CBX concentrations and BCLs Figure , †. As eL) or transverse (RTT) directions of propagation.Optical rise time (RT) has been previously used as an inverse correlate of cellular excitability , with increased sensitivity at 30 μM CBX. These results are consistent with our data obtained in mice with narrow perinexal widths and presumably increased EpC  is reduced, the slow component of the delayed rectifier potassium current (IKs) is increased, and the rapid component of the delayed rectifier potassium current (IKr) is decreased  and increasing sodium driving force ([Na+]o). Further, even greater sodium channel inactivation by rapid pacing does not significantly decrease CV even with GJ uncoupling sufficient to reduce CV.It is well accepted that CV decreases when pacing rate increases, due to sodium channel inactivation  is a correlate of peak INa. This is supported by studies which demonstrated that decreasing INa, max can decrease dV/dtmax and CV (Shaw and Rudy, max (Spach et al., Previous studies suggest that IOur group previously demonstrated that changing bulk interstitial volume can modulate CV (Veeraraghavan et al., +]o and [K+]o can modulate cardiac conduction with pharmacologic inhibition is similar to findings in mice heterozygous null for Cx43 (George et al., This study relies entirely upon using pacing protocols as well as pharmacological GJ inhibitors. However, the findings that altering o, [Na+]o, and heart rate have important effects on GJ mediated conduction slowing. The magnitude of ionic changes, while small, were in accordance with the original hypothesis that the changes in [K+]o and [Na+]o would not substantially alter CV as previously demonstrated in mice with normal Cx43 expression levels, but GJ uncoupling can produce solution dependent CV changes. The implications of this study are that relatively small physiologic changes in extracellular ionic composition do not significantly perturb cardiac electrophysiology under “normal” conditions, but these same changes may have significant effects during cardiac stress as a result of GJ remodeling or increased heart rate.We present evidence that altering [KAll authors are fully aware of the content of the manuscript and have contributed to its writing. ME and SP were responsible for the entire direction of the project. SG was responsible for the design of the solution fluids and the TEM imaging. MZ was responsible for the immunoblotting, analysis and reporting of Cx43. JS was responsible for supervising and the immunoblotting, analysis and reporting of Cx43.This work was supported by an R01-HL102298 awarded to SP, and a VTCRI Medical Research Scholar Award to ME.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To assess the effect of catheter ablation on atrial fibrillation (AF) symptoms and quality of life (QoL).Patients with AF scheduled for ablation were recruited. Pulmonary vein isolation (PVI) was performed and complex fractionated atrial electrogram (CFAE)±linear ablation undertaken in patients in AF despite PVI. QoL and AF symptoms were assessed using SF-36 V2 and Atrial Fibrillation Effect on Quality-of-Life (AFEQT) questionnaires before and 3 months after ablation. Change in QoL scores after ablation was correlated with clinical parameters and the extent of ablation. Magnitude of QoL change was compared between AFEQT and SF-36 physical component summary (PCS) and mental component summary (MCS) scores and correlated with arrhythmia outcome.80 patients were studied. Summative and individual health scores for both AFEQT  and SF-36  improved significantly in patients who maintained sinus rhythm after ablation, but not in those with recurrent AF. Improvement in AFEQT (25.4±19) was significantly greater than change in PCS  and MCS  scores and correlated more closely with arrhythmia outcome .Patients who maintained sinus rhythm after ablation had a significant improvement in AF symptoms and QoL; however, no improvement was observed in patients with recurrent AF. QoL change after ablation did not correlate with baseline clinical parameters or ablation strategy. AF specific QoL scales are more responsive to change and correlate better with ablation outcome. Previous research has shown that catheter ablation improves symptoms and quality of life (QoL) in patients with AF irrespective of arrhythmia outcome and that this improvement is sustained for several years.Summative and individual health domain scores for both Atrial Fibrillation Effect on Quality-of-Life (AFEQT) (AF specific) and SF-36 V2 (generic) questionnaires improved significantly in patients who maintained sinus rhythm after ablation; however, there was no improvement in patients with recurrent atrial fibrillation (AF).Magnitude of QoL change after ablation did not correlate with clinical parameters (including AF type) or ablation strategy.Change in AFEQT was significantly greater than change in SF-36 scores and correlated more closely with arrhythmia outcome following ablation.The similar degree of QoL improvement after ablation in paroxysmal and persistent AF supports the continued use of catheter ablation in both patient groups.AF specific QoL scales  are more sensitive to change after ablation and correlate better with arrhythmia outcome. Therefore, studies assessing QoL in patients with AF should use AF specific rather than generic QoL scales.et alaim of this study was to assess the effect of catheter ablation on AF symptoms and QoL in patients with paroxysmal or persistent AF using the AFEQT and SF-36 V2 questionnaires.Atrial fibrillation is the most common arrhythmia in clinical practice affecting up to 2% of the general population and is associated with significant morbidity and mortality.Study participants were recruited from patients scheduled to undergo their first catheter ablation procedure for symptomatic AF. In line with usual practice, class I and III antiarrhythmic drugs were discontinued five half-lives prior to ablation and only restarted in patients with recurrent AF after ablation.This study complies with the Declaration of Helsinki and was granted a favourable ethical opinion by the National Research Ethics North West Committee (REC reference: 11/NW/0476). Written informed consent was obtained from all patients recruited to the study.QoL and AF symptoms were assessed at baseline and 3 months after ablation using the SF-36 V2 and AFEQT questionnaires. Questionnaires were completed without input from study personnel. SF-36 V2 consists of 36 items that assess eight health domains: physical functioning, role limitations because of physical problems, bodily pain, general health perception, vitality, social functioning, role limitations because of emotional problems and mental health. In addition to these eight subscales, physical component summary (PCS) and mental component summary (MCS) scores are also generated, which are normalised to an overall population mean of 50±10.All patients underwent pulmonary vein isolation (PVI) using one of three ablation technologies: (1) PVAC multielectrode circumferential ablation catheter (Medtronic Ablation Frontiers), (2) Arctic Front Advance 28 mm cryoballoon (Medtronic CryoCath), and (3) Wide-area circumferential ablation guided by the CARTO 3 cardiac mapping system (Biosense Webster). Electrical isolation of the pulmonary veins was confirmed using standard pacing manoeuvres.Additional complex fractionated atrial electrogram (CFAE) and/or linear ablation was performed in patients who remained in AF after PVI, according to the degree of signal complexity and fractionation in the left atrium.A detailed analysis of the left atrium and inter-atrial septum was performed to identify CFAEs , which were then ablated. This process was completed when no residual CFAE sites could be identified or when sinus rhythm was restored by ablation.A combination of ‘roof line’ (connecting right and left upper pulmonary vein ostia), ‘mitral line’ , and ‘inferior line’ (connecting right lower pulmonary vein to coronary sinus) were constructed depending on the degree of signal complexity in each region. Linear ablation was performed until electrograms were no longer recordable or double potentials were evident along the length of each line. Differential pacing manoeuvres were not performed routinely to further confirm conduction block. All patients in AF at the end of the ablation procedure underwent electrical cardioversion.Clinical outcome was determined by symptom review, 12-lead ECG and 72 h Holter monitoring 3 months after ablation and was divided into two categories: (1) ‘Sinus’ rhythm (no arrhythmia symptoms and no documented AF episodes >30 s); (2) AF recurrence (arrhythmia symptoms and documented AF episodes >30 s).2 test for categorical variables.Continuous variables are expressed as mean±SD. Patient characteristics were compared between paroxysmal and persistent AF groups using Student's independent t test for continuous variables and Pearson's χBaseline QoL scores  were compared and correlated with clinical parameters using Pearson's correlation. Individual and summative health domains of the AFEQT and SF-36 V2 questionnaires were compared at baseline and 3 months after ablation using Student's paired t tests.Change