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Background: Type 2 diabetes (T2D) is an independent risk factor for the development of nonalcoholic steatohepatitis (NASH. However, there is a need for non-invasive tests (NIT) to identify at-risk NASH patients requiring intense lifestyle or clinical intervention. Aim: The aim of this study was to access clinical performance metrics of NIS4™ either alone or in combination with other NITs in patients characterized as nondiabetic, pre-diabetic and T2D and with either at-risk of NASH (NAS≥4 and F≥2) or advanced fibrosis (F≥3). Methods: Patients in the RESOLVE-IT DIAG cohort presented with one or more risk factors for NAFLD and had corresponding NIT data: NIS4™ (miR-34a-5p, α2M, YKL-40, HbA1c), Fibrosis-4 (FIB-4), NAFLD activity score (NFS), and FibroScan™ (FS). NIT data were used to simulate different testing strategies to identify patients with at-risk NASH or advanced fibrosis. Results: The 467 patients were divided into 3 subgroups with 200 (43%) non-T2D, 67 (14%) pre-diabetic and 200 (43%) T2D with a prevalence for at-risk NASH ranging 42-71%, 58-78% and 66-84%, respectively. The corresponding prevalence for advanced fibrosis in the same groups was 18-27%, 34-40%, 42-49%, respectively. Generally, the performance was impacted by variability in sample size and prevalence which mainly affected PPV and NPV. Under these circumstance NIS4™ together with FS or FIB-4 provided a strong overall balanced performance (Acc: 66-82%, Sen: 50-80%, Spe: 58-94%). Conclusion: In this cohort, the sequential use of NIS4™ together with another blood-based test FIB-4 or an imaging-based FibroScan™ test provided an overall more balanced performance for identification of at-risk NASH or advanced fibrosis for nondiabetic and T2D patients. Therefore, NIS4™ has the potential to be a valuable clinical tool for also identifying diabetic patients at higher risk of negative outcomes. Disclosure C. Rosenquist: Employee; Self; GENFIT. Y. Hajji: None. J. Magnanensi: Employee; Self; GENFIT. N. Stankovic-valentin: Employee; Self; GENFIT, Stock/Shareholder; Self; GENFIT. S. Hosmane: Stock/Shareholder; Self; GENFIT, Stock/Shareholder; Spouse/Partner; Vertex Pharmaceuticals Incorporated. A. J. Sanyal: Advisory Panel; Self; Novo Nordisk, Consultant; Self; Akero Therapeutics, Inc., Alnylam Pharmaceuticals, Inc., Amgen Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Bristol-Myers Squibb Company, Genentech, Inc., Gilead Sciences, Inc., Intercept Pharmaceuticals, Inc., Inventiva Pharma, Lilly Diabetes, Lipocine Inc., Merck & Co., Inc., NGM Biopharmaceuticals, Novartis Pharmaceuticals Corporation, Pfizer Inc., Regeneron Genetics Center, Siemens Corporation, Other Relationship; Self; AstraZeneca, UpToDate, Stock/Shareholder; Self; GENFIT, HemoShear Therapeutics, LLC, Indalo Therapeutics, Inc., Tiziana Life Sciences plc.

