In this study we sought to investigate the relationship between inflammation and tumor growth in vivo using the PET tracer 11C-(R)-PK11195 PET and a comprehensive MR protocol, including high temporal resolution DCE-MRI in 15 patients. An intertumor comparison of 11C-(R)-PK11195 binding potential (BPND) and DCE-MRI derived vascular biomarkers  across the 3 different tumor growth cohorts was undertaken. Tissue of 8 tumors was examined with immunohistochemistry markers for inflammation (Iba1), neoplastic cells (S-100 protein), vessels (CD31), the PK11195 target translocator protein (TSPO), fibrinogen for vascular permeability, and proliferation (Ki-67). Results were correlated with PET and DCE-MRI data.Nineteen patients with sporadic VS  underwent prospective imaging with dynamic 11C-(R)-PK11195 BPND , and higher mean tumor Ktrans . Immunohistochemistry confirmed the imaging findings and demonstrated that TSPO is predominantly expressed in macrophages. Within growing VS, macrophages rather than tumor cells accounted for the majority of proliferating cells.Compared with static tumors, growing VS displayed significantly higher mean 11C-(R)-PK11195 specific binding and DCE-MRI derived parameters can be used as imaging biomarkers of inflammation and vascular permeability in this tumor group.We present the first in vivo imaging evidence of increased inflammation within growing sporadic VS. Our results demonstrate that  Inflammation and vascular permeability are related to growth in sporadic vestibular schwannomaPET markers of inflammation and DCE MRI permeability metrics can detect growth in sporadic VSMacrophages account for the majority of proliferating cells in sporadic VSVS has an unpredictable natural history. Some tumors show sustained growth, while others remain stable or even shrink. An understanding of the mechanism of growth may have prognostic and predictive potential and lead to the development of novel therapeutic approaches. Previous evidence based on tissue analysis suggested that inflammation and angiogenesis may be relevant to VS growth, but no in vivo studies have ever been performed. Using an established PET tracer for inflammation and DCE-MRI derived vascular biomarkers, we have provided the first in vivo evidence that increased inflammation and vascular permeability correlate with growing VS. The results of this study suggest further investigation of these imaging biomarkers as predictors of tumor growth, and consideration of inflammation as a therapeutic target in sporadic VS.1 and occurs most commonly as a sporadic lesion. Sporadic VS is thought to be caused by inactivation of the NF2 gene and loss of the suppressor protein merlin.2 Although regarded as a benign tumor in the World Health Organization (WHO) classification, VS can cause significant morbidity in terms of both the tumor itself and the treatment thereof. A specific challenge in the management of VS is that only about one-third of sporadic VS tumors grow, while two-thirds of them remain stable or less commonly shrink.3 At present there is no method for identifying which tumors will grow and which will not. Treatment options include therefore conservative management of static tumors and surgery or radiotherapy (often stereotactic radiosurgery [SRS]) for growing tumors. Growing tumors, left untreated, will eventually produce brainstem and cerebellar compression, cranial nerve dysfunction, and hydrocephalus, with potential risks including trigeminal and bulbar dysfunction, stroke, blindness, and death. Treatment-related morbidity, which includes this same list but in addition the specific risk of facial nerve paralysis, is less when tumors are diagnosed and treated while they remain small.4 SRS is generally accepted to be a treatment option only for tumors smaller than 3cm in diameter.4 Predicting growth in individual VS at presentation would enable early intervention and therefore optimize outcomes.Vestibular schwannoma (VS) is a benign tumor arising from the vestibular component of the VIII cranial nerve. It accounts for approximately 8% of intracranial tumors in adults, with an incidence of 1 per 1000005 While cyst formation may contribute to volume increase in some instances,6 it has been suggested that inflammation and angiogenesis may also play a pivotal role.7 In neurofibromatosis type II (NF2)–associated VS, angiogenesis has proven to be sufficiently critical to tumor growth that it provides a specific therapeutic target with demonstrable response to the anti-angiogenic agent bevacizumab.8 Angiogenesis may also play a role in sporadic VS growth9–11 with vascular endothelial growth factor expression and other angiogenic factors correlating with microvessel density,10 tumor volume,10 and tumor growth rate.12 These studies, however, are inevitably based on tissue specimens. As such, their generalizability to nongrowing tumors that do not typically undergo surgery is less certain. There is therefore a need for an in vivo metric such as an imaging biomarker to enable characterization of all VS patient groups and to facilitate longitudinal studies.The mechanisms that trigger and maintain growth in VS remain unclear. Previous studies have shown a poor association between classical markers of neoplasia such as cellular proliferation indices and tumor growth.13 but the role of inflammation in benign neoplasms including VS has been less widely investigated. Macrophages are commonly found in VS especially within Antoni B areas,14 and their presence correlates with duration of symptoms,11 tumor size,5 and rapid growth.7 Detection of inflammation with positron emission tomography (PET) has been the subject of considerable research, including the development of several PET ligands targeting the 18 kDa translocator protein (TSPO).15 The full range of TSPO function is unknown, but the molecule is expressed by inflammatory cells and its level increases significantly following their activation.15–17 TSPO expression has not been previously studied in sporadic VS.The immune response is integral to growth and invasion in malignant tumors,19 GBCA concentration can be quantified and modeled through approaches such as the extended Tofts model20 to derive a number of key tissue microvascular parameters, including Ktrans, vp, and ve. Whereas vp and ve represent the proportion of each image voxel occupied by blood plasma and extravascular-extracellular space (EES), respectively, the volume transfer constant Ktrans is a composite parameter reflecting tissue blood flow, vessel surface area, and vessel permeability.20 While a limitation in the widespread usage of DCE-MRI in clinical practice has been variation in acquisition protocols and modeling approaches across different studies,18 DCE-MRI derived parameters have been shown to be reproducible21 and correlate in tissue validation studies with both vascularity markers23 and histological estimates of the EES.25 Vascular biomarkers derived from DCE-MRI have been utilized in other CNS tumors, including NF2-related tumors,26 but their potential role in sporadic VS has not been previously studied.Dynamic contrast enhanced (DCE) MRI non-invasively quantifies tissue microvascular structure by measuring the pharmacokinetics of intravenously administered gadolinium-based contrast agents (GBCAs).11C-(R)-PK11195 and investigated vascular parameters, including vascular permeability within these tumors, using dual-injection DCE-MRI. Tissue analysis in the group of participants who subsequently underwent surgery was used to validate the PET and DCE-MR imaging results.We hypothesized that tumor growth in sporadic VS is driven by intratumoral inflammation and that inflammation could therefore represent a biomarker of tumor growth and a therapeutic target. We investigated inflammation using the TSPO PET ligand 11C-(R)-PK11195 binding, such as benzodiazepines or steroids. Due to the spatial resolution of the PET camera, only patients with sporadic VS larger than 7.5 mm diameter  in the cerebellopontine angle were recruited into the study. All patients underwent at least 2 MRI acquisitions to establish tumor growth; this included the study scan in 2 cases, where definite growth was evident. The study MRI scan in addition to the results of previous MR imaging were reviewed by the multidisciplinary team and tumors were classified as static, growing, or shrinking. The median length of follow-up from diagnosis was 2.44 years  for the static cohort, 3.02 years  for shrinking, and 0.71 years  for growing tumors, reflecting the prompt decision to treat in this group. The growth classification was based primarily upon clinical decision making in these patients, with tumors classified as static or shrinking being offered a period of observation and growing tumors being recommended for microsurgery or SRS. Volumetric measurements of tumor size were made for both the study MRI scan and the preceding clinical scan to confirm different behavior between the tumor cohorts -PK11195. PET images were reconstructed using an OSEM (ordered-subset expectation maximization) 3D iterative method29 and corrected for attenuation, scatter, random coincidences, dead time, and detector normalization as previously described30 .31 (see MRI data were acquired on a 1.5-tesla whole body scanner using a dedicated head coil (Philips Achieva). The following axial MR images were obtained: high-resolution 3D T1-weighted (T1W) gradient echo sequence with full brain coverage  and balanced steady state gradient echo imaging of skull base . Fifteen patients also underwent contrast enhanced imaging, while 4 patients were unable to undergo contrast imaging due to concerns about uncertain renal function. DCE-MRI data were collected using a dual-injection technique as previously described11C-(R)-PK11195 binding potential (BPND), representing the ratio of specifically bound radioligand over the nondisplaceable one in tissue at equilibrium, were calculated using the simplified reference tissue model with atlas-defined cerebellar gray matter providing a “pseudoreference” tissue input function.32 To address potential concerns about use of a reference region adjacent to the pathology under study for modeling, relative standardized uptake values (SUVs) were also calculated as the average tumor tissue activity over the final 30 minutes divided by either cerebellar gray matter (SUVT/Gm) or a manually defined region of centrum semiovale bilaterally (SUVT/WM)  for vascularity (SRTMV), which takes into account the tracer activity in the vasculature33  see . To specre33 see .N.34 Using the arterial input function derived from low-dose high-temporal (LDHT) DCE, the LDHT-DCE tissue concentration time course curves were fitted to the extended Tofts model.20 Estimated values of Ktrans, ve, and vp were calculated on a pixel-by-pixel basis. The generated maps were coregistered to the T1W structural images.The signal intensity time curves were converted to tissue contrast concentration curves