Hepatic NADH reductive stress underlies common variation in metabolic traits.

The cellular NADH/NAD+ ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we report the in vivo application of Lactobacillus brevis (Lb)NOX1, a bacterial water-forming NADH oxidase, to assess the metabolic consequences of directly lowering the hepatic cytosolic NADH/NAD+ ratio in mice. By combining this genetic tool with metabolomics, we identify circulating α-hydroxybutyrate levels as a robust marker of an elevated hepatic cytosolic NADH/NAD+ ratio, also known as reductive stress. In humans, elevations in circulating α-hydroxybutyrate levels have previously been associated with impaired glucose tolerance2, insulin resistance3 and mitochondrial disease4, and are associated with a common genetic variant in GCKR5, which has previously been associated with many seemingly disparate metabolic traits. Using LbNOX, we demonstrate that NADH reductive stress mediates the effects of GCKR variation on many metabolic traits, including circulating triglyceride levels, glucose tolerance and FGF21 levels. Our work identifies an elevated hepatic NADH/NAD+ ratio as a latent metabolic parameter that is shaped by human genetic variation and contributes causally to key metabolic traits and diseases. Moreover, it underscores the utility of genetic tools such as LbNOX to empower studies of 'causal metabolism'.

Hepatic gap junctions amplify alcohol liver injury by propagating cGAS-mediated IRF3 activation.

Alcohol-related liver disease (ALD) accounts for the majority of cirrhosis and liver-related deaths worldwide. Activation of IFN-regulatory factor (IRF3) initiates alcohol-induced hepatocyte apoptosis, which fuels a robust secondary inflammatory response that drives ALD. The dominant molecular mechanism by which alcohol activates IRF3 and the pathways that amplify inflammatory signals in ALD remains unknown. Here we show that cytoplasmic sensor cyclic guanosine monophosphate-adenosine monophosphate (AMP) synthase (cGAS) drives IRF3 activation in both alcohol-injured hepatocytes and the neighboring parenchyma via a gap junction intercellular communication pathway. Hepatic RNA-seq analysis of patients with a wide spectrum of ALD revealed that expression of the cGAS-IRF3 pathway correlated positively with disease severity. Alcohol-fed mice demonstrated increased hepatic expression of the cGAS-IRF3 pathway. Mice genetically deficient in cGAS and IRF3 were protected against ALD. Ablation of cGAS in hepatocytes only phenocopied this hepatoprotection, highlighting the critical role of hepatocytes in fueling the cGAS-IRF3 response to alcohol. We identified connexin 32 (Cx32), the predominant hepatic gap junction, as a critical regulator of spreading cGAS-driven IRF3 activation through the liver parenchyma. Disruption of Cx32 in ALD impaired IRF3-stimulated gene expression, resulting in decreased hepatic injury despite an increase in hepatic steatosis. Taken together, these results identify cGAS and Cx32 as key factors in ALD pathogenesis and as potential therapeutic targets for hepatoprotection.

ADAMTSL2 protein and a soluble biomarker signature identify at-risk non-alcoholic steatohepatitis and fibrosis in adults with NAFLD.

BACKGROUND & AIMS: Identifying fibrosis in non-alcoholic fatty liver disease (NAFLD) is essential to predict liver-related outcomes and guide treatment decisions. A protein-based signature of fibrosis could serve as a valuable, non-invasive diagnostic tool. This study sought to identify circulating proteins associated with fibrosis in NAFLD. METHODS: We used aptamer-based proteomics to measure 4,783 proteins in 2 cohorts (Cohort A and B). Targeted, quantitative assays coupling aptamer-based protein pull down and mass spectrometry (SPMS) validated the profiling results in a bariatric and NAFLD cohort (Cohort C and D, respectively). Generalized linear modeling-logistic regression assessed the ability of candidate proteins to classify fibrosis. RESULTS: From the multiplex profiling, 16 proteins differed significantly by fibrosis in cohorts A (n = 62) and B (n = 98). Quantitative and robust SPMS assays were developed for 8 proteins and validated in Cohorts C (n = 71) and D (n = 84). The A disintegrin and metalloproteinase with thrombospondin motifs like 2 (ADAMTSL2) protein accurately distinguished non-alcoholic fatty liver (NAFL)/non-alcoholic steatohepatitis (NASH) with fibrosis stage 0-1 (F0-1) from at-risk NASH with fibrosis stage 2-4, with AUROCs of 0.83 and 0.86 in Cohorts C and D, respectively, and from NASH with significant fibrosis (F2-3), with AUROCs of 0.80 and 0.83 in Cohorts C and D, respectively. An 8-protein panel distinguished NAFL/NASH F0-1 from at-risk NASH (AUROCs 0.90 and 0.87 in Cohort C and D, respectively) and NASH F2-3 (AUROCs 0.89 and 0.83 in Cohorts C and D, respectively). The 8-protein panel and ADAMTSL2 protein had superior performance to the NAFLD fibrosis score and fibrosis-4 score. CONCLUSION: The ADAMTSL2 protein and an 8-protein soluble biomarker panel are highly associated with at-risk NASH and significant fibrosis; they exhibited superior diagnostic performance compared to standard of care fibrosis scores. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of liver disease worldwide. Diagnosing NAFLD and identifying fibrosis (scarring of the liver) currently requires a liver biopsy. Our study identified novel proteins found in the blood which may identify fibrosis without the need for a liver biopsy.