in QoL scores after ablation were correlated with clinical parameters using Pearson's correlation (continuous variables) and independent t test  and were compared between different ablation strategies using one-way analysis of variance (ANOVA). Parameters with p<0.05 were entered into a multivariate linear regression model using stepwise selection to assess their independent and combined ability to predict change in QoL after ablation. After multivariate analysis, parameters with p<0.10 were retained. Standardised coefficients (β) and corresponding p values are reported. The magnitude of QoL change after ablation was compared between AFEQT, PCS and MCS scores using one-way ANOVA and correlation coefficients for the three QoL scores and ablation outcome were compared using Fisher r-to-z transformation. All tests were two-tailed and p<0.05 was considered statistically significant.The clinical characteristics of the 80 consecutive patients recruited to the study are shown in There was a significant increase in the summative and individual health domain scores for both AFEQT  and SF-36 V2  questionnaires in patients who maintained sinus rhythm 3 months after ablation A and 2A.However, there was no significant change in summative or individual scores from either questionnaire in patients with recurrent AF B and 2B.In univariate analysis, there was no significant correlation between clinical parameters and change in QoL scores after ablation . Of noteAFEQT scores increased significantly after PVI, PVI+Linear and PVI+Linear+CFAE ablation indicating an improvement in QoL . AFEQT sBaseline AFEQT scores correlated closely with PCS scores  and moderately with MCS scores . However, there was no correlation between PCS and MCS scores . Hypertension was associated with a lower baseline AFEQT score with a trend towards a lower MCS score . Older aet alet al.The main finding of this prospective study is the significant improvement in AF symptoms and QoL in patients who maintained sinus rhythm 3 months after ablation and the contrasting lack of improvement in patients with recurrent AF. This finding was consistent across all individual and summative components of the AFEQT and SF-36 V2 questionnaires. The mean increase in AFEQT score of 30 points in patients who maintained sinus rhythm is consistent with a marked improvement in QoL.Previous studies have shown that AF specific QoL scales are more responsive to QoL change in this population and correlate better with ablation outcome.9–11et alHowever, Bulkova et alMantovan et alThis study showed no correlation between gender and QoL, in contrast to Reynolds et al.Hypertension was associated with a higher symptom burden and reduced QoL secondary to AF in this study, which has not been previously reported. Persistent AF was associated with lower physical health scores than paroxysmal AF, as shown by Bulkova First, the choice of ablation strategy after PVI was not randomised and was dependent on the operator and the degree of signal complexity in the left atrium. Second, this was a prospective cohort study without a randomised control group comparing AF ablation to best non-ablative management (rate vs rhythm control). However, the superiority of ablation over antiarrhythmic drugs in improving QoL has been demonstrated consistently. Third, AF recurrence was determined by patients’ symptoms, 12-lead ECG and 72 h Holter monitoring 3 months after ablation and not by continuous ECG monitoring. Although, we cannot completely exclude asymptomatic AF episodes in the ‘sinus rhythm’ group, we consider this unlikely given previous AF symptoms. Fourth, change in QoL was assessed 3 months after ablation in line with the first outpatient clinic review and therefore, the longer-term effect of ablation on QoL is unknown. Finally, patients enrolled in this study had been referred for catheter ablation and therefore, represent a select cohort with a high AF symptom burden. Extrapolation of the benefits demonstrated to a wider population requires caution.Patients who maintained sinus rhythm after ablation had a significant improvement in AF symptoms and QoL. No improvement was observed in patients with recurrent AF. QoL did not correlate with either baseline clinical parameters or extent of ablation.AF specific QoL scales are more responsive to change than generic measures and correlate better with arrhythmia outcome.


C) is incompletely understood. The expression of PrPC in hematopoietic stem cells and immune cells suggests a role in the development of these cells, and in PrPC knockout animals altered immune cell proliferation and phagocytic function have been observed. Recently, a spontaneous nonsense mutation at codon 32 in the PRNP gene in goats of the Norwegian Dairy breed was discovered, rendering homozygous animals devoid of PrPC. Here we report hematological and immunological analyses of homozygous goat kids lacking PrPC (Ter/TerPRNP) compared to heterozygous (PRNP+/Ter) and normal (PRNP+/+) kids. Levels of cell surface PrPC and PRNP mRNA in peripheral blood mononuclear cells (PBMCs) correlated well and were very low in PRNPTer/Ter, intermediate in PRNP+/Ter and high in PRNP+/+ kids. The PRNPTer/Ter animals had a shift in blood cell composition with an elevated number of red blood cells (RBCs) and a tendency toward a smaller mean RBC volume (P = 0.08) and an increased number of neutrophils (P = 0.068), all values within the reference ranges. Morphological investigations of blood smears and bone marrow imprints did not reveal irregularities. Studies of relative composition of PBMCs, phagocytic ability of monocytes and T-cell proliferation revealed no significant differences between the genotypes. Our data suggest that PrPC has a role in bone marrow physiology and warrant further studies of PrPC in erythroid and immune cell progenitors as well as differentiated effector cells also under stressful conditions. Altogether, this genetically unmanipulated PrPC-free animal model represents a unique opportunity to unveil the enigmatic physiology and function of PrPC.The physiological role of the cellular prion protein (PrP C) was first described as the substrate for PrP scrapie (PrPSc)  mice do not replicate prions, nor do they develop prion disease  in the C expression (PRNPTer/Ter) compared to heterozygotes (PRNP+/Ter) and normal (PRNP+/+) goats.Here, we report results of hematological and immunological analyses of goat kids without PrPPRNP genotypes  which is based upon the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes.The animals included in this study were of the Norwegian Dairy Goat Breed obtained from a research herd of around 100 animals at the Norwegian University of Life Sciences. Based on daily monitoring and enhanced health surveillance through a national goat health monitoring service, the health status of this flock is considered to be very good. Scrapie outbreaks have never occurred and there have been no cases of caprine arthritis encephalitis, Johne's disease or caseous lymphadenitis during the last 5 years  , PRNP+/Ter (n = 16), and PRNP+/+ (n = 24) were included.Blood was drawn from the jugular vein into heparinized tubes and tubes without anticoagulant from age matched goat kids at 3–4 weeks of age. For hematological and clinical chemistry analyses, goat kids carrying the genotypes Hematological analyses were performed with an Advia® 2120 Hematology System using Advia 2120 MultiSpecies System Software and clinical chemistry analyses were performed with Advia 1800 Chemistry System .PRNPTer/Ter (n = 8) and PRNP+/+ (n = 8) goat kids by gradient centrifugation  at 1760 × g. Red blood cells (RBC) were lysed by brief exposure to sterile water prior to counting and trypan blue viability assessment using a Countess Automated Cell Counter (Life Technologies).Peripheral blood mononuclear cells (PBMCs) were isolated from PRNPTer/Ter and one PRNP+/+, were necropsied at 3 months of age. Macroscopic examinations were performed routinely. Fresh bone marrow imprints and smears were made and evaluated after staining with May Grünewald Giemsa. Histological slides were prepared from formalin fixed and paraffin-embedded tissues and stained with hematoxylin and eosin (HE) prior to examination by light microscopy.For morphological studies, two age-matched male kids, one Total RNA was isolated from PBMCs using the Qiagen RNeasy mini plus kit (Qiagen) following the manufacturer's instructions. RNA concentration and purity was analyzed using NanoDrop-1000 Spectrophotometer (Thermo Fisher Scientific), and quality was assessed using RNA Nano Chips on an Agilent 2100 Bioanalyzer (both from Agilent Technologies). RNA was stored at −80°C. cDNA was synthesized using the SuperScript III Reverse Transcriptase, RNase Out, dNTP mix and Random Primers  at the following conditions: 5 min at 65°C, >1 min on ice, 5 min at 25°C, 1 h at 50°C, and 15 min at 70°C.PRNP as target gene  and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as reference gene . cDNA corresponding to 1 ng RNA was used per reaction. The samples had a total volume of 20 μl, and were run on a LightCycler 480 System (Roche). Conditions: 5 min at 95°C; 40 cycles of 10 s at 95°C, 10 s at 60°C, and 10 s at 72°C; and