using measured values of R1ND parametric map derived using both the SRTM and SRTMV models for sampling the mean (BPmean) and maximum (BPmax) BPND value of the entire tumor. Similarly manually defined object masks were applied to the LDHT parametric maps to allow estimation of whole tumor Ktrans, ve, and vp values.Tumors were manually delineated on the coregistered T1W postcontrast image to create an object mask. For the 4 patients who had undergone imaging without contrast, the tumor was delineated on the noncontrast T1W structural image. Care was taken when delineating the tumor to avoid partial volume effects from nearby structures or surrounding CSF. The object mask was projected onto the BP35 , Schwann cells (anti-S100), TSPO expression (anti-TSPO), microvessel area (anti-CD31), vascular permeability (anti-fibrinogen), and proliferation (anti–Ki-67) was assessed quantitatively on the tissue sections of 8 tumors using immunoperoxidase immunohistochemistry following established protocolst-test was used. The intertumor correlation between imaging and immunohistochemistry-derived parameters are reported as Pearson’s product moment correlation coefficient (r) or Spearman’s rho in the case of nonlinear associations. For linear associations, the results of linear regression are reported as adjusted R2 estimates on included figures.Stata v11 was used for all statistical analyses. Normality and homogeneity of variance for all individual data parameters were assessed using the Shapiro–Wilk and Levene tests, respectively. One-way ANOVA with Bonferroni correction was used to compare the parametric PET and DCE-MRI derived values across the 3 tumor growth groups . For nonparametric data  the Kruskal–Wallis test with adjustment for multiple comparisons was used. For comparison of parametric immunohistochemistry-derived parameters between growing and static tumors a 2-tailed P > 0.05; see 3; the volume of growing lesions was significantly larger than static or shrinking tumors (P < 0.05). Confirmatory volumetric measurements demonstrated that growing tumors displayed a significantly higher annual adjusted growth rate compared with the static  and shrinking  groups (see Demographic details of the 19 patients who completed the PET and core MRI acquisitions are detailed in oups see .11C-(R)-PK11195 BPND tumor values for the 3 growth groups are shown in max  and BPmean  compared with static tumors. SUV values similarly demonstrated that growing tumors displayed significantly higher mean SUVT/GM  compared with static tumors (see 11C-(R)-PK11195 BPND values and both SUVT/WM  and SUVT/GM . Use of BPND values derived from the SRTMV model (see P = 0.011) and mean  specific binding (BPND-BV) compared with static tumors. Growing lesions also had higher BPmax and BPmean values than shrinking tumors, but that difference was not statistically significant .Maximum and mean mors see . Correlaodel see  similarl11C-(R)-PK11195 mean BPND -PK11195 BPND within the tumor.Tumor size at the time of PET scanning was strongly correlated with Ktrans values than static tumors . Correlation analysis of derived PET and DCE-MRI parameters demonstrated that mean tumor Ktrans positively correlated with both mean BPND  and mean ve . There was no significant difference in mean tumor ve between tumor growth groups  and mean  Iba1+ macrophage count compared with static VS , and between max Iba1+ cell count and max BPND . Mean TSPO optical density (OD) positively correlated with mean BPND  . Mean TSPO OD was higher in the growing group (P = 0.100), but this result was not statistically significant due to the presence of one static tumor with high TSPO OD. As shown in t-test, P = 0.014) and positively correlated with DCE-MRI derived mean tumor vp -PK11195 PET and DCE-MRI derived parameters is shown in 11C-(R)-PK11195 specific binding. The close relationship between immunohistochemistry and both the P < 0.001; see P < 0.001), which accounted for >50% of the proliferating cells within these lesions , as was the percentage of non-inflammatory Ki-67+/Iba1− positive cells -PK11195, a PET marker of inflammation,36 occurs within sporadic VS and that it is increased within growing tumors. Tissue analysis suggested that intratumoral macrophages were the major source of TSPO and therefore the PET signal, and that growing tumors were predominantly composed of inflammatory cells. Our findings suggest that inflammation may have a key role in VS growth and expand upon earlier studies which showed increased expression of macrophage surface markers within larger and fast growing tumors.7 TSPO PET imaging enables the study in vivo of inflammatory burden and characterization of those VS that would not typically be surgically resected for the first time.This is the largest PET study to our knowledge in sporadic VS and the first to document a positive association between tracer uptake and tumor growth. The study demonstrated that specific binding of 11C-(R)-PK11195 has been extensively characterized as a PET tracer for neuroinflammation and less so for peripheral macrophages. Its high lipophilicity ensures that delivery is not solely dependent on endothelial permeability16 and use of the simplified reference tissue model in this study differentiated specific binding or binding potential (BPND) from relative tracer uptake.37 TSPO is expressed in the endothelial and smooth muscle cells of blood vessels,38 but our tissue analysis suggested intratumoral inflammatory cells were the principal source of TSPO in growing tumors supporting the interpretation that increased 11C-(R)-PK11195 reflected increased inflammatory burden. TSPO expression was quantified using OD as a surrogate value of protein density. This approach proved more accurate in correlating PET imaging data and TSPO expression in tissue than the measurement of TSPO positive cells as it reflects the levels of TSPO available in tissue to bind the imaging probe 11C-(R)-PK11195.3039 We therefore hypothesized that growing tumors express pro-inflammatory cytokines and chemokines to a critical threshold, and it is the resulting recruitment of inflammatory cells, rather than Schwann cell proliferation, which in turn leads to their continued and sometimes rapid growth. Further larger studies are needed to fully characterize these pro-inflammatory molecules and understand their respective roles in both static and growing VS.Recent studies have documented upregulation of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor–α, and leukocyte adhesion molecules such as intercellular adhesion molecule 1 in VS compared with normal vestibular nerve.Ktrans values, a measure of vascular permeability,26 and a strong association between this metric and 11C-(R)-PK11195 binding. Several methods have been proposed to evaluate vessel permeability in tissue sections, including extravascular fibrinogen assessment. Fibrinogen is a large plasma glycoprotein (340 kDa) which is only detectable in areas that have significant endothelial disruption.41 Multiple previous studies have demonstrated fibrinogen as an accurate marker of vessel permeability40–42 that correlates with other permeability markers such as immunoglobulin G.41 Supporting immunohistochemistry demonstrated both increased vascular surface area and extravascular fibrinogen staining within growing VS, suggesting a link between vascular permeability and recruitment of inflammatory cells.44Analysis of concomitantly acquired DCE-MRI data in 15 patients demonstrated that growing tumors displayed higher 11C]-methionine in sporadic VS have been inconclusive due to limited tracer uptake irrespective of tumor size or growth.45–47 These results may be due in part to the inherently low proliferative and thereby low metabolic activity of these tumors.48 Our data demonstrate consistent cellular proliferation rates in the neoplastic cell population irrespective of tumor behavior, with the only variation being an increased population of proliferating inflammatory cells within growing tumors. Inflammatory and vascular biomarkers may therefore be more relevant in sporadic VS than cellular proliferation.Previous studies that have investigated PET tracers such as 18-fluorodeoxyglucose or [11C-(R)-PK11195 specific binding and inflammatory cell number. The correlation between the PET assessment and inflammatory cell density was preserved and so this did not detract from the potential of 11C-(R)-PK11195 as a biomarker, but the reason for high immunohistochemical TSPO labeling in this case could not be clarified. Previous studies have shown that tumor size itself is a predictive marker of VS growth,49 although this is not universally accepted. Separating the relative contributions of tumor size and growth rate to 11C-(R)-PK11195 specific binding from our data is difficult as the growing tumors were also statistically significantly larger. Larger studies with dedicated follow-up imaging should be undertaken to address the prognostic benefit of 11C-(R)-PK11195 uptake or DCE derived parameters in predicting VS growth.Our study is the first to provide combined imaging and tissue demonstration of both increased inflammation and vascular permeability in growing sporadic VS. One patient in our study displayed disproportionately high TSPO expression through immunohistochemical analysis relative to the This study presents evidence of the critical role that inflammatory cell migration plays in the growth of sporadic VS and is the first to provide an estimate of the inflammatory contribution to static tumors as well as the growing tumors that have previously been characterized ex vivo. We present a PET biomarker of inflammation alongside candidate DCE-MRI derived vascular biomarkers in the first combined study in this patient group. Our imaging and immunohistochemistry findings of both increased inflammation and vascular permeability within these tumors should prompt further research into the role these pathophysiological mechanisms play in VS growth and consideration of these processes as novel therapeutic targets.This project has been funded by both Cancer Research UK (CRUK) and the Engineering and Physical Sciences Research council (ESPRC) through the Cancer Imaging Centres grant (reference: C8742/A18097). FR and DM work leading to these results has received funding from the Seventh Framework Programme (FP7/2007-2013) under grant agreement nHEALTH-F2-2011-278850 (INMiND); Mr Ricky Williams has been funded by a private donation from Mr Robert Ward and his family.Supplementary Table 1Click here for additional data file.Supplementary Table 2Click here for additional data file.Supplementary Table 3Click here for additional data file.Supplementary Table 4Click here for additional data file.Supplementary Figure S1Click here for additional data file.Supplementary Figure S2Click here for additional data file.Supplementary MethodsClick here for additional data file.Supplementary Figure LegendsClick here for additional data file.