Summary Background Non-invasive tests that can identify patients with non-alcoholic steatohepatitis (NASH) at higher risk of disease progression are lacking. We report the development and validation of a blood-based diagnostic test to non-invasively rule in and rule out at-risk NASH (defined as non-alcoholic fatty liver disease [NAFLD] activity score [NAS] ≥4 and fibrosis stage ≥2). Methods In this prospective derivation and global validation study, blood samples, clinical data, and liver biopsy results from three independent cohorts with suspected NAFLD were used to develop and validate a non-invasive blood-based diagnostic test, called NIS4. Derivation was done in the discovery cohort, which comprised 239 prospectively recruited patients with biopsy-confirmed NASH (NAFLD NAS ≥3; fibrosis stage 0–3) from the international GOLDEN-505 phase 2b clinical trial. A complete matrix based on 23 variables selected for univariate association with the presence of at-risk NASH and avoiding high multi-collinearity was used to derive the model in a bootstrap-based process that minimised the Akaike information criterion. The overall diagnostic performance of NIS4 was externally validated in two independent cohorts: RESOLVE-IT diag and Angers. The RESOLVE-IT diag cohort comprised the first 475 patients screened for potential inclusion into the RESOLVE-IT phase 3 clinical trial. Angers was a retrospective cohort of 227 prospectively recruited patients with suspected NAFLD and clinical risk factors for NASH or fibrosis stage 2 or more according to abnormal elastography results or abnormal liver biochemistry. Both external validation cohorts were independently analysed and were combined into a pooled validation cohort (n=702) to assess clinical performance of NIS4 and other non-invasive tests. Findings The derived NIS4 algorithm comprised four independent NASH-associated biomarkers (miR-34a-5p, alpha-2 macroglobulin, YKL-40, and glycated haemoglobin; area under the receiver operating characteristics curve [AUROC] 0·80, 95% CI 0·73–0·85), and did not require adjustment for age, sex, body-mass index (BMI), or aminotransferase concentrations. Clinical cutoffs were established within the discovery cohort to optimise both rule out and rule in clinical performance while minimising indeterminate results. NIS4 was validated in the RESOLVE-IT diag cohort (AUROC 0·83, 95% CI 0·79–0·86) and the Angers cohort (0·76, 0·69–0·82). In the pooled validation cohort, patients with a NIS4 value less than 0·36 were classified as not having at-risk NASH (ruled out) with 81·5% (95% CI 76·9–85·3) sensitivity, 63·0% (57·8–68·0) specificity, and a negative predictive value of 77·9% (72·5–82·4), whereas those with a NIS4 value of more than 0·63 were classified as having at-risk NASH (ruled in) with 87·1% (83·1–90·3) specificity, 50·7% (45·3–56·1) sensitivity, and a positive predictive value of 79·2% (73·1–84·2). The diagnostic performance of NIS4 within the external validation cohorts was not influenced by age, sex, BMI, or aminotransferase concentrations. Interpretation NIS4 is a novel blood-based diagnostic that provides an effective way to non-invasively rule in or rule out at-risk NASH in patients with metabolic risk factors and suspected disease. Use of NIS4 in clinical trials or in the clinic has the potential to greatly reduce unnecessary liver biopsies in patients with lower risk of disease progression. Funding Genfit.