melting curve with 5 s at 95°C, 1 min at 65 and 97°C. Relative expression levels were calculated using an externally run standard curve for the PBMCs generated from one PRNP+/+ animal, run in duplicates, with one randomly selected PRNP+/+ as positive control (in-run).Quantitative-PCR was conducted with LightCycler 480 Sybr Green I Master mix (Roche), with Immunophenotyping in flow cytometry was performed as previously described  (200 μg/ml), recombinant ovine interleukin (IL)-2   using 10 μl beads per 107 cells. The purity was consistently measured between 90 and 95% by flow cytometry using anti-CD14 mAbs . CD14+ monocytes were seeded in a 96 well non-adherent plate (Corning Costar Ultra-Low Attachment multiwell plates) in complete medium  at 2 × 105 cells per well and incubated for 24 h at 37°C and 5% CO2 in the presence of 5000 U/ml recombinant bovine granulocyte macrophage colony stimulating factor (rbGM-CSF) that had been expressed in 293T cells as described , pHrodo red E. coli or pHrodo red Zymosan (both from Life Technologies) in separate wells. Phagocytosis was terminated after 30 min of incubation  by placing the plate on ice. The particle uptake was measured in a Gallios flow cytometer. The monocyte gate was adjusted to exclude free particles using cell-free particle-only samples as control.CD14To confirm that the particles had been phagocytized, monocytes were centrifuged onto slides at 1000 rpm for 5 min. The cells were fixed in acetone, blocked with goat serum and immunolabeled with mAb against human CD68 (Dako). The cytospots were incubated with secondary antibodies, Alexa 488 goat-anti-mouse IgG1 or Alexa 594 goat-anti-mouse IgG1, mounted in Prolong Gold Antifade reagent with DAPI  and analyzed by standard fluorescence and confocal microscopy.P < 0.05 was considered statistically significant and shown as * and ** in figures. GraphPad prism version 6.04 software was used for statistical analyses.All data was analyzed for statistical significance with the Mann-Whitney test and data reported with medians. A level of C on PBMCs from PRNPTer/Ter goats was confirmed by three different anti-PrPC mAbs , B cells (B-B2+), and T cells (CD3+) (data not shown). Anti-PrPC mAbs staining of whole blood revealed that PrPC was not expressed on the surface of granulocytes in goats of either genotype, while its presence was confirmed in the lymphocyte gate in +/+PRNP goats  was not significantly different between the groups, although the distribution of values suggested a clear tendency of lower MCV in the PRNPTer/Ter animals compared to the PRNP+/+ and PRNP+/Ter groups (Table The number of RBCs was increased in ds Table  as compaps Table .The groups did not differ in hematocrit (HCT) values Table , suggestPRNPTer/Ter (n = 8) and PRNP+/+ (n = 24) animals a marked poikilocytosis was observed, as expected in young kids. The neutrophil granulocytes were mature and showed no signs of left shift in any of the genotypes. No reticulocytes were present in any of the smears (data not shown).In blood smears from PRNPTer/Ter animals as well as an increase in the monocyte number in PRNPTer/Ter and PRNP+/Ter animals compared to the PRNP+/+ group , B cells (B-B2+), T cells (CD3+) as well as the gamma-delta (WC1+) and CD8+ subsets of T cells were quantified by flow cytometry  and PRNPTer/Ter (n = 1) goat kids were analyzed. However, there were no differences in myeloid/erythroid ratio between the two genotypes, and the precursor cells had a normal appearance. In addition, there was no observable difference in degree of apoptosis (data not shown). Altogether, no evident morphological changes were found within the bone marrow of the PRNPTer/Ter goat kid.To investigate whether the observed difference in hematological profile could be reflected by morphological changes in bone marrow, smears, and imprints of bone marrow from 3 months old C might have a functional impact on white blood cells, we performed phagocytosis and proliferation studies to assess two major functional properties of leukocytes. Positively selected CD14+ monocytes from peripheral blood were cultured for 24 h to stabilize cells following isolation, and supplemented with GM-CSF to activate the cells and prevent apoptosis , or zymosan-covered yeast cells (Saccharomyces cerevisiae) for 30 min. Fluorescence and confocal microscopy of cytospots confirmed the cellular uptake of particles .By investigating complete blood counts of 3–4 weeks old goat kids we demonstrated that C are only clearly evident under various stressful conditions, such as tissue damage, infection, or anemia , as these cells showed reduced regenerative capacity when undergoing serial transplantations (Zhang et al., PRNPTer/Ter animal compared to a normal control. More comprehensive and cell-specific investigations are however needed to clarify whether there are changes in bone marrow cellular composition in growing PRNPTer/Ter kids, and further studies are needed to reach a final conclusion regarding possible differences between genotypes with respect to stem cell phenotypes in animals, both under normal and stressful conditions.Studies in PrPC expression in peripheral blood mononuclear leukocytes but not granulocytes of PRNP+/+ animals, similar to previous reports that have led to a particular interest for PrPC functions in immune cells, several of which have suggested PrPC involvement in immune cell functions (Dodelet and Cashman, PRNPTer/Ter kids, while flow cytometric analyses did not confirm any difference in CD14+ monocyte numbers, and relative numbers of other major circulating mononuclear cell subsets also appeared unaffected by PrPC loss. Similar results, including increased neutrophil counts, have been found in KO cattle, although a tendency of more numerous γδ T cells in a group of four PrPC cattle compared to four WT cattle, was found (Richt et al., C KO models but in a double KO, namely PrPC and Doppel KO mice, no alterations in the immune cell populations were noted (Genoud et al., C.We detected high PrPC is expressed in regulatory and memory T cells (Li et al., C increase during T-cell proliferation (Cashman et al., C display reduced proliferation rates (Mabbott et al., in vitro with mitogen or cytokines to assess alternative proliferation pathways but detected no difference in the proliferation rates between the genotypes. This correlates with the findings in transgenic cattle (Richt et al., PRNPTer/Ter group consistent through all stimulations could suggest that proliferative differences may be present in more restricted lymphocyte subsets. Furthermore, we quantified the uptake of bacteria, yeast and latex particles by activated blood monocytes in order to approach different activation pathways of phagocytosis (Flannagan et al., C deficiency had any influence on the proportion or efficiency of monocytes to phagocytize any of these particle types. Earlier studies on phagocytosis have been performed with cells from PrPC KO mice and have given conflicting results. In an in vitro study of phagocytosis of E. coli by bone marrow-derived macrophages, the activity was enhanced in cells from PrPC KO mice (Wang et al., C KO mice were shown to phagocytize latex beads at a lower rate than macrophages from WT mice (Uraki et al., C is a negative modulator of phagocytosis, as peritoneal macrophages from PrPC KO mice phagocytized apoptotic cells at a higher rate than cells from WT mice in vitro and in vivo, a result confirmed by two different mouse strains. The results from the latter study have later been linked to polymorphisms in PrPC flanking genes involved in phagocytosis, rather than the loss of PrPC in itself (Nuvolone et al., C functions from PrPC KO models. Our results from naturally PrPC-devoid animals are not in support of a principal mechanistic role of the prion protein neither in lymphocyte proliferation nor in phagocytosis.It has been shown that PrPC-deficient goat kids displayed a generally healthy constitution, moderate shifts in red blood cell and possibly granulocytic cell lineages but not in other major circulating hematopoietic cells. No impairment of major immune cell functions was detected. These results point toward a role for PrPC related to maturation and release of selected cell lineages from the bone marrow, and underscore previous observations that PrPC loss appears to have limited physiological or pathological consequences for the animal, at least under conventional living conditions and in the absence of significant stressors. The present natural goat model offers a unique opportunity to study the function of PrPCin and ex vivo without the confounding factors of genetic engineering, which may hopefully help to shed light on the elusive nature of this protein and its related diseases.In summary, this study showed that naturally occurring PrPPB, AE, MT, MB, MR designed the study. MR, PB, MB performed the experiments and MR, PB, MT, AE, MB analyzed the data. AL assisted with the monocyte isolation protocol and experiments and HB contributed to the analysis of hematology data, blood smears and bone marrow imprints, and smears. MR, MB, HB, AL, IO, MT, AE, PB wrote or critically reviewed the manuscript.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.