During HLC differentiation we observed the occurrence of epithelial cells with a phenotype divergent from the typical hepatic polygonal shape- we refer to these as endoderm derived epithelial cells (EDECs). These cells do not express the mature hepatocyte marker ALB or the progenitor marker AFP. However they express the cholangiocyte markers SOX9, OPN, CFTR as well as HNF4α, CK18 and CK19. Interestingly, they express both E Cadherin and Vimentin, two markers that are mutually exclusive, except for cancer cells. EDECs grow spontaneously under low density cell culture conditions and their occurrence was unaffected by interfering with the above mentioned signalling pathways.During embryonic development bipotential hepatoblasts differentiate into hepatocytes and cholangiocytes- the two main cell types within the liver. Cell fate decision depends on elaborate interactions between distinct signalling pathways, namely Notch, WNT, TGFβ, and Hedgehog. Several  In vitro differentiation of human pluripotent stem cells (hPSCs) into hepatocyte like cells (HLCs) or cholangiocyte like cells (CLCs) provide valuable tools for modelling hepatogenesis, studying liver-associated diseases, assessing toxicology and for drug screenings. Several protocols have been established to obtain one or the other cell type [ell type –10. The ell type –6 or CLCell type –10.in vivo [Bipotential hepatoblasts give rise to hepatocytes and cholangiocytes in vivo –13. HepaJAGGED1 (JAG1) or NOTCH2 mutations causes Alagille Syndrome, a disease that manifests in the liver by a reduction of bile ducts in combination with cholestasis [Notch signalling is crucial for the development of cholangiocytes. Impaired Notch signalling due to lestasis –16. Bilelestasis , 18. Thelestasis . Notch slestasis . Intereslestasis .Susceptibility to Notch signalling depends on transforming growth factor (TGF) β-signalling. As cells of the periportal mesenchyme are major sources for TGFβ secretion, a gradient with decreasing concentrations forms along the periportal-parenchyme axis , 21. Celet al. recently demonstrated that β-catenin is not necessary for biliary development but that its overexpression perturbs cholangiocyte differentiation as well as bile duct morphogenesis [Additionally, wingless-type MMTV integration site family (WNT) signalling has been proposed to be involved in hepatic cell fate specification, however, to date contradictory results preclude an unambiguous assignment of its exact role in this process. Several studies have indicated that WNT-β-catenin signalling promotes cholangiocyte and not hepatocyte fate , 23, whiogenesis .Finally, Hedgehog (Hh) is involved in the complex signalling orchestra that regulates hepatic cell fate. Bipotential hepatoblasts produce and respond to Hh ligands. This dual capacity is retained in cholangiocytes, while healthy hepatocytes lose the ability to produce Hh ligands or to react to its signals. However, upon liver injury they regain the ability to produce Hh ligands .in vitro, we often observed cells with an epithelial but non-polygonal morphology lacking HLC characteristics occurring at areas of the dish where cell density is low. Here we set out to characterize these cells and applied distinct pathway inhibitors with the aim to reduce their appearance during HLC differentiation and maybe increase the homogeneity of HLC populations.During HLC differentiation The use of iPSC lines for this study was approved by the ethics committee of the medical faculty of Heinrich-Heine University under the number 5013.The human ESC line H1 was purchased from WiCell Research Institute , human iPSCs were generated as described in , 27.2 after splitting at the HE stage. On day 10 the differentiation was continued with HLC medium: 82% Leibovitz 15 medium, 8% fetal calf serum, 8% Tryptose Phosphate Broth, 1% Glx, 1% P/S  with 1 μM Insulin (Sigma), 10 ng/ml hepatocyte growth factor (HGF) (Peprotech), 20 ng/ml Oncostatin M (OSM) 209 a.a. (Immunotools), 25 ng/ml Dexamethasone (DEX) (Sigma) (hPSCs were cultured on matrigel (Corning) coated plates with Stem MACS (Miltenyi) or TSR E8 medium (Stemcell Technologies). Medium was changed on a daily basis. Spontaneously differentiated cells were removed manually if necessary. For differentiation, cells were split onto matrigel coated plates and kept in the stem cell medium for another 16-24h. Afterwards, HLC differentiation was performed as described previously [ (Sigma) . During In order to switch cell fate after HE stage, several signaling pathways were inhibited or activated with small molecules listed in Cells were fixed with 4% paraformaldehyde for 15 min. Unspecific binding sites were blocked by incubating 2 h at room temperature with blocking buffer . Antibodies were diluted in blocking buffer diluted 1:2 with 1x PBS . PrimaryUp to 500,000 cells were lysed in 500 μl Trizol and RNA was isolated with the Direct-zol™ RNA Isolation Kit (Zymo Research) according to the user’s manual. On-column DNase digestion was performed. 500 ng of RNA were transcribed into cDNA using the TaqMan Reverse Transcription (RT) Kit (Applied Biosystems). In the case of H1 derived EDECs also cRNA obtained from Affymetrics Array preparation was transcribed into cDNA using the TaqMan RT kit with random hexamers instead of oligo-dT primers. Primers for qRT-PCR were purchased from MWG . Real tiMicroarray experiments were performed employing the Affymetrix PrimeView chip . Details of data analysis are given in Supplementary materials and methods.Cytochrome P450 3A4 (CYP3A4) activity was measured in technical triplicates with the respective P450 Glo assay from Promega, according to the recommendations. Supernatants were stored from every step of the differentiation process and urea content was measured in technical duplicates of biological duplicates with the QuantiChrom Urea Assay (Bioassay systems) according to the manufacturer’s recommendations. Results are depicted as mean values with standard error of the mean (SEM) in case of the CYP3A4 assay and standard deviation in case of urea measurement. P-Values were calculated with two-tailed student’s t-tests .in vitro differentiation of hPSCs into hepatocyte like cells (HLCs), we frequently observed cells with atypical morphology, predominantly at the borders of densely grown colonies. As these cells only occur at regions of low cellular density, we reasoned that they require only lose cell-cell contact in combination with enough space for growth. The cells are of endodermal origin and have an epithelial morphology but are much larger than HLCs or cholangiocyte like cells (CLCs). In order to characterize these endoderm derived epithelial cells (EDECs), we tried to produce a pure population by reducing cellular density during HLC differentiation. To this end, we differentiated two iPSC lines as well as H1 ESCs into bipotential hepatoblasts by following our recently published protocol for hepatocyte differentiation until the stage of hepatic endoderm (HE) [During erm (HE)  and humaerm (HE) , respectThe two iPSC lines as well as H1 ESCs were differentiated into HLCs and EDECs and stained for expression of characteristic markers at the respective end-stages. HLCs expressed Albumin (ALB) and Alpha Fetoprotein (AFP) as well as Cytokeratin 19 (CK19) and HNF4α , while ETo test whether EDECs are related to cholangiocytes, we analysed the expression of characteristic markers for this cell type by immunocytochemistry. The cholangiocyte specific transcription factor SOX9 and the multifunctional protein osteopontin (OPN) were almost not present in HLCs at the protein level . HoweverIn contrast, EDECs expressed SOX9 as well as weak levels of OPN and they were highly positive for CFTR which is clearly localized within the cell membrane as expected in cholangiocytes .Vimentin , which is characteristic of epithelial cells . Howevertin VIM,  an interal cells . GFAP, aal cells .HE cells which have the potential to differentiate into HLCs, CLCs and also EDECs were positive for all investigated markers that are known to be expressed in the immature stages of either HLCs or EDECs, namely AFP, CK19, HNF4α, SOX9, and ECAD . HoweverALB, CYP3A4) as well as immature (AFP) hepatocyte markers were significantly up-regulated in HLCs compared to EDECs and HE cells  activity as well as urea synthesis in order to check if EDECs might have hepatocyte-associated functions. HFF- and H1 derived HLCs had the highest levels of CYP3A4 activity . In bothInterestingly, similar cell types are present in rat fetal liver where three distinct populations, comprising cells either expressing AFP, ALB and CK19, or AFP and ALB or only CK19 have been associated with distinct lineage commitment and re-population capacities .Notch signalling has been described during liver development as the most important signalling pathway for the determination of cholangiocyte fate in contrast to hepatocyte , 18. Thec/EBPα we observed similar regulation for all genes analysed in both cell lines, but because of the limited number of samples analysed, these expression changes are not significant  were expressed at higher levels in HLCs than in EDECs. This was unexpected as it has been described that hepatocyte differentiation does not require Notch signalling. HLCs are however still immature and the presence of molecules belonging to the Notch signalling pathway might indicate that they are still able to switch cell fate towards CLCs or EDECs. In particular, the presence of DLK1 has been described as being characteristic for bipotential hepatoblasts [Surprisingly, members of the Notch signalling pathway  or EDECs  were assigned to distinct gene ontology (GO) terms . InteresAnalysis of differentially expressed genes between EDECs and EDECs treated with γ-secretase inhibitor I revealed that untreated cells expressed more genes related to development and differentiation in general  Tables. We next wanted to determine whether interference with one of the other major signalling pathways that have been described as essential for cell fate decision making between hepatocytes and cholangiocytes can revert the EDEC phenotype and push the cells into the HLC direction. To this end, we applied several small molecules to either induce or repress WNT, Hh, and TGFβ signalling . As withOverall, it seems that splitting and replating pluripotent stem cell derived bipotential hepatoblasts at low density at the HE stage of the HLC differentiation process leads to the accumulation of EDECs. None of the applied pathway interferences was able to revert or prevent this transition. However, cell fate decision is a tightly orchestrated process with a high level of synergy between different signalling pathways. Thus, it is possible that the right interplay between the various pathways at the right time point is capable of altering cell fate. In order to determine the full potential of these cells and classify them according to already known categories of hepatic stem/progenitor cells, transplantation experiments with rats or mice after partial hepatectomy will be necessary.As cells with EDEC morphology often appear during HLC differentiation in regions of lower cell density, it is essential to keep the cells during the whole differentiation process at a high density. The accumulation of EDECs within a population of HLCs might interfere with disease modelling or drug screenings specifically related to hepatocytes.S1 FigTwo iPSC lines and one ESC line were differentiated into HE cells and stained for characteristic hepatocyte or cholangiocyte markers.