Background and Aim: T2D is an independent risk factor for the development of nonalcoholic steatohepatitis (NASH). Amongst NASH patients, those with elevated disease activity (NAS≥4) and fibrosis (F≥2) upon scoring of a liver biopsy are at higher risk of progressing to negative clinical outcomes. There is a lack of diagnostic tools to identify these “at-risk𠇍 NASH patients that need clinical intervention. Here we compare the ability of NIS4, a novel blood test, to other available tests for detection of “at-risk” NASH in a T2D population. Methods: The blood and liver biopsy samples from 714 patients (446 non-T2D + 268 T2D) with metabolic risk factors for NASH were used. Disease activity (NAS) and fibrosis stage (F) were evaluated by a single expert pathologist. “At-risk” NASH was defined as NASH with NAS≥4 and F≥2. The diagnostic performance of NIS4 to identify patients with “at-risk” NASH in T2D subpopulation was assessed through ROC analysis and compared versus existing tests and statistical comparisons were done by DeLong testing. Results: In the total cohort (T2D prevalence = 38%), AUROC of NIS4 was 0.83 [95% CI: 0.80 - 0.86] for identifying “at-risk” NASH. The prevalence of “at-risk” NASH was higher in T2D (61%) vs. non-T2D (45%). AUROC was comparable in T2D and non-T2D: 0.80 [0.75 - 0.85] vs. 0.83 [0.80 - 0.87]. In T2D, a head to head comparison (AUROC; 95% CI) showed that NIS4 [0.80; 0.75-0.85] significantly outperformed all other tests for the identification of “at-risk” NASH: APRI [0.74; 0.68-0.80], FIB4 [0.70; 0.64-0.77], ELF [0.70; 0.64-0.77], FibroTest [0.68; 0.61-0.74], and NFS [0.60; 0.52-0.67]. Conclusion: Amongst T2D patients with suspected NASH, the prevalence of “at-risk” NASH is high. This highlights the need for active surveillance amongst T2D patients. For that purpose, NIS4 outperforms other tests and is a promising non-invasive tool to identify and potentially track T2D patients who need clinical intervention to manage their disease. Disclosure B. Staels: None. V. Ratziu: Other Relationship; Self; Gilead Sciences, Inc. S. Francque: Consultant; Self; GENFIT. S.A. Harrison: Advisory Panel; Self; Gilead Sciences, Inc. P. Bedossa: Consultant; Self; GENFIT. A. Roudot: Employee; Self; GENFIT. Z. Majd: Employee; Self; GENFIT. J. Brozek: Employee; Self; GENFIT. F. Ben-Sudrik: Employee; Self; GENFIT. P. Birman: Employee; Self; GENFIT. D.W. Hum: Employee; Self; GENFIT. S. Hosmane: Employee; Self; GENFIT. P. Chaumat: Employee; Self; GENFIT. R. Hanf: Employee; Self; GENFIT. B. Cariou: Board Member; Self; Novo Nordisk A/S, Regeneron Pharmaceuticals. Consultant; Self; GENFIT, Sanofi-Aventis. Research Support; Self; Amgen Inc., Pfizer Inc. Speaker’s Bureau; Self; Abbott, Akcea Therapeutics, Merck Sharp & Dohme Corp. A. Sanyal: Consultant; Self; Ardelyx, Boehringer Ingelheim Pharmaceuticals, Inc., Exhalenz, Gilead Sciences, Inc., Hemoshear, Intercept Pharmaceuticals, Inc., Lilly, Mallinckrodt Pharmaceuticals, Nimbus Therapeutics, Nitto Denko, Novartis Pharmaceuticals Corporation, Pfizer Inc. Employee; Self; Sanyal Bio. Research Support; Self; Bristol-Myers Squibb Company, Conatus Pharmaceuticals Inc., Echosens, Galectin Therapeutics Inc., Immuron Ltd, Merck & Co., Inc., Salix Pharmaceuticals, Sequanna. Stock/Shareholder; Self; Akarna Therapeutics, GENFIT, Natural Shield, NewCo LLC, Tiziana. Other Relationship; Self; Elsevier, UpToDate.

Microglia are rapidly activated in the central nervous system (CNS) in response to a variety of injuries, including inflammation, trauma, and stroke. In addition to modulation of the innate immune response, a key function of microglia is the phagocytosis of dying cells and cellular debris, which can facilitate recovery. Despite emerging evidence that axonal debris can pose a barrier to regeneration of new axons in the CNS, little is known of the cellular and molecular mechanisms that underlie clearance of degenerating CNS axons. We utilize a custom micropatterned microfluidic system that enables robust microglial-axon co-culture to explore the role of Toll-like receptors (TLRs) in microglial phagocytosis of degenerating axons. We find that pharmacologic and genetic disruption of TLR4 blocks induction of the Type-1 interferon response and inhibits phagocytosis of axon debris in vitro. Moreover, TLR4-dependent microglial clearance of unmyelinated axon debris facilitates axon outgrowth. In vivo, microglial phagocytosis of CNS axons undergoing Wallerian degeneration in a dorsal root axotomy model is impaired in adult mice in which TLR4 has been deleted. Since purinergic receptors can influence TLR4-mediated signaling, we also explored a role for the microglia P2 receptors and found that the P2X7R contributes to microglial clearance of degenerating axons. Overall, we identify TLR4 as a key player in axonal debris clearance by microglia, thus creating a more permissive environment for axonal outgrowth. Our findings have significant implications for the development of protective and regenerative strategies for the many inflammatory, traumatic, and neurodegenerative conditions characterized by CNS axon degeneration.