Entamoeba histolytica is the protozoan agent responsible for human amoebiasis. Trophozoites are highly phagocytic cells and the lysobisphosphatidic acid (LBPA) is involved in endocytosis. LBPA interacts with EhADH protein (an ALIX family member) also participating in phagocytosis, as it is referred in the research article Identification of the phospholipid lysobisphosphatidic acid in the protozoan Entamoeba histolytica: an active molecule in endocytosis   Specifications TableValue of the data•Docking analyses to predict the interacting sites between EhADH protein and phospholipid ligand LBPA.•In silico mutation of EhADH interacting site with LBPA as an important tool to further support the docking predictions and perform the experiments with a higher precision.•Molecular dynamic simulation of the mutated conformers to confirm in silico the specificity of interaction sites between EhADH and LBPA.1Data presented here show: i) the in silico predicted interaction site between LBPA and EhADH , ii) the22.11.In silico mutations were performed on residues indicated inTable 1.2.Molecular dynamics simulation of mutated EhADH Bro1 domain.The 3D molecular model of EhADH was built with I-TASSER server http://clusterhibrido.cinvestav.mx/) using the NAMD 2.8 software Molecular dynamics (MD) simulation of mutated Bro1 domain was performed on Cluster hybrid-Xiuhcoatl (


Arterial Spin Labeling (ASL) perfusion image series have recently been utilized for functional connectivity (FC) analysis in healthy volunteers and children with autism spectrum disorders (ASD). Noise reduction by using nuisance variables has been shown to be necessary to minimize potential confounding effects of head motion and physiological signals on BOLD based FC analysis. The purpose of the present study is to systematically evaluate the effectiveness of different noise reduction strategies (NRS) using nuisance variables to improve perfusion based FC analysis in two cohorts of healthy adults using state of the art 3D background-suppressed (BS) GRASE pseudo-continuous ASL (pCASL) and dual-echo 2D-EPI pCASL sequences. Five different NRS were performed in healthy volunteers to compare their performance. We then compared seed-based FC analysis using 3D BS GRASE pCASL in a cohort of 12 children with ASD  and 13 typically developing (TD) children  in conjunction with NRS. Regression of different combinations of nuisance variables affected FC analysis from a seed in the posterior cingulate cortex (PCC) to other areas of the default mode network (DMN) in both BOLD and pCASL data sets. Consistent with existing literature on BOLD-FC, we observed improved spatial specificity after physiological noise reduction and improved long-range connectivity using head movement related regressors. Furthermore, 3D BS GRASE pCASL shows much higher temporal SNR compared to dual-echo 2D-EPI pCASL and similar effects of noise reduction as those observed for BOLD. Seed-based FC analysis using 3D BS GRASE pCASL in children with ASD and TD children showed that noise reduction including physiological and motion related signals as nuisance variables is crucial for identifying altered long-range connectivity from PCC to frontal brain areas associated with ASD. This is the first study that systematically evaluated the effects of different NRS on ASL based FC analysis. 3D BS GRASE pCASL is the preferred ASL sequence for FC analysis due to its superior temporal SNR. Removing physiological noise and motion parameters is critical for detecting altered FC in neurodevelopmental disorders such as ASD. Functional connectivity (FC) analysis to compute functionally connected networks (FCNs) has become a major imaging approach to investigate the brain's organization and function. Moreover, comparing different cohorts such as elderly subjects to young adults, or healthy control groups to psychiatric populations, have identified patterns of altered connectivity within specific FCNs. In the past few years, however, it has become evident that there are several potential confounding factors that may lead to spurious findings when not properly addressed. Physiological noise such as fluctuations in respiratory and cardiac cycles or head movements can influence BOLD signal intensities in fMRI. This is particularly relevant since different study cohorts could exhibit different patterns or amounts of such confounding factors . Accordingly, using nuisance variables to account for noise related signal fluctuations in BOLD-fMRI based FC analysis has been shown to be imperative to minimize or avoid potential confounding effects of motion or other physiological factors  on network connectivity measures  datasets have been recently used to compute FCNs  and a standard 2D EPI BOLD sequence . For comparison, a separate cohort of 10 healthy volunteers  underwent resting state fMRI scans using a dual-echo 2D EPI pCASL sequence  to simultaneously acquire ASL and BOLD data. All datasets were first realigned to account for spatial motion displacements (for ASL separately for label and control images). Five different regression models using different sets of nuisance variables [here termed Noise Reduction Strategies (NRS)] were then performed:NRS1: no nuisance variables for noise reduction.- NRS2: 6 motion parameters  and their 1st derivatives.- NRS3: same as NRS2 plus additional regressor for Framewise Displacement (FD). FD was computed following the procedure described by Power et al. , nor between groups for BOLD . There was a small difference showing slightly higher motion in 3D pCASL than 2D pCASL .- r et al. . RotatioNRS4: White matter and CSF fluctuations .- NRS5: NRS3 + NRS4.- All adult neurotypical participants in this study gave written informed consent according to a research protocol approved by the UCLA Institutional Review Board. Inclusion criteria of healthy volunteers included no history of psychiatric or neurological disorders, and no contraindications to MRI scan. Scans were performed on a 3T Siemens TIM Trio scanner, using body coil as the transmitter and 12-ch head coil as the receiver. We acquired ASL and BOLD data in 10 healthy young participants  with a 3D background-suppressed (BS: 85% suppression) GRASE pCASL sequence   using the posterior cingulate cortex (PCC) as a seed   and IQ  and Chi-square test with Yates correction for small samples for gender .To investigate the effects of NRS on seed-based FC analysis in a clinical cohort, we compared FC differences between a group of 12 children with ASD  and an age and IQ matched group of 13 typically developing (TD) children . Subjects and parents provided written consent according to the guidelines specified by the UCLA Institutional Review Board. Clinical diagnosis of ASD was confirmed with the Autism Diagnostic Observation Schedule . This analysis will reveal the effects of different NRSs on the outcome of ASL-FC differences between ASD and TD. To minimize the effects of differences in head motion between the groups, the ASD and TD groups were also matched for the amount of motion: mean frame-wise displacement (FD) for ASD was 0.453 ± 0.238 and TD 0.392 ± 0.241 .We used the 3D BS GRASE pCASL sequence to acquire CBF data in these two groups given its favorable temporal characteristics shown in the above analyses. Preprocessing was identical as described above and NRS4 and NRS5 were compared to NRS1 in these cohorts. FC from the PCC-seed was computed for each subject and NRS. Within group analyses included comparisons between NRSs using voxel-wise paired-sample p < 1e−10. Dice Similarity Coefficients (DSCs) to the template BOLD-DMN  = 25.53, p < 1e-10, tNRS0vsNRS4 = 5.34, p = 0.0005; FBOLDdual−echo = 12.49, p < 1e−10, tNRS0vsNRS4 = 2.69, p = 0.025], while motion parameter regression showed a tendency to slightly increase FC . The same trend was observed for 3D GRASE pCASL ; however, FC values were overall lower. For 2D pCASL there was little to no correlation between the two ROIs and no significant effect in the ANOVA . Mean values of correlation coefficient across subjects for all NRSs and datasets are displayed in Figure Analysis of the connectivity between PCC and ACC/mPFC ROIs in both BOLD datasets showed a general decrease of FC after WM/CSF regression . An opposite effect was observed for 2D dual-echo pCASL where regression of WM/CSF signals (NRS4&5) slightly reduced