(TIF)Click here for additional data file.S2 FighPSC were differentiated into HLCs and EDECs. (A) CYP3A4 activity assay (B) QuantiChrome Urea Assay.(TIF)Click here for additional data file.S3 FigGlobal expression patterns of genes involved in Notch (A) and Hedgehog (B) signalling were analysed in HLCs and EDECs with and without Notch inhibitor. Genes were colour-coded according to their function. Asterisks mark the genes that are expressed above threshold in at least the EDEC sample or the EDEC sample with inhibitor.(TIF)Click here for additional data file.S4 FigGlobal expression patterns of genes involved in WNT (A) and TGFβ (B) signalling were analysed in HLCs and EDECs with and without Notch inhibitor. Genes were colour-coded according to their function. Asterisks mark the genes that are expressed above threshold in at least the EDEC sample or the EDEC sample with inhibitor.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableThe genes included in the different sets of the venn diagram shown in (XLS)Click here for additional data file.S5 TableGenes expressed either in HLCs or in EDECS (regardless of inhibitor treatment) from the venn diagram  were use(XLSX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.S11 Table(XLSX)Click here for additional data file.


Liver cancer is the third rank amongst the common malignancies, causing maximum death in the patientsdiagnosed with cancers. Currently available biomarkers are not enough sensitive for early diagnosis of hepatocellularcarcinoma (HCC). This makes difficult management of HCC. With the aim of finding new generation of proteomic-basedbiomarkers, the represented study was designed to characterize the differentially expressed proteins at different stagesof HCC initiation and at progression. This could lead to find potential biomarkers for early detection of HCC. In this experimental study, we report induction of HCC by administrating chemical carcinogensin male Wistar rats. Disease progression was monitored by histological evaluation. Serum proteomic analyses such as2 dimensional (2D)-electrophoresis, MALDI-TOF-MS/MS and Western blot have been used to analyze and characterizethe differentially expressed proteins during HCC development. HCC initiation and tumorigenesis were observed at one and four months post carcinogen treatment,respectively. One of the differentially-expressed proteins, namely, cytosolic phospholipase A2delta was significantlyup-regulated at very early stage of HCC development. Its expression continued to increase during cancer progressionand hepatotumorigenesis stages. Its elevated expression has been confirmed by Western blot analysis. Consistent tothis, analyses of the sera in the clinically confirmed liver cancer patients showed elevated expression of this protein,further validating our experimental results.This study suggests that elevation in the expression of cytosolic phospholipase A2delta is associated withprogression of HCC. Hepatocellular carcinoma (HCC) is globally the fifthmost common cancer with a high rate of morbidity andthe third type of cancer causing maximum death among the patients diagnosed with cancers , 2. EtioIn the present study, using animal model, we aimed to find out differentially expressed proteins that are associatedwith HCC initiation and progression to introduce as potential biomarker(s) or to target as therapeutic agent at very early stage of liver cancer initiation.in vivo which has previously been developed in our laboratory to study HCC. Further, the obtained data are validated with sera of clinically approved liver cancer patients. The experimental study involves analysis of rodent model Liver cancer was chemically induced in 4-6 weeks old male Wistar rats weighing 80-100 g, by administrating chemical carcinogens DEN and 2-AAF as reported by our group earlier. Animal experimentation was performed following approval from Jamia Hamdard  Institutional Animal Ethics Committee formed for the Purpose of Control and Supervision of Experiments on Animals (project number 908). The protocol for HCC development in rats wasessentially the same as previously described instruction  and checked employing light microscope for histological changes. The Bradford’s method was used to measure protein concentration . Depleti30 µg of total serum protein was fractionated on 10% poly acrylamide gel at 80 V and it was transferred to polyvinylidene difluoride (PVDF) membrane employing Hoefer Western blotting apparatus . Immunodetection was performed using 1:500 diluted primary antibody  in Tris-bufferedsaline (TBS) overnight at 4°C and 1:4000 diluted HRP conjugated anti-rabbit secondary antibody  for three hours. The protein expression was visualized with diaminobenzidine  and LuminataTM Forte Western HRP Substrate  system. Analyses of clinically approved liver cancer patients sera  and controls  were carried out after receiving the approval of Jamia Hamdard Institutional Ethics Committee (JHIEC). The informed consent was obtained from all participating subjects. The HCC patients were clinically approved and were under various therapies. The experiments were carried out in triplicate and data are revealed as means ± standard error of the mean (SEM). The significance of differences (control vs. treated groups) was analyzed employing One-way ANOVA pursued by Dunnett test and they were considered statistically significant when P<0.05. HCC was induced by administrating chemical carcinogens (DEN and 2-AAF) in male Wistar rats, as previously reported by us . Histolo2 delta, gi|109470683, [Rattus norvegicus], using NCBI database search by MASCOT software  numbers to protein spots and compared differentially expressed proteins -10. One software . Detail2 delta expression in the serum of carcinogen treated rats vis-a-vis age-matched controls. Expression of cytosolic phospholipase A2 delta was elevated at one month after carcinogen treatment and it was continued to increase during cancer progression until tumor stage at four months post carcinogen treatment. Expression of ß-actin was considered as internal control  regulating growth of primary and transformed hepatocytes. The interrelationship among cytosolic phospholipase A2-α and TGF-ß signaling pathways has been reported in primary hepatocytes of rats and human HCC; thus cytosolic phospholipase A2-α is an important factor regulating TGF-ß signaling pathway and controlling proliferation of hepatocytes and hepatocarcinogenesis (2-α regulates biosynthesis of prostaglandins through arachidonic acid cleavage, frommembrane phospholipids (2 delta in the serum of HCC rats and in human patients with liver cancer. This suggests that is one of the important factors associated with HCC initiation and progression leading to hepatotumorigenesis. Elevation of cytosolic phospholipase A2 delta expression in livercancer might be associated with dysregulation of lipidmetabolism and liver damage, causing cancer initiation in tissue at precancerous stage, while the epithelial cells are actively proliferating.The importance of cytosolic phospholipase Aenzymes in cancer progression is of the considerable interests, as these enzymes play important role in the pathways associated with progression of cancer. This enzyme family controls cell proliferation, differentiation, survival and motility in almost all tissues. Their increased expression results in dysregulation and facilitates unlimited growth of tumors and metastasis for cancer cells. Significant role of this family members has been implicated in tumor progression and tumorigenesis . This faε and –ζ . Cytosolembranes  of virtuembranes . Aberrane, liver and coloe, liver. A studyfication . No much in vivo . Among tan cells . Cytosologenesis . Cytosoholipids , throughholipids . The proholipids . This paholipids . Arachidholipids , is a nholipids , tumor cholipids , 15. We 2 delta concentration, alone or in consolidation with other conventional markers, may provide critical knowledge for the early noninvasive disclosure of HCC. Moreover, cytosolic phospholipase A2 delta might also be served as a potential target to find out the status and progression of liver cancer. Taken together, the present study suggests that evaluation of cytosolic phospholipase A