Microglia are rapidly activated in the central nervous system (CNS) in response to a variety of injuries, including inflammation, trauma, and stroke. In addition to modulation of the innate immune response, a key function of microglia is the phagocytosis of dying cells and cellular debris, which can facilitate recovery. Despite emerging evidence that axonal debris can pose a barrier to regeneration of new axons in the CNS, little is known of the cellular and molecular mechanisms that underlie clearance of degenerating CNS axons. We utilize a custom micropatterned microfluidic system that enables robust microglial-axon co-culture to explore the role of Toll-like receptors (TLRs) in microglial phagocytosis of degenerating axons. We find that pharmacologic and genetic disruption of TLR4 blocks induction of the Type-1 interferon response and inhibits phagocytosis of axon debris in vitro. Moreover, TLR4-dependent microglial clearance of unmyelinated axon debris facilitates axon outgrowth. In vivo, microglial phagocytosis of CNS axons undergoing Wallerian degeneration in a dorsal root axotomy model is impaired in adult mice in which TLR4 has been deleted. Since purinergic receptors can influence TLR4-mediated signaling, we also explored a role for the microglia P2 receptors and found that the P2X7R contributes to microglial clearance of degenerating axons. Overall, we identify TLR4 as a key player in axonal debris clearance by microglia, thus creating a more permissive environment for axonal outgrowth. Our findings have significant implications for the development of protective and regenerative strategies for the many inflammatory, traumatic, and neurodegenerative conditions characterized by CNS axon degeneration.

The “carbons apart” tetracarbon carborane nido-2,6-(R)2-4,12-(SiMe3)2-2,4,6,12-C4B8H8 (R = SiMe3 (I), n-butyl (II)) and several of its B-alkylated derivatives react with Mg metal in THF solvent to produce magnesacarboranes (IV−VI and XI) in yields ranging from 57% to 74%. The magnesacarboranes were characterized by chemical analysis and infrared and 1H, 11B, and 13C NMR spectroscopy and by single-crystal X-ray diffraction. Two types of cages were found, one in (THF)2Mg(SiMe3)4(B-Me)C4B7H7 (IV) and the other in (L)2Mg(SiMe3)2(R)2(B-Y)C4B7H7 (L = THF, R = SiMe3, Y = t-Bu (V); L = THF, R = SiMe3, Y = H (VI); (L)2 = TMEDA, R = n-Bu, Y = H (XI)). Both cages showed the presence of electron-precise C and B atoms, as well as electron-deficient fragments. Approximate density functional ab initio molecular orbital calculations showed that the dianionic C4B8 cage can exist in a number of energy-equivalent isomeric forms that can be trapped by a metal ion such as Mg. The reactions of I with the group 1 metals followe...

The sodium/lithium compounds closo-exo-Li(THF)-1-Na(THF)-2-(SiMe3)-3-(R)-2,3-C2B4H4 (R = SiMe3, Me, H), were found to react with several different hafnium reagents to produce half- and full-sandwich and mixed-ligand chlorohafnacarboranes in good yields. The reaction of the sodium/lithium compounds with HfCl4 in 2:1 molar ratios produced the full-sandwich complexes 1-Cl-1-(THF)-2,2‘-(SiMe3)2-3,3‘-(R)2-4‘,5,5‘,6-Li(THF)n-[1,1‘-commo-Hf(2,3-C2B4H4)2] (R = SiMe3, n = 2 (I); R = Me, n = 1 (II); R = H, n = 1 (III)) in yields of 77%, 65%, and 71%, respectively. When R = SiMe3, a 1:1 molar ratio of carborane to HfCl4 gave the half-sandwich species [Li(THF)2][1,1,1-(Cl)3-closo-1-Hf-2,3-(SiMe3)2-2,3-C2B4H4] (V) in 77% yield. On the other hand, both 2:1 and 1:1 reaction ratios of carborane (R = Me) to Cp*HfCl3 gave only the mixed-ligand sandwich compound 1,1‘-(Cl)2-2-(SiMe3)-3-(Me)-4,5-Li(THF)-1,1‘-commo-Hf[(η5-C5Me5)(η5-2,3-C2B4H4)] (IV) in 82% yield. All compounds were characterized by infrared spectroscopy, 1H, 1...