global mean CBF , as shown in Table Along with the increase in temporal SNR for 3D GRASE pCASL, global mean CBF was also slightly higher for NRS4&5 than those observed for NRS1–3 [Comparing correlation maps seeded from the PCC revealed differences in long-range connections to the frontal cortex after noise reduction in both groups (TD and ASD children). Specifically, differences between NRS4 and NRS5 revealed increased long-range and decreased local correlations within the DMN, which are in accordance with the general observations of motion-regression effects  representing noise from physiological noise sources and head movements in two separate BOLD and pCASL implementations, our study showed that accounting for physiological noise and motion-induced effects could indeed alter the connectivity strength and hence spatial maps of the DMN. For BOLD rs-fMRI, these effects have been described and were replicated in our study. For ASL, so far little has been known regarding how noise reduction affects ASL signal and ASL based FC analysis.Our findings generally align well with what has been described with regard to noise reduction effects in BOLD based FC analysis. First, regression of physiological noise related nuisance variables from WM and CSF signal fluctuations . It remains to be determined whether using larger samples or lower statistical thresholds will make 2D ASL based FC analysis feasible. Furthermore, as discussed above, the BOLD images acquired at the second echo of the dual echo ASL sequence used in our study showed highly similar network maps, correlation strength and behavior to NRS as the conventional BOLD sequence. On the other hand, the FC strength decrease between PCC and the frontal ROI in 3D BS GRASE pCASL mirrors the effects observed for BOLD. Not surprisingly, the CBF-FC was generally lower than that of BOLD in both ASL implementations, in agreement with other studies comparing ASL and BOLD FC , it may also benefit the separation between functional networks in autism. These alterations in FC due to noise reduction were also observed in direct group comparisons, leading to marked changes in observed group differences, both in terms of hyper- and hypoconnectivity. While all NRS  yielded altered connectivity in somatomotor network in ASD, NRS4&5 revealed reduced long-range FC from PCC to the dorsal portion of the prefrontal cortex and parahippocampal gyri, with suppression of hyperconnectivity with lateral-temporal areas. Overall, noise reduction altered the pattern of temporal lobe hyperconnectivity highlighting instead long-range hypoconnections to frontal areas and the medial temporal lobes . Similar motion related effects on FC were found in a study using independent component analysis to identify BOLD-DMN subnetworks in ASD . First, changes in FC strength and spatial maps of the DMN with regard to physiological nuisance variables  and head movement related nuisance variables were replicated in two separate BOLD datasets, one with a conventional EPI implementation and another based on data acquired in a 2D dual-echo pCASL sequence. Second, analysis of NRS effects on FC analysis of CBF data demonstrated that 3D BS GRASE pCASL shows similar behavior as that observed for BOLD. The favorable noise properties of 3D BS GRASE pCASL as compared to 2D dual-echo pCASL and the improved tSNR after noise reduction render this pCASL implementation more suitable for CBF based FC analyses showing similar networks (Dai et al., Finally, applying the full spectrum of NRS in a cohort of children with ASD and typically developing controls we observed that 3D BS GRASE pCASL based FC analysis yielded results that are in accordance with effects of head motion and group differences between ASD and TD children observed in BOLD-DMNs. These findings underline the complex changes in functional organization in ASD and the impact that different preprocessing steps could have on the research findings (Nair et al., KJ designed the experiment, performed the analyses and wrote the manuscript. ER analyzed the data and RS designed the experiment and contributed analysis tools. MD wrote the manuscript. DW conceptualized the study and wrote the manuscript.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.


Our main objective of this study was to determine how Human Immunodeficiency Virus (HIV) avoids induction of the antiviral Type I Interferon (IFN) system. To limit viral infection, the innate immune system produces important antiviral cytokines such as the IFN. IFN set up a critical roadblock to virus infection by limiting further replication of a virus. Usually, IFN production is induced by the recognition of viral nucleic acids by innate immune receptors and subsequent downstream signaling. However, the importance of IFN in the defense against viruses has lead most pathogenic viruses to evolve strategies to inhibit host IFN induction or responses allowing for increased pathogenicity and persistence of the virus. While the adaptive immune responses to HIV infection have been extensively studied, less is known about the balance between induction and inhibition of innate immune defenses, including the antiviral IFN response, by HIV infection. Here we show that HIV infection of T cells does not induce significant IFN production even IFN I Interferon production. To explain this paradox, we screened HIV proteins and found that two HIV encoded proteins, Vpu and Nef, strongly antagonize IFN induction, with expression of these proteins leading to loss of expression of the innate immune viral RNA sensing adaptor protein, IPS-1 (IFN-β promoter stimulator-1). We hypothesize that with lower levels of IPS-1 present, infected cells are defective in mounting antiviral responses allowing HIV to replicate without the normal antiviral actions of the host IFN response. Using cell lines as well as primary human derived cells, we show that HIV targeting of IPS-1 is key to limiting IFN induction. These findings describe how HIV infection modulates IFN induction providing insight into the mechanisms by which HIV establishes infection and persistence in a host. Global infection by Human Immunodeficiency Virus (HIV), the cause of Acquired Immune Deficiency Syndrome or AIDS, has reached a pandemic scale. This global burden of HIV infection and the lack of an efficacious HIV vaccine underscore the need for a deeper understanding of the host-pathogen interactions involved in HIV infection, especially the immune response to HIV infection , 2. As aWhile the mechanisms by which long term HIV infection leads to a compromised adaptive immune response is increasingly well characterized, much less is known of how HIV modulates the innate immune response. After any virus is introduced into a human host, receptors of the innate immune system will bind to pathogen associated molecular patterns (PAMPs) present as structural components or replication intermediates of the virus , 10. ComIn particular, the quintessential innate immune cytokines for limiting the replication of viruses, and thus key in eliminating all types of viruses, are Type I Interferons (IFNs) , 14. RecOur goal was to understand why HIV is not blocked by the huge array of ISGs and system of IFN that should stop progression of infection. To understand if our host innate immune system is able to detect HIV infection and generate proper innate immune responses, we looked at induction of IFN in cells infected with HIV. Here we show how IFN infection by infected CD4+ T cells is directly modulated by HIV infection.6) were infected with 400 ng of p24 protein by spinoculation in a flat-bottomed 24-well plate in 400 μl volume of media  containing virus and 10 μg/ml polybrene. The plate was centrifuged at 1200xg for 2 hr at 25°C. Then cells were washed and incubated in 5 ml of media for 48 hours. The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: CEM CD4+ Cells from Dr. J.P. Jacobs [Infectious HIV-1 (NL4.3) was produced by transient transfection of 293FT , 23 cell6 cells.For primary cell infections, peripheral blood mononuclear cells were purified from healthy human blood by Ficoll-Paque PLUS  separation. Primary CD4+ T cells were then isolated by negative selection using the CD4+ T Cell Isolation Kit II (Miltenyi Biotec) and an AutoMACS cell separator according to the manufacturer’s instructions. Cells were costimulated