Atypical presentations of diabetes mellitus (DM) have been reported innon-European ethnic populations under various names. It is unclear whetherthose names are used for the same or different clinical phenotypes. Unclearterminology may lead to inappropriate treatment and an underestimation ofthe burden caused by atypical diabetes phenotypes overlapping with classictypes of diabetes. This review aimed to describe the terms used for atypicalforms of diabetes and to investigate whether the terms are used for similaror different phenotypes.PubMed and Scopus were searched for relevant publications in French orEnglish available before 15 September 2015 using the terms: ”Atypicaldiabetes”, “Malnutrition Related Diabetes Mellitus(MRDM)”, “Fibro-calculus pancreatic diabetes (FCPD)”,Protein deficient Pancreatic Diabetes (PDPD)”, “Africandiabetes”, “Ketosis prone-type 2 diabetes”,“tropical diabetes”, “Flatbush diabetes”,“J-type diabetes”. Titles, abstracts screening and qualityassessment were performed by two independent authors. Observational studiesaddressing atypical diabetes in humans aged 14 years and above wereincluded. One author extracted data from selected articles.22 articles among 350 identified articles were retained for data extraction.Two atypical diabetes phenotypes were identified, each of them with avariety of names but similar definitions. One phenotype occurred in verythin people less than 30 years of age, typically from poor socio-economicbackgrounds and requires insulin for life. It differs from type 1 diabetesin the tolerance of high blood glucose without ketosis in the absence ofexogenous insulin. The second phenotype resembles type1 diabetes as itpresents with ketosis at onset but responds well, as type2 diabetes, to oralhypoglycemic drugs after initial stabilization with insulin. It occurs inindividuals who are usually over 30 years of age, with normal or overweightand absence of auto antibodies mainly found in type 1 diabetes.The scarce existing literature used various terms for similar diabetesphenotypes. Agreement on nomenclature for the various forms of diabetesusing the above reported characteristics are needed in populations whereatypical forms of diabetes exist as well as better characterization ofphenotypes and genotypes to inform evidence based treatment. Diabetes mellitus is a highly heterogeneous disease, the classification of which haschanged as knowledge about its clinical and pathophysiological features have evolved-5.In 19Since the early 1990s, the term atypical diabetes mellitus has been used to refer to somerare types of diabetes mellitus such as Maturity Onset Diabetes of the Young (MODY). MODY isLADA and MODY are well characterized and have long been recognized globally as atypicalforms of diabetes. There are, however, other forms of atypical diabetes which do notclearly fit within the existing classifications. These forms appear to be more prevalentin populations of African and Asian ancestry. One such rare and atypical presentation ofdiabetes mellitus known as ketosis prone type 2 diabetes, has been described in peopleof non-white ancestry in European and American countries -12. HoweTerms such as “atypical diabetes”, “Malnutrition Related DiabetesMellitus (MRDM)”, “Fibro-calculus pancreatic diabetes (FCPD)”, Proteindeficient Pancreatic Diabetes (PDPD)”, “African diabetes”,“Ketosis prone-type 2 diabetes”, “tropical diabetes”,“Flatbush diabetes”, “J-type diabetes” among others have beenused to describe diabetes phenotypes which do not clearly fit the type1 or type 2diabetes definitions and which are not yet well understood. These terms are not,however, clearly defined in the literature, and it is unclear whether the same term isused to describe different phenotypes in different contexts or conversely, whetherdifferent terms are used to describe similar phenotypes in different contexts. This lackof clarity is problematic because it may lead to inappropriate diabetes classificationat diagnosis, difficulties in the application of diabetes treatment guidelines (based onclassic types of diabetes) and the provision of appropriate educational materialsglobally and particularly in underserved settings.The aim of this study, whose protocol has been published in PROSPERO  is to syPubMed and Scopus databases were searched to retrieve relevant publicationsrelated to the review objectives. The following key words were used to searchrelevant articles: Flatbush diabetes, ketosis-prone diabetes, tropical diabetes,malnutrition-related diabetes, fibrocalculous pancreatic diabetes, chronicpancreatic diabetes, protein deficiency pancreatic diabetes and Africandiabetes. The search strategy was adjusted as appropriate for each database. Thesearch strategy is detailed in the Annexes S1 and S2 in Observational studies including prospective or retrospective cohort studies,case–control, cross-sectional studies and case series published before thedate of download (15 September 2015) on human non-European ethnic participantsaged 14 years and above were included. We included studies in English or Frenchreporting on atypical diabetes mellitus expressed as used key words. Studieswith unavailable abstract, genetic studies, case reports and studies focusing onclassic types of diabetes such as type 1 and type 2 diabetes mellitus,gestational diabetes mellitus, secondary diabetes, LADA and MODY wereexcluded.Titles and abstracts of retrieved bibliographic records were screened by twodifferent authors (CB and DS). Disagreements were resolved by discussion amongthe two and consensus. The full text of each potentially eligible study wasretrieved through HINARI and the library of Edinburgh University. Full textswere critically appraised independently by two authors (CB and DS). The qualityof individual studies was assessed using the Health Evidence Bulletins –Wales checklist for assessing the quality of observational studies . This toData extraction was done by one author (CB) using a specially designed worksheetincluding terms used to describe atypical forms of diabetes, its definitionsclinical and biochemical characteristic. Study characteristics including date ofpublication, country and study method  wereextracted from individual studies. Terms identified that used the samedefinition and similar clinical characteristics were grouped in one phenotype.Narrative report was done by CB and reviewed by SM, ID, RM and SW.A total of 282 articles from PubMed and 68 from Scopus were identified. Most of the articles included (12 out of the 22) were published before 2000 asshown in In 1983, Mohan and collaborators used the term “tropical pancreaticdiabetes (TPD)” for diabetes mellitus occurring in individuals aged 15 to30 years who were underweight or wasted and required insulin but who did notexperience ketosis on discontinuation of insulin treatment; this phenotypeis also referred to as ketosis resistant diabetes ,17.Subs2) at diabetes onset, insulinrequirement from diagnosis for blood glucose control and absence of significantketonuria [The above phenotype under various terminologies was described in some studies asa subclass of malnutrition related diabetes (MRDM), the term recommended by WHOin 1985 ,24. Howeetonuria . MRDM haetonuria ,24. Diffetonuria ,21,22,25Ketosis prone diabetes (KPD) has been defined as a subgroup of TPD with theaddition of increased susceptibility to developing ketosis . HoweverFrom the above definitions numerous names were used for similar phenotypes whichcould collectively be described as either malnutrition related diabetes (MRDM)or ketosis prone diabetes whose characteristics are summarized in MRDM has been described under the following names: “tropical pancreaticdiabetes”, “chronic tropical pancreatic diabetes”,“fibro-calculus pancreatic diabetes”, “protein deficientpancreatic diabetes”, “J type diabetes” and “phasicinsulin dependent diabetes (PIDDM)”. Most of these publications werebefore 2000, apart from some papers from Ethiopia describing atypicalpresentation of insulin requiring diabetes . The comThe only study identified describing the prevalence of MRDM estimated that itoccurred in 6% of Indian diabetic patients ≤30 years old . The ageMRDM, as described in 1985 by the WHO, had two sub-types: protein deficientpancreatic diabetes (PDPD) and fibrocalculus pancreatic diabetes, with thelatter characterized by impairment of exocrine pancreas and changes on pancreasimaging such as calcification in the pancreas or fibrosis, biliary ductdilatation and decreased size and irregular surface of the pancreas ,22,23. VKey features of MRDM phenotype described above are in line with the1985 WHOdefinition: onset below the age of 30 years, living in poverty, lack of ketosisin presence of very high blood glucose, low BMI, relative presence of featuresof under nutrition, requirement of high dose of insulin to control bloodglucose, relative beta cell impairment with fibrocalcific pancreatic changes ina subset of patients.In contrast, ketosis prone diabetes differs from MRDM in age at onset, higher BMIand presence of ketosis at onset. The age at diagnosis of KPD is reported to belater (in 4th decade) compared to that in type 1 DM ,20,26 whThis systematic review identified heterogeneous studies describing characteristics ofpeople with atypical diabetes. Two main phenotypes have been identified that havebeen described using a variety of names. The first phenotype that we have identifiedas MRDM has some similar characteristics to the type 1 diabetes. It occurs in leanpeople living in poor socioeconomic conditions whose diagnosis is made at an olderage (third decade) than most people with type 1 diabetes, who have features of undernutrition, absence of beta cell antibodies, who may have pancreatic calcification onimaging and who do not develop ketosis. Characteristics of MRDM have been reportedmainly by a few historical clinic based studies with small sample sizes from LMICwith noticeable scarcity of data from Africa. The second phenotype, KPT2DM typicallyoccurs in normal weight or overweight people who present with unprovoked ketosis atonset but in whom insulin may be withdrawn and replaced with other diabetestreatments after some months of glucose stabilization and has been reported to occurin Asian countries and in people of African ancestry in developed countries.There is a scarcity of contemporary data on prevalence of MRDM. The existingliterature from clinics based research in urban tertiary settings suggests that itis rare . For exaIn order to ensure that appropriate treatment guidelines are being followed and tobuild responsive and appropriate health systems, it is important to understand thecontribution of different types of diabetes to a nation‘s overall diabetesburden. The situation is particularly challenging for health system planners inrapidly developing low- and middle-income countries (LMIC), who are alreadygrappling with an increasing prevalence of type 2 diabetes, driven by a substantialincrease in the prevalence of obesity and sedentary lifestyles.MRDM typically occurs in the 3rd decade of life, which is older than the age at whichtype 1 diabetes typically occurs. Childhood under nutrition might be one of thereasons why diabetes mellitus is increasing in the young population in LMIC. Forexample Shen and collaborators reported that pre-diabetes and diabetes were commonin young, lean South Asians in a multi-center population based study . FurtherLow body mass index (BMI) at onset was recorded to be frequent in MRDM and consideredas a criterion for MRDM diagnosis by some studies. However one study recorded thatbody mass index was similar in people diagnosed with MRDM and classic type 1 DM.This discrepancy might be due to different study populations at different stage ofdisease at diagnosis and underlying BMI distributions in populations. However lowBMI at onset in such poor patients may be due to many factors such as long durationof diabetes symptoms, as MRDM patients have been recorded to tolerate high bloodglucose, exocrine pancreatic failure with nutrient malabsorption leading to wastingor persistent malnutrition from childhood sustained by hunger and