We describe a compartmentalized circular microfluidic platform that enables directed cell placement within defined microenvironments for the study of axon–glia interactions. The multi-compartment platform consists of independent units of radial microchannel arrays that fluidically isolate somal from axonal compartments. Fluidic access ports punched near the microchannels allow for direct pipetting of cells into the device. Adjacent somal or axonal compartments can be readily merged so that independent groups of neurons or axons can be maintained in either separate or uniform microenvironments. We demonstrate three distinct modes of directed cell placement in this device, to suit varying experimental needs for the study of axon–glia interactions: (1) centrifugation of the circular platform can result in a two-fold increase in axonal throughput in microchannels and provides a new technique to establish axon–glia interactions; (2) microstencils can be utilized to directly place glial cells within areas of interest; and (3) intimate axon–glia co-culture can be attained via standard pipetting techniques. We take advantage of this microfluidic platform to demonstrate a two-fold preferential accumulation of microglia specifically near injured CNS axons, an event implicated in the maintenance and progression of a number of chronic neuroinflammatory and neurodegenerative diseases.

Chemotherapy induced peripheral neuropathy is a common and dose-limiting side effect of anticancer drugs. Studies aimed at understanding the underlying mechanism of neurotoxicity of chemotherapeutic drugs have been hampered by lack of suitable culture systems that can differentiate between neuronal cell body, axon or associated glial cells. Here, we have developed an in vitro compartmentalized microfluidic culture system to examine the site of toxicity of chemotherapeutic drugs. To test the culture platform, we used paclitaxel, a widely used anticancer drug for breast cancer, because it causes sensory polyneuropathy in a large proportion of patients and there is no effective treatment. In previous in vitro studies, paclitaxel induced distal axonal degeneration but it was unclear if this was due to direct toxicity on the axon or a consequence of toxicity on the neuronal cell body. Using microfluidic channels that allow compartmentalized culturing of neurons and axons, we demonstrate that the axons are much more susceptible to toxic effects of paclitaxel. When paclitaxel was applied to the axonal side, there was clear degeneration of axons; but when paclitaxel was applied to the soma side, there was no change in axon length. Furthermore, we show that recombinant human erythropoietin, which had been shown to be neuroprotective against paclitaxel neurotoxicity, provides neuroprotection whether it is applied to the cell body or the axons directly. This observation has implications for development of neuroprotective drugs for chemotherapy induced peripheral neuropathies as dorsal root ganglia do not possess blood-nerve-barrier, eliminating one of the cardinal requirements of drug development for the nervous system. This compartmentalized microfluidic culture system can be used for studies aimed at understanding axon degeneration, neuroprotection and development of the nervous system.

We describe a novel valve-based microfluidic axon injury micro-compression (AIM) platform that enables focal and graded compression of micron-scale segments of single central nervous system (CNS) axons. The device utilizes independently controlled "push-down" injury pads that descend upon pressure application and contact underlying axonal processes. Regulated compressed gas is input into the AIM system and pressure levels are modulated to specify the level of injury. Finite element modeling (FEM) is used to quantitatively characterize device performance and parameterize the extent of axonal injury by estimating the forces applied between the injury pad and glass substrate. In doing so, injuries are normalized across experiments to overcome small variations in device geometry. The AIM platform permits, for the first time, observation of axon deformation prior to, during, and immediately after focal mechanical injury. Single axons acutely compressed (~5 s) under varying compressive loads (0-250 kPa) were observed through phase time-lapse microscopy for up to 12 h post injury. Under mild injury conditions (55 kPa) ~73% of axons continued to grow, while at moderate (55-95 kPa) levels of injury, the number of growing axons dramatically reduced to 8%. At severe levels of injury (95 kPa), virtually all axons were instantaneously transected and nearly half (~46%) of these axons were able to regrow within the imaging period in the absence of exogenous stimulating factors.