for 2 days prior to infection by culturing in RPMI Medium 1640 (Invitrogen) supplemented with 10% FBS, Pen/Strep, and 20 units/ml of interleukin-2 (Roche) in the presence of soluble anti-CD28 and plate-bound anti-CD3 antibodies as previously described . InfectiDuring harvesting, cells were pelleted by centrifugation then 100 μl of supernatant was removed and added to 900 μl of 2% Triton-X-100. Supernatants were stored at 4°C then assayed by ELISA for IFN (PBL VeriKine Interferon Detection Kits) or p24 levels (Beckman Coulter). For flow cytometry, cell pellets were fixed in 2% paraformaldehyde, permeabilized using 0.02% Tween 20, and then stained for intracellular p24 protein detection using KC57-Phycoerythrin (Beckman Coulter). Cell pellets were frozen at -80°C before use in western analysis.For Vesicular Stomatitis Virus  was used at an MOI of 0.1 to infect CEM cells. Aliquots of cells were monitored for GFP expression by flow cytometry and supernatants were assayed for IFN production by ELISA.This study did not include human subjects. Peripheral blood mononuclear cells were obtained through the UCLA Blood Bank in an anonymous fashion. Cells from anonymous sources are not considered human subjects, and are thus not subject to Institutional Review Board review.Purified HIV virion lysate was purchased from Advanced Biotechnologies Inc (Cat #10-119-100). Biochemical treatments were done with RNaseA, RNaseV1, and Alkaline Conditions according to manufactures protocol . After treatment the nucleic acid components were purified by standard phenol:chloroform extraction. Transfections of the lysates are as described below. Cells were transfected with 1 ng of lysate per well of a 24 well dish. Experiments were repeated on three separate occasions and representative data is shown.HEK 293T cells or murine embryonic fibroblasts (MEFs) were transfected with IFN-β luciferase and Renilla luciferase plasmids at a 100 ng:50 ng ratio for a set of triplicates in three wells of a 24 well dish using Fugene 6 (Roche). RNA or lysates were transfected using Lipofectamine 2000 (Invitrogen) according to manufacturers instructions. All transfections done solely with DNA were done with Fugene 6 (Roche) according to the manufacturers instructions. ISRE and κB luciferase were used at a 200 ng:50 ng ratio for a set of triplicates in three wells of a 24 well dish. Inducers (e.g. IPS-1) and blockers (e.g. Vpu or Nef) were used at 200 ng:1000ng ratio for a set of triplicates in three wells of a 24 well dish. Transfections were allowed to proceed for 18 to 24 hours, harvested and analyzed using a Dual-Luciferase Reporter Assay (Promega) according to manufacturer’s protocol. Infections with Vesicular Stomatitis Virus (VSV) were done 12 hours after transfection for 18 to 24 hours using an MOI of 0.01. Afterwards cells were fixed and analysed by flow cytometry for levels of GFP. Experiments were repeated on at least three separate occasions and representative data is shown.HEK 293T cells were transfected as above but were lysed in SDS-Loading buffer for whole cell lysates. Infected CEM T cells were treated in a similar fashion. Cell lysates were separated and western blotted for the protein as well as tubulin. Signal for each protein was detected by digital capture of HRP catalyzed light release using a BioRad ChemiDoc XRS System with Quantity One Software. The individual band intensities were quantitated and each target protein was normalized to the level of tubulin signal present. Experiments were repeated on five separate occasions for IPS-1 and three occasions for other molecules being assayed.XhoI to cleave only the site within the nef open reading frame [SalI-BamHI region of this vector was then replaced with the corresponding region of a previously described 3' HIV clone that is deficient in vpu and nef [The NLΔvpuΔnef clone (referred to as Delta Vpu/Nef in figures) was produced by partially digesting the denv(Wt) plasmid with ng frame . An inac and nef , to prodIn an effort to show genuine quantitative data, we do not show fold induction for any experiment but instead show the actual values obtained in an experiment normalized to the internal standard. We feel this gives a more genuine representation of data and that the data is not masked through the use of fold induction values, especially in reporter assays. Consequently, the values in different experiments may vary but the interpretation remains the same and is more consistent with the biology of each individual experiment.To begin understanding how HIV modulates global ISG function, we started with a classical measurement of IFN-α release after HIV infection of CD4+ T cells. We used the CD4+ T cell line CEM that responds with IFN-α release upon Vesicular Stomatitis Virus (VSV) infection as monitored by ELISA . We wereTo determine if the necessary PAMPs were present in HIV to induce IFN via the innate immune system we used lysates from purified HIV virions that were transfected into cells with reporters for IFN-β promoter activity. Transfection of HIV virion lysates strongly induces IFN-β promoter activity  confirmiVpu or Nef ORF strongly inhibits IFN induction, while other ORFs such as Env and Tat did not have significant effects . We normalized infection of these viruses and analysed the cells for protein stability and IFN induction state . Upon inFinally, we went on to show that the observation of HIV modulation of IPS-1 mediated IFN induction in CEM T cells could also be seen in primary CD4+ T cells. Using primary CD4+ T cells infected with either wildtype or mutant HIV, we see that HIV that is mutant in both Vpu and Nef replicates at a rate far below wildtype HIV . HoweverOur studies have added to the foundation for a general framework for the host- pathogen interaction in regulation of the IFN response to HIV during infection of T cells. First, we show that though little to no IFN is actually produced upon infection of T cells by HIV, host cells are indeed capable of sensing HIV RNA to initiate innate immune responses via IFN induction. We have further shown that the HIV proteins Nef and Vpu can cause IPS-1 degradation, thereby inhibiting RLR-mediated interferon induction. Furthermore, ablation of Vpu and Nef expression in a mutant HIV restores IFN release from T cells during infection. Our current studies are focused on determining if Vpu and Nef degrade IPS-1 using similar mechanisms as in CD4 degradation. Nevertheless, the targeting of IPS-1 by more than one ORF of HIV is a testament to the importance of abrogating this system before IFN begins working to limit the viral infection. Overall, affecting the RLR/IPS-1 pathways will be critical to the spread of virus and the establishment of a pathogenically effective founder population of infected cells in the local microenvironment of infection .The role of IFN in control of HIV infection and during the development of AIDS has several important facets to consider. Previous studies into HIV pathogenesis have focused on the role of plasmacytoid dendritic cells (pDCs) in response to HIV , 41. In The same thing may be true for HIV infection, in which IFN and other innate immune responses by HIV infected cells such as T-cells and macrophages may play more important roles than pDCs in inhibiting HIV replication and prevent the establishment of HIV persistent infection. . These vIt is now possible to begin modelling the host-pathogen signalling network of innate immune response to HIV infection . PositivWithout induction of IFN, ISGs such as the important antiviral protein APOBEC3G are not potentiated to initiate an anti-HIV state in infected cells allowing HIV to spread unhindered. The dynamics of this network will affect HIV replication, especially establishment of infection during primary HIV infection and ultimately persistence of the HIV infection. Also, this network will impact the replication of other viruses within or in close proximity to HIV infected cells as secondary infections become a major problem for HIV-infected patients. Further studies on the interactions between host innate immune molecules and HIV viral products will fill the gap between primary HIV infection and the well characterized adaptive immune responses. These studies will move to providing insight towards the development of novel approaches against this important human pathogen.