poverty. Althoughthere is a relationship between low body mass index and MRDM, there is a need toclarify whether MRDM contributes to weight loss.One study addressing MRDM highlighted the low binding of insulin to blood cells,which reflects insulin resistance and that is consistent with longitudinal evidencewhich supports the correlation between low birth weight and cardiovascular risksincluding insulin resistance known to cause diabetes . The patP < 0.05) as well as decreased insulin binding tored and white blood cells together with lack of insulin in the atypical phenotype[To the best of our knowledge, only one study has described complications of the MRDMphenotype: a case-control study with a small sample, reported higher mean level ofalbuminuria in that phenotype than in IDDM (Type 1) and NIDDM (type2)  compared to semi-urban andurban area . This suKetosis-prone type 2 diabetes includes features of both type 1 and type 2 diabetes.It resembles type 1 DM by the presence of ketosis at diagnosis; and type 2 DMin terms of later age and association with overweight at onset as well as preservedinsulin secretion. Ketosis-prone type 2 diabetes has been described in AfricanAmerican populations and African immigrants to Europe. However cases have also beenreported in European ancestry populations . Very feDiabetes classification has evolved continuously however its presentation isheterogeneous in LMICs where there is limited information available about lesscommon forms of diabetes. The lack of consensus on the naming and classification ofdiabetes occurring in abnormally thin young people in poor socioeconomic areas mayoverestimate type 1 DM prevalence and may result in inappropriate management of thesubset of individuals with atypical diabetes. Further translational research tocharacterize diabetes, using reported characteristics in populations living inpoverty with prevalent childhood malnutrition, is required.Ketosis-prone diabetes phenotype, “type 2 DM-like with unprovoked ketosis atonset”, appears to exist in association with modernization and urbanization.It is unclear whether this can be considered as a subtype of type 2 DM and managedaccordingly. A systematic review of evidence on the pathophysiology of thisphenotype and further studies on adequate sample size to identify potential reasonsto ketosis at diabetes onset and the duration of insulin requirement are required toprovide evidence for its management.


The primate cerebral cortex is broadly organized along hierarchical processing streams underpinned by corresponding variation in the brain’s microstructure and interareal connectivity patterns. Fulcher et al. recently demonstrated that a similar organization exists in the mouse cortex by combining independent datasets of cytoarchitecture, gene expression, cell densities, and long-range axonal connectivity. Using the T1w:T2w magnetic resonance imaging map as a common spatial reference for data-driven comparison of cortical gradients between mouse and human, we highlighted a common hierarchical expression pattern of numerous brain-related genes, providing new understanding of how systematic structural variation shapes functional specialization in mammalian brains. Reflecting on these findings, here we discuss how open neuroscience datasets, combined with advanced neuroinformatics approaches, will be crucial in the ongoing search for organization principles of brain structure. We explore the promises and challenges of integrative studies and argue that a tighter collaboration between experimental, statistical, and theoretical neuroscientists is needed to drive progress further. Comment on: Fulcher BD, Murray JD, Zerbi V, Wang XJ. Multimodal gradients across mouse cortex. Proc Natl Acad Sci U S A. 2019;116:4689-4695. doi: 10.1073/pnas.1814144116. PubMed PMID:30782826; PubMed Central PMCID:PMC6410879. https://www.ncbi.nlm.nih.gov/pubmed/3078282613 Progress in this direction requires closer collaboration between scientists with experimental, statistical, and theoretical modeling expertise. In this commentary, we highlight key results from our recent paper,4 framing them in the context of the modern neuroscience landscape.A core question in neuroscience is how the brain’s structure enables the rich cognitive function that defines our conscious experience. Clinically, such knowledge would provide a mechanistic basis from which to design treatments to minimize cognitive deficits in disease, or to enhance cognitive function in healthy individuals. Technologically, it may open the door to neuromimicry: brain-inspired design of new algorithmic frameworks for artificial intelligence. Recent unprecedented investments in neuroscience research have yielded large volumes of data ranging from intricate whole-brain maps of the microscopic properties of neural circuits to neural dynamics measured across a wide range of spatial and temporal scales. Despite this deluge of data, we have relatively few organizing principles to explain the patterns, make predictions, and design new experiments to test them.4 we assembled multiple datasets as spatial maps of the mouse cortex: (1) gene expression,5 (2) cell type densities,7 (3) axonal connectivity,8 (4) cytoarchitecture,9 and (5) magnetic resonance imaging (MRI) structural maps.10 We demonstrated a surprising commonality of spatial variation between these diverse measurements, which together ordered mouse brain areas along a putative functional hierarchy, from primary somatosensory through to transmodal prefrontal areas. Our integrative approach provides clues about the multifaceted structural underpinning of hierarchical functional specialization11 and allowed us to produce data-driven cortical organization schemes by representing each cortical area as a multimodal set of properties, such as that shown in In Fulcher et al.,5 and its associated informatics challenges,13 the culture of data sharing in neuroscience has become widespread. For computational and theoretical scientists, this provides new opportunities to leverage what are large financial investments in cutting-edge experimental protocols to discover new properties of brain organization, including those that extend across datasets that would not traditionally be compared. This line of research is not without substantial challenges: compared with prototypical collaborations of experimental and theoretical scientists, analyses of open data involve an unusual level of disconnect between the process of data collection and statistical analysis. This requires much greater care on the part of the data analyst to proactively ensure that their analyses respect the subtleties of the dataset and any limitations in its measurement. For example, recent work comparing studies analyzing transcriptional data from the Allen Human Brain Atlas14 found a high degree of inconsistency in how researchers subsequently processed the data and demonstrated that these choices can have a large impact on the final results and their interpretation.15Our study is an example of integrative research that bridges the specialized expertise of many individual laboratories through data sharing. Following pioneering large-scale experimental work by the Allen Institute16 studies of comparative anatomy (most commonly performed between different primate species) require a cortical parcellation mapped between species. Both the choice of parcellation and its mapping require subjective choices on the part of the researcher.18 In Fulcher et al.,4 we circumvented the need to define a mouse-human homology of cortical areas, instead using the noninvasive MRI contrast map, T1w:T2w, as a common spatial reference for interspecies comparison. As well as confirming the increase of T1w:T2w from dysgranular to eulaminate areas in mouse, as in primate and human,11 our results revealed a compelling consistency in the transcriptional patterning of brain-related genes relative to T1w:T2w. Mapping ortholog genes between mouse and human, we found significant mouse-human correspondence: Spearman ρ = 0.44 (P = 1 × 10−4), a result that was reproduced across a wide variety of gene sets: all genes, brain-expressed genes, astrocyte-enriched genes, neuron-enriched genes, and oligodendrocyte-enriched genes. As T1w:T2w is commonly interpreted as an MRI imaging marker for myelin content,19 interspecies correspondence may be expected on the basis of common variation of genes coding for myelin content. Whereas myelin-related gene expression did show interspecies correspondence, e.g., Mobp  and Mbp , the overall correlation was driven by functionally diverse genes, including the striking interspecies correspondences of N-methyl-D-aspartate (NMDA) receptor signaling genes such as Grin3a  and the interneuron marker Pvalb .Properties of brain organization that are shared across scales and species are strong candidates for providing an evolutionary advantage by enabling cost-efficient information processing. Although some such conserved properties are derived from abstractions of the brain as topological networks,The agreement is striking given (1) measurement noise in both datasets, (2) differences in measurement modalities between mouse (high-throughput in situ hybridization) and human (post-mortem microarray from 6 adults), and (3) vast differences in spatial scale. Although we developed new quality-control methods for the data, we note that the atlas-based expression measurements can be very noisy and that a noisy measurement in one species would manifest as a reduced correlation with T1w:T2w. In this respect, our data are again consistent with a meaningful interspecies correspondence: a high correlation in one species consistently matched the same direction in the other species. This mouse-human conservation of gene expression gradients suggests that common structural features may shape hierarchical functional specialization in both mammalian brains.20 This research has painted a consistent picture of the relative uniformity of the mouse brain compared with the highly differentiated primate cortex. In our study, correlations between T1w:T2w and other spatial maps were consistently weaker in mouse relative to macaque and human: for hierarchical level , cytoarchitectural type , and the leading principal component of gene transcription .11 In characterizing the most salient mouse-human differences in brain structure, we also flagged candidate genes for further investigation that show the most difference between mouse and human, including NMDA receptor signaling genes Grin2b/GRIN2B , Grin2d/GRIN2D , and Grin3b/GRIN3B .Whereas our results demonstrate a surprising consistency in