Nonalcoholic fatty liver disease (NAFLD) is rapidly becoming the most prevalent cause of liver disease worldwide and afflicts adults and children as currently associated with obesity and insulin resistance. Even though lately some advances have been made to elucidate the mechanism and causes of the disease much remains unknown about NAFLD. The aim of this paper is to discuss the present knowledge regarding the pathogenesis of the disease aiming at the initial steps of NAFLD development, when inflammation impinges on fat liver deposition. At this stage, the Kupffer cells attain a prominent role. This knowledge becomes subsequently relevant for the development of future diagnostic, prevention, and therapeutic options for the management of NAFLD. We are facing a rampant epidemic of nonalcoholic fatty liver disease (NAFLD) which afflicts adults and children and is frequently associated with obesity and insulin resistance. In Europe and in the United States, it is estimated that 3 out of 10 adults developed NAFLD . As NAFLThe estimation of NAFLD impact at population level poses two questions. (1) Realistically, should we assess NAFLD recurrently in the population? (2) Is there a good, easy, and not costly way of doing it? At a first glance, NAFLD could be seen as a response mechanism to deal with increased amounts of lipids intake, representing a protective mechanism. To identify the disease in early stages, we would need liver biopsies, the recognized gold standard for NAFLD diagnosis. Obviously, the invasive nature of such procedure excludes its widespread use. The more widely used ultrasound, however, will not inform us of the grade of liver inflammation. Magnetic resonance imaging and computed tomography even though more sensitive are expensive and cannot distinguish between simple steatosis and the severe form of NAFLD termed nonalcoholic steatohepatitis (NASH) . AlternaTriglycerides (TG) accumulation is an efficient energy storage mechanism. When compared to carbohydrates  or proteins , TG provides higher caloric intake 9 kcal/g) [ kcal/g .Physiologically, TG are a way of balanced energy storage. However, over the last decades, the excessive consumption of food rich in fat and sugars, associated with excessive caloric intake, has led to alterations in lipids and glucose metabolism. The current recommendations for adults are that 20% to 35% of the daily calories ingested are derived from dietary lipids. Nevertheless, this value is estimated to be around 40% in western diets . The imb de novo lipogenesis (DNL)) and lipid disposal (via fatty acid oxidation and/or production VLDL) in the liver [Overconsumption of fat and sugar ends up in ectopic lipids accumulation, with the liver being one of the prime targets in this process. Hepatic accumulation results from a disparity between lipids availability  [ In vitro experiments also demonstrated that incubation with palmitate (SFA) lead to cell toxicity, with ROS production and cell death, contrary to incubation with oleate (MUFA). Oleate alone increased TG accumulation without cell damage and in combination with palmitate and promoted lipid droplet formation abolishing the palmitate effect [Hepatic steatosis occurs primarily by the formation of lipid droplets  considers (TLRs) , contribs (TLRs) . In vitre effect . Anothere effect . Similare effect , 22.The exact mechanisms by which SFA-induced lipotoxicity occur are not well established. It is known that these FFA promote the release of several proinflammatory cytokines and trigger endoplasmic reticulum (ER) and oxidative stress . ER is rIncreased oxidative stress was observed in NASH patients and in animal model in association with accumulation of oxidized lipids . Taken tHighlighting the role of diet composition, it was shown that NAFLD and NASH patients had a dietary intake richer in SFA and cholesterol and poorer in polyunsaturated FFA and fibers . A lowerEvidence suggests that high sugar intake combined with lipid oversupply exacerbates liver pathology. Fructose is a popular monosaccharide used industrially in the form of high-fructose corn syrup  to sweeten foods and beverages . ConsumpIn humans, all fructose that is ingested is metabolized by the liver . In cont per se plays a role in triggering inflammation, possibly through the disruption in gut microbiota and increased mucosal permeability. Spruss et al. [α, portal lipopolysaccharide (LPS), and myeloid differentiation primary response gene (MyD)88 have observed these mice [Fructoses et al.  showed tese mice .Taken together, these results show fructose as an important dietary contributor to NAFLD pathogenesis and severity , being mThe severe form of NAFLD disease is termed as NASH and is characterized by the presence of hepatocellular injury, lobular and/or portal inflammation, and frequently, deposition of collagen fibers . AlthougRecent evidences place the liver and its local response to metabolic and immune mediated disturbances in the gut-adipose tissue axis . Understβ and proIL-18 into mature forms [The innate immune system recognizes immunogenic signals through pattern recognition receptors (PRRs). Bacterial products such as LPS, lipoproteins, flagellins, and peptidoglycans are amongst the PAMPs recognized by these receptors. Endogenous DAMPs like heat shock proteins, high mobility group box 1 (HMGB1), and breakdown products of extracellular matrix liberated from tissue damage and cell death also signal through PRRs . PRRs hare forms . By contκB, a critical modulator of liver immune responses [TLR signaling and inflammatory cytokines like IL-1 regulate the activation of the transcription factor NF-esponses .β (TRIF) to induce type 1 interferon (IFN) through Myd88 independent pathway [κB signaling [β) signaling in a Myd88 dependent path in a model of hepatic fibrosis [β secretion by KCs is required for fibrosis development. However, absence of TLR4 on KCs did not affect the fibrogenic process in this model [TLRs are found in several cellular components like plasma membrane and endosomes and the majority associates with a common adaptor molecule, MyD88 through Toll/IL-1 receptor (TIR) domain. Trafficking and location of TLRs within the cell are important for ligand binding and downstream signaling transduction. TLR4 is located mainly in the plasma membrane and associates with the LPS binding protein CD14 and myeloid differentiation- (MD)-2 molecule to recognize LPS. TLR4 can also bind to TIR-domain containing adapter inducing interferon- pathway . Endosomignaling . Ameliorignaling , 52, 53.ignaling . Increasignaling . Markedlignaling . TLR4, wβ secretion by KCs [TLR9 is located in the endosomal compartment and binds to an unmethylated CpG motif that is very prevalent in bacterial DNA. TLR9 knockout mice under a choline-deficient amino acid defined (CDAA) diet for 22 weeks, known to induce NASH with characteristic steatosis, inflammation, and fibrosis, showed amelioration of disease through suppression of IL-1n by KCs . These rβ, and reactive oxygen species (ROS) among others have been suggested as NLPR3 activators. Danger signals released from fatty acid-treated hepatocytes were also shown to induce inflammasome activation in liver mononuclear cells [The most studied inflammasome is NLRP3  that comprises the NOD-like receptor NLRP3, the apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc), and the effector molecule procaspase 1. Inflammasome activation is regarded as a two-step process in which the first signal, frequently TLR signaling, upregulates inflammasome expression and the second signal triggered by an inflammasome ligand results in activation . LPS, DNar cells , 47. Ascar cells . On the ar cells , 57, disar cells . Anotherar cells . Augmentar cells . It is iar cells .κB, hepatic steatosis, and rise in IL-6, IL-1β, and TNFα gene expression [High fat diets in mice lead to increased hepatic expression of NF-pression .β, and IL-1α knockout mice showed attenuation of liver pathology induced by high fat diets [κB [Supporting a role for inflammasome in NASH, IL-1 receptor (IL-1R), IL-1at diets . Activatiets [κB . IL-1 foiets [κB .α overexpression is viewed as the hallmark of inflammation in obesity and NAFLD pathology and a major link to insulin resistance. Upon binding to its receptor, TNFα can activate proapoptotic or antiapoptotic signaling cascades leading to NF-κB activation, thus regulating cell viability, inflammation, metabolism, and other cytokine productions [α or TNFα receptor knockout obese mice have improved insulin sensitivity compared to wild-type controls [α, through NF-κB both promotes and is activated by insulin resistance and is involved in liver inflammatory and metabolic alterations [α antagonizes the anti-inflammatory cytokine adiponectin. Adiponectin is produced by adipocytes and sensitizes cells to insulin. It also promotes fatty acid oxidation with marked anti-inflammatory and antilipogenic effects in the liver [α, on the other hand, correlate with NAFLD disease activity as measured by histological parameters in NAFLD patients. Likewise, TNFα and TNFα receptor gene expression was increased in hepatic and adipose tissues in NASH patients [α neutralizing monoclonal antibody. However, highlighting the complexity of inflammatory signaling, infliximab treatment did not improve insulin sensitivity in human obese insulin resistant individuals. Furthermore, pentoxifylline, a phosphodiesterase inhibitor, prevented TNFα production with only modest amelioration of insulin resistance in small studies of NASH patients [TNFductions . It is ocontrols . TNFα, terations . Additiohe liver . Increaspatients . Experimpatients .Visceral adipose tissue of obese individuals secretes increased amounts of IL-6 when compared to subcutaneous fat. Increased levels of plasma IL-6 were associated with augmented inflammation and fibrosis in NAFLD patients . Adipocyα deficient mice displayed reduced hepatocarcinogenesis after high fat diet and upon carcinogenesis induction by diethylnitrosamine (DEN) treatment [Interestingly, IL-6 and TNFreatment . On the reatment . In thisreatment .The liver is structurally and functionally heterogeneous. Parenchymal cells, that is, hepatocytes, are the most numerous and comprise 60% of the total liver cells and 80% of the volume of liver. Nonparenchymal cells (NPCs) represent around 20% of the liver cells and include sinusoidal endothelial cells (20% of liver cells), KCs (approximately 15% of liver cells), and hepatic stellate cells (5–8% of liver cells). Other immune cell populations mostly of natural killer T cells (NKTs) comprise a minor fraction of NPCs . HepatocKCs are the resident macrophages of the liver comprising the largest tissue specific population of macrophages in the body. The major immune function of KCs in healthy liver is to phagocyte and present pathogens entering from portal vein and arterial circulation, constituting one of the first lines of defense of the organism. Under these circumstances, they present mostly a “tolerogenic” phenotype preventing undesirable immune responses to the common gut-derived antigens they are constantly in contact with .γ and LPS induce differentiation into the M1 phenotype marked by the release of proinflammatory cytokines like TNF-α, IL-1, and IL-12. Alternative activation into M2 phenotype is more heterogeneous as different stimuli (such as IL-4 or IL-10) can induce different phenotypes. Typically, increased expression of arginase 1, secretion of immune-modulatory cytokines , and involvement in tissue repair phase are considered as indicators of M2 differentiation. The different origin of the cells together with the functional plasticity of macrophages can explain the phenotypic and functional heterogeneity of KCs observed upon different triggers of liver pathology.KCs are differently distributed in the liver and it is possible to find two different populations regarding its localization. Large KCs are localized in the periportal zone and have increased phagocytosis and increased production of biological mediators. These large KCs can be identified by the expression of CD163, a scavenger receptor. KCs can be also identified by the general macrophage marker F4/80 or by CD68, which is present in all KCs regardless of their location . The KC β, TNFα, CCL2, and CCL5, contributing to a paracrine activation of protective or apoptotic signaling pathways in hepatocytes and the recruitment of other immune cells [KCs are known to be involved in the control of inflammatory responses in NAFLD. In the early stages of the disease, hepatic macrophages expand rapidly and secrete cytokines and chemokines such as IL-1ne cells . TLR4 plne cells . per se and high-energy diets can be harmful to the liver. Evidences show that overload of lipids and cholesterol derivatives activate KCs in models of fatty liver disease and steatohepatitis [α and IFNγ [−/− bone marrow donors had improved inflammation and steatosis phenotype [ In vitro assays demonstrated that these fat-laden KCs were more responsive to LPS induced activation when compared to KCs from lean mice [ in vitro stimulation of mouse KCs with the SFA palmitic acid was shown to upregulate expression of TLR receptors [KCs are not only responsive to inflammatory signals but also to metabolic fluctuations . As refeepatitis . A diffeand IFNγ   and migrate to sites of injury. Besides secreting extracellular matrix proteins, HSCs secrete cytokines, growth factors that promote regeneration of hepatic epithelial cells, and angiogenic factors that modulate endothelial cell and hepatocyte proliferation. Prolonged activation of HSC causes fibrosis and during fibrosis regression the number of these cells is greatly reduced by induction of cellular senescence and apoptosis and return to quiescent state [Myofibroblast that migrate and accumulate at sites of liver injury in response to autocrine and paracrine signals produced by neighboring cells constitute the cellular source of fibrosis during chronic liver diseases . Althougnt state .α, IL-1β, and IL-6), genes mediating ROS generation, and genes coding for fibrogenic growth factors (TGFβ1), vasoactive substances, and adipokines (Leptin) were shown to be critically involved in liver fibrogenesis [β1 polymorphism may confer susceptibility to NASH progression to fibrosis [β signaling in HSC stimulates activation, synthesis of extracellular matrix protein, and inhibition of its degradation [β and platelet derived growth factor (PDGF) that constitutes a potent mitogenic factor for HSC [α lead to activation of NF-κB signaling pathway in HSC promoting cell survival [ in vivo mouse models of fibrosis. Among these genes were the NF-κB-regulated genes Il-6, and tissue inhibitor of metalloproteinase 1 (timp1) [Genes regulating hepatocellular apoptosis and/or necrosis, genes regulating the inflammatory response to injury  . Timp1 i (timp1) .Macrophages can produce Mmps and contribute in a later stage of the wound healing process to fibrosis regression  Figure . In a weAutophagy is a catabolic mechanism whereby unnecessary or dysfunctional cellular components are degraded in the lysosome. Autophagy-related (ATG) genes are necessary for the formation of the phagophore and autophagosomes, the initial steps in autophagy . Autophaβ-catenin signalling is crucial for hepatic progenitor cell (HPC) differentiation and engagement into hepatocellular fate. Macrophage depletion during hepatocyte regeneration was shown to remove the stimuli for hepatocyte differentiation and HPC differentiated preferentially into cholangiocyte, forming biliary structures [Phagocytosis of hepatocyte debris by liver macrophages/KCs was also shown to promote Wnt signaling upregulation. Wnt/ructures .Macrophage plasticity may differ according to the nature of the stimuli leading to pathology. It is unknown whether impairments on macrophage phagocytosis and differentiation into matrix degrading prone-macrophages underlie fibrosis or hepatocellular differentiation triggered by NASH . Of noteNAFLD is a rising concern that goes together with the increasing prevalence and incidence of obesity being a current epidemic problem. NAFLD pathogenesis is linked with insulin resistance, adipose tissue distribution, and dietary and genetic factors, which are risk factors not only for NASH but also for diabetes and associated pathologies.From this review, it is clear that much remains to be understood regarding the mechanism of the disease. The lack of knowledge in relation to this pathogenesis becomes a hurdle in the path towards novel approaches for the prevention and treatment of the disease. Up to our days, prevention can only be based on calorie restriction and favorable dietary composition as well as exercise. More effective lifestyles/therapeutic methods should be addressed not only to prevent fat deposition, but primarily to avoid subclinical inflammation, where KCs play a prominent role. How the interdependent effects of diet microbiota inflammation directly impact liver dysmetabolism is an issue that remains to be elucidated. Nevertheless, from an interventional standpoint, this vicious cycle needs to be broken. If this hypothesis is proven to be correct, we should start to integrate the impact on intestinal microbiota composition and subsequent inflammatory responses in the nutritional value of foods, as a proxy for predicting the potential to evoke dysmetabolic states that are determinants of NAFLD development.