how transcriptional gradients vary with T1w:T2w, they also provide insights into key differences in the degree of specialization in the mouse cortex. Previous comparative studies of mouse and primate have focused on quantifying microstructural variation between areas with the most disparate function, e.g., between V1 and frontal cortex in mouse and between V1 and lateral prefrontal cortex in rhesus monkey.5 with direct measures of interneuron cell densities6 allowed us to validate gene markers for interneuron cell types. We demonstrated that using gene expression data as a proxy for cell-type density can be highly accurate, as shown for the strongest correspondence we observed between the density of parvalbumin-containing interneurons6 and the independently measured expression of Pvalb5 across cortical areas in Vip gene expression was also strong, ρ = 0.76 (P = 3 × 10−7), but was much weaker for somatostatin (SST) cell density with Sst expression, ρ = 0.24 (P = 0.1). The lack of agreement for Sst may be due to a breakdown in the relationship between Sst expression and SST cell density: Sst expression decreases in adults and is regulated by neural activity.21 Our results suggest that the inference of cell density from gene expression atlases can be accurate, but a direct correspondence can be confounded by the many factors regulating gene transcription, as well as additional factors complicating straightforward inference of protein levels from mRNA.22 Continuing the search for cell types and their marker genes,24 and validating them against independent cell density measurements,7 will equip us with tools to infer the spatial distributions of cell types, including their contribution to psychiatric phenotypes.Bringing together measurements of gene expression25 that is commonly approximated as a set of discrete brain areas partitioned by hard boundaries. Our analysis brought together many open datasets of spatial maps reported at the level of the Allen Reference Atlas areas, but neuroscience datasets are now being measured and shared at cellular resolution.26 These data open the door to future research overcoming the limitations of discrete parcellations, and moving toward a continuous characterization of the fine-scale spatial patterning of the brain’s structural and functional properties. The next generation of brain atlases will likely contain a far more subtle representation of the brain’s spatial organization that takes into account multimodal information measured at each voxel,27 likely yielding a combination of continuous gradient-like variation28 as well as sudden changes consistent with interareal boundaries.At the macroscale, the brain is a continuous physical system1 Seen through this lens, the multimodal statistical relationships we present in Fulcher et al.4 will be important for constraining new mechanistic models of brain dynamics that bridge spatial scales of brain organization by capturing whole-brain activity dynamics in terms of putative physiological mechanisms. Whole-brain models, whether formulated physically  or phenomenologically , have overwhelmingly made the simplifying assumption of spatially uniform parameters when simulating brain dynamics. New models are beginning to incorporate inter-regional heterogeneity, allowing model parameters to vary with an empirical measurement such as spine count29 or T1w:T2w.30 Models including this simple 1-dimensional heterogeneity yield better out-of-sample fits to experimental functional-connectivity measurements and the hierarchical organization of intrinsic timescales,30 providing evidence that hierarchical variation in local circuits plays a key role in shaping cortical dynamics. Physiologically based models, as opposed to phenomenological models, stand to gain the most from the rich new neuroscience datasets, as their formulation and parameters can be directly constrained and verified against data.25Linking neuroscience big data to scientific understanding requires statistical analysis to uncover and characterize interesting patterns and theories that explain diverse observations in a coherent conceptual framework.4 that assemble many diverse datasets across multiple species will play a key role in uncovering the principles of how the brain’s organization enables a diverse functional repertoire.The modern landscape of neuroscience has been shaped by advances in neuroinformatics, data-sharing standards, and greater contributions from statistical and computational modeling in generating guiding hypotheses for new experiments. Integrative studies like Fulcher et al.


Expanding bile leaks after blunt liver trauma require more aggressive treatment than contained bile leaks. In this retrospective study approved by institution review board, we analyzed if non-invasive contrast-enhanced magnetic resonance cholangiography (CEMRC) using hepatocyte-specific contrast agent (gadoxetic acid disodium) could detect and characterize traumatic bile leaks. Between March 2012 and December 2014, written informed consents from 22 included patients  with a median age of 24.5 years  were obtained. Biliary tree visualization and bile leak detection on CEMRC acquired at 10, 20, 30, 90 minutes time points were independently graded by three radiologists on a 5-point Likert scale. Intraclass Correlation (ICC) was computed as estimates of interrater reliability. Accuracy was measured by area under receiver operating characteristic curves (AUROC). Biliary tree visualization was the best on CEMRC at 90 minutes (score 4.30) with excellent inter-rater reliability (ICC = 0.930). Of 22 CEMRC, 15 had bile leak . The largest AUROC of bile leak detection by three radiologists were 0.824, 0.914, 0.929 respectively on CEMRC at 90 minutes with ICC of 0.816. In conclusion, bile leaks of blunt liver trauma can be accurately detected and characterized on CEMRC. Its frequency has been reported to range from 3% in low grade liver trauma to about 15% in high grade trauma10. However, the frequency may have been underestimated because biliary injuries are initially asymptomatic and manifestation of symptoms is always delayed for weeks until biliary injuries are complicated with superinfection or biliary tree obstruction5. Expanding bile leaks associated with liver capsular rupture require more aggressive treatment as compared to contained bile leaks, similar to liver injuries with extracapsular vascular extravasation11. Therefore, complicated biliary injuries often require further diagnostic work up as well as complex therapeutic surgical or endoscopic treatment procedures14. These result in a higher medical cost, prolonged hospital stay or higher mortality rates in more severe cases14.Biliary injuries can occur at the time of blunt liver trauma16. However, the lack of anatomical details on planar images can sometimes misinterpret radioactive tracer in duodenum or normal anatomical variant of biliary tree as bile leaks15. Delayed imaging up to twenty four hours is required for biloma confirmation and that is not practical among trauma patients15. The site of biliary injuries and extent of bile leak can be directly evaluated with endoscopic retrograde cholangiography and percutaneous transhepatic cholangiography3. These diagnostic procedures are standard reference for biliary injuries and can also provide therapeutic intervention7. In addition, intrahepatic fluid accumulation can be aspirated and analyzed for bile content, or it can be contrasted by percutaneous cavitography for evaluation if the intrahepatic fluid communicates with biliary tree18. However, all these invasive procedures can pose additional risks to recently injured patients. Therefore, they are rarely the first-line diagnostic procedure for a suspected bile leak.Hepatobiliary iminodiacetic acid scan can serve as a test for bile leaks detection20. Extravasation of this biliary contrast medium from biliary tree to the intrahepatic or perihepatic biloma is readily detected and easily differentiated from other fluid collections21. Consequently, the gadoxetic acid disodium contrast-enhanced magnetic resonance cholangiography (CEMRC) can be used to evaluate hepatic excretory function and anatomy of biliary tree22. However, most of the published reports on CEMRC are focused on liver tumors and post-operative bile leak23. Little has been mentioned about its acquisition time and efficacy for blunt biliary injuries detection.With the advent of magnetic resonance imaging (MRI) and introduction of hepatocyte specific contrast agents, MRI has revolutionize hepatobiliary imaging. About 50% of the intravenously injected hepatocyte-specific MRI contrast agent such as gadoxetic acid disodium is excreted by hepatocytes into biliary system shortening the T1 effect of bile on MRITherefore, optimization of CEMRC for detecting and characterizing traumatic bile leaks was performed in this study. Our purpose is to analyze the diagnostic performance of CEMRC for traumatic bile leak and to compare different characteristics of bile leak (expanding or contained) with clinical parameters, specific treatment and complication.A total of 22 patients were included in this study. All had recent major blunt liver trauma ≥grade III injuries according to scoring scheme of American Association for the Surgery of Trauma (AAST). The initial contrast-enhanced CT showed focal traumatic distortion of liver architecture. Three patients had other associated abdominal organs trauma such as spleen (n = 2), kidney (n = 1), duodenum (n = 1) and pancreas (n = 1). The overall median injury severity score was 29 .Of 22 patients, 15 (68.2%) patients were determined to have bile leaks at expert-panel consensus decision. Among 15 of them, 7 were type I and 8 were type II bile leaks on CEMRC. Type I bile leak was confined, type II bile leak was expanding in the presence of liver capsule disruption. Additional examinations such as endoscopic retrograde cholangiography (n = 3), percutaneous cavitography and bile stained aspirates (n = 5) were available in this group. Of the other 7 patients who were determined to have no bile leak at expert-panel consensus decision, one was checked on by laparotomy to exclude biliary injury, two were confirmed by a negative result on percutaneous cavitography and aspirates analysis. Four others who recovered rapidly were clinically followed up. They did not develop any biliary complication and were cleared of biliary injury at panel decision.In Table The median time to perform CEMRC after blunt liver trauma for the group with a positive result of bile leak did not differ significantly from the group with a negative result , as well as for the group with type I bile leak from the group with type II bile leak .The scores on bile leak detection among three readers increased from CEMRC acquired at 10 minutes to CEMRC acquired at 90 minutes Fig. . The AURAs summarized in Table Among 22 patients, 8 underwent catheter drainage, 14 were treated with observation. The eight catheter drainage procedures included three endoscopic retrograde biliary drainage and five percutaneous catheter drainage. Of these 8 patients, seven  had bile leak. The other one patient who underwent catheter drainage did not have bile leak and the aspirate yielded old blood. Of the 14 patients treated with observation, eight  had bile leak.The difference of treatment choice between groups with and without bile leak was not significant. However, as shown in Table 24. However, other fluid collections of the liver are difficult to be differentiated from bile leak because all are hyperintense on T2-weighted images21.With the advent of magnetic resonance imaging, the diagnostic performance of T2-weighted magnetic resonance cholangiography which capitalizes on the long T2-relaxation property of bile is comparable to that of endoscopic retrograde cholangiography for biliary cancer, biliary stone and biliary anomaly27 although the acquisition can range from 20 to 180 minutes25. The best acquisition time of CEMRC using Gd-EOB-DTPA among trauma patients has never been investigated. Our main concern in this study is to optimize the acquisition time and expedite the examination for trauma patients. We found that CEMRC of 30-minute and 90-minutes acquisitions have higher scores of biliary tree visualization on a 5-point scale, as compared to CEMRC of 10-minute and 20-minute acquisitions. Furthermore, the reliability test of visualization scores among three radiologists was the best on CEMRC of 90-minute acquisition.On the contrary, by using gadoxetic acid disodium as the contrast agent, CEMRC can easily identify the T1-shortening bile leak. It has been reported that CEMRC acquired 20 minutes after administration of Gd-EOB-DTPA is sufficient for obtaining hepatobiliary phase imagesOur study showed that bile leak was also best detected on CEMRC acquired at 90-minute with the highest AUROC. The diagnostic yield for bile leak is better when the biliary tree is more adequately filled up with excreted contrast medium. Similarly, depiction of bile leak was also the best reliable with a very good ICC on CEMRC acquired at 90-minutes among three radiologists.28, our results showed that bile leak was significantly associated with active liver hemorrhage on arrival CT. The severe lacerations of major liver trauma not only injure the hepatic vessels, its shearing force can also disrupt bile ducts and portal veins at the portal triad. Active arterial bleeding or pseudoaneurysm is often treated by angioembolization31 for patients who are transiently responsive to fluid resuscitation. We have found that angioembolization is associated with bile leak. This is because angioembolization may compromise the vascular supply to the walls of bile ducts, causing a possibility of local epithelial ischemia and delayed healing at the site of biliary injuries, therefore resulting in delayed bile leak28.Not only a high risk of biliary injuries can occur in patients with major liver traumaTraumatic biliary injuries with type I bile leak can resolve spontaneously. In contrast, the type II bile leak is associated with a higher level of total serum bilirubin and catheter drainage. Even though one patient with expanding bile leak underwent endoscopic retrograde biliary stenting and percutaneous transhepatic biliary drainage, he still developed biliary stricture. Moreover, patients with type II bile leak usually have a longer length of hospital stay than those with type I bile leak.25 which is mediated by the same transporter system of bilirubin transport.Biliary injury is often underestimated because the diagnostic tools are too invasive for asymptomatic patients. We have proven that non-invasive CEMRC is feasible to detect and characterize bile leak in trauma patients. However, good quality CEMRC depends on liver excretion function of Gd-EOB-DTPAIn this study, we only included patients with major blunt liver trauma who were co-operative and compliant with the MRI safety requirements. The implanted cardiac devices or embedded metallic foreign bodies after trauma can prohibit patients from undergoing CEMRC. Small sample size is therefore the main limitation of this study. The second limitation is that we categorized patients into groups with and without bile leak by expert-panel consensus because verification by the ideal endoscopic retrograde cholangiography is too invasive for asymptomatic patients. The third limitation is that we did not have patients with very high serum level of bilirubin. The total bilirubin level of our patients ranged from 0.50 to 3.60 mg/dL. We do not know whether CEMRC acquired at 90-minute is still optimal for bile leak detection if bilirubin level exceeds 3.60 mg/dL. The fourth limitation is that we did not acquire CEMRC at longer than 90 minutes time points because MRI scheduling is always tight and we cannot afford to decrease MRI throughput per day. Fortunately, excretion of Gd-EOB-DTPA can be visualized on CEMRC acquired at 90-minutes in all patients in this study. We did not investigate whether or not CEMRC at 60 minutes time point would have yielded a similar results as 90 minutes. This has left us an opportunity for clarification in the next study.In conclusion, CEMRC is feasible to provide both anatomical and functional information for traumatic bile leaks among major liver trauma patients with a history of active liver bleeder treated by angioembolization. Bile leak is most optimally detected on CEMRC acquired at 90 minutes time point after intravenous injection of Gd-EOB-DTPA. Blunt liver trauma patients with type II (expanding) bile leak are associated with higher level of total serum bilirubin, more likely to undergo catheter drainage and a longer length of hospital stay than patients with type I (contained) bile leak.This retrospective study on CEMRC for biliary injuries was approved by Chang Gung Memorial Hospital’s institution review board and all methods were performed in accordance with the relevant guidelines (RE: 99–3856B). A written informed consent was obtained from every participant. From March 2012 to December 2014, a total of 25 consecutive patients met the inclusion criteria . Among them  after blunt liver trauma at the time when the patients had been evaluated by attending surgeons as stable to be transferred to MRI scanning room. All examinations were performed with a 3 T MR system . Each patient received intravenous bolus injection of 25 μmol/kg of body weight of gadoxetic acid disodium  at a flow rate of 2 mL/sec, followed by 10 mL saline flush. Images of CEMRC in axial plane were acquired at 10, 20, 30 and 90 minutes time points respectively using three dimensional gradient-echo (GRE) T1-weighted volume interpolated breath-hold examination (VIBE) technique. Coronal plane images of T1-weighted VIBE were acquired at 30 and 90 minutes. Besides CEMRC, axial images of T1-weighted VIBE at arterial phase, portal venous phase and equilibrium phase as well as axial T2-weighted images with and without fat saturation were also obtained. The imaging techniques was summarized in Supplementary Table The CEMRC of different acquisition times were separated into different sets of images. Each set of images was randomly given a record number without chronological order and transferred to a picture archiving and communication system for analysis. Three radiologists  who did not have access to the entire dataset were also blinded to the clinical course and final diagnosis. In a research meeting, these three radiologists were briefed on the properties of gadoxetic acid disodium, scoring scheme of biliary tree visualization and imaging features of bile leak on CEMRC.They were asked to independently score biliary tree visualization as well as presence or absence of bile leak. The scores of biliary tree visualization was recorded on a 5-point Likert scale . Bile leak on CEMRC was defined as extravasation of liver-excreted contrast medium from bile duct. The detection of bile leak was recorded independently on a five-point Likert scale .The final decision on whether bile leak was present or absent was determined by an expert-panel who had full knowledge of all CEMRC, available additional examinations, laboratory data and clinical information of the patients. This expert-panel comprised two abdominal radiologists and one trauma surgeon who had more than 20 years of experience in trauma radiology. None of them was involved or had influence over the scoring of biliary tree visualization and bile leak detection. All data were studied by this expert-panel group to reach a consensus of whether or not bile leak was present or absent. Characterization of the bile leak based on morphology of extravasated biliary contrast medium was further categorized by the expert-panel. The bile leak was characterized as type I if it was confined, and characterized as type II if it was expanding in the presence of liver capsule disruption.All patients were followed up for a median of 19.8 months .Their admission CT reports were reviewed for AAST grading of liver trauma and active extravasation of vascular iodinated contrast medium. Their admission notes were reviewed for serum liver enzymes and bilirubin levels on arrival as well as initial treatment for liver trauma (observation or angioembolization). Their medical records were reviewed for the choice of specific treatment for biliary injuries, biliary-related complications and the length of hospital stay.Statistical analyses were computed with a software package (IBM SPSS Statistics version 21). Intraclass Correlation Coefficient (ICC) of scores on biliary tree visualization and bile leak detection were computed to estimate interrater reliability. It was categorized as poor (ICC < 0.5), fair (ICC of 0.5–0.7), good (ICC of 0.7–0.8), very good (ICC of 0.8–0.9) or excellent (ICC of 0.9–1.0). Accuracy of bile leak detection was measured by the area under receiver operating characteristic curve (AUROC). Differences of data between the groups of (1) bile leak versus no bile leak, and (2) type I versus type II bile leak were compared. Categorical data were compared using Fisher’s exact test. Continuous data were compared with Mann-Whitney test. The differences were considered significant if the 2-tailed p-values were less than 0.050.Figure S1Table S1