Targeting clinical epigenetic reprogramming for chemoprevention of metabolic and viral hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is the fastest-growing cause of cancer-related mortality with chronic viral hepatitis and non-alcoholic steatohepatitis (NASH) as major aetiologies. Treatment options for HCC are unsatisfactory and chemopreventive approaches are absent. Chronic hepatitis C (CHC) results in epigenetic alterations driving HCC risk and persisting following cure. Here, we aimed to investigate epigenetic modifications as targets for liver cancer chemoprevention. DESIGN: Liver tissues from patients with NASH and CHC were analysed by ChIP-Seq (H3K27ac) and RNA-Seq. The liver disease-specific epigenetic and transcriptional reprogramming in patients was modelled in a liver cell culture system. Perturbation studies combined with a targeted small molecule screen followed by in vivo and ex vivo validation were used to identify chromatin modifiers and readers for HCC chemoprevention. RESULTS: In patients, CHC and NASH share similar epigenetic and transcriptomic modifications driving cancer risk. Using a cell-based system modelling epigenetic modifications in patients, we identified chromatin readers as targets to revert liver gene transcription driving clinical HCC risk. Proof-of-concept studies in a NASH-HCC mouse model showed that the pharmacological inhibition of chromatin reader bromodomain 4 inhibited liver disease progression and hepatocarcinogenesis by restoring transcriptional reprogramming of the genes that were epigenetically altered in patients. CONCLUSION: Our results unravel the functional relevance of metabolic and virus-induced epigenetic alterations for pathogenesis of HCC development and identify chromatin readers as targets for chemoprevention in patients with chronic liver diseases.

Successful use of azithromycin for Escherichia coli-associated renal allograft malakoplakia: a report of two cases.

Malakoplakia is a chronic granulomatous disease associated with incomplete clearance of bacterial pathogens. A multimodal approach to therapy includes antimicrobials with intracellular activity, reduction in immunosuppression, and debulking of lesions. Azithromycin has an intracellular mechanism of action and enhanced Gram-negative activity compared to other macrolides. Despite some in vitro data to support its use, there are no clinical breakpoints or epidemiological cut-off values for most Enterobacterales from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) or the Clinical and Laboratory Standards Institute (CLSI). We present two cases, previously unreported, of Escherichia coli associated renal allograft malakoplakia successfully treated with azithromycin.

Ablation of Hepatocyte Smad1, Smad5, and Smad8 Causes Severe Tissue Iron Loading and Liver Fibrosis in Mice.

A failure of iron to appropriately regulate liver hepcidin production is central to the pathogenesis of hereditary hemochromatosis. SMAD1/5 transcription factors, activated by bone morphogenetic protein (BMP) signaling, are major regulators of hepcidin production in response to iron; however, the role of SMAD8 and the contribution of SMADs to hepcidin production by other systemic cues remain uncertain. Here, we generated hepatocyte Smad8 single (Smad8fl/fl ;Alb-Cre+ ), Smad1/5/8 triple (Smad158;Alb-Cre+ ), and littermate Smad1/5 double (Smad15;Alb-Cre+ ) knockout mice to investigate the role of SMAD8 in hepcidin and iron homeostasis regulation and liver injury. We found that Smad8;Alb-Cre+ mice exhibited no iron phenotype, whereas Smad158;Alb-Cre+ mice had greater iron overload than Smad15;Alb-Cre+ mice. In contrast to the sexual dimorphism reported for wild-type mice and other hemochromatosis models, hepcidin deficiency and extrahepatic iron loading were similarly severe in Smad15;Alb-Cre+ and Smad158;Alb-Cre+ female compared with male mice. Moreover, epidermal growth factor (EGF) failed to suppress hepcidin in Smad15;Alb-Cre+ hepatocytes. Conversely, hepcidin was still increased by lipopolysaccharide in Smad158;Alb-Cre+ mice, although lower basal hepcidin resulted in lower maximal hepcidin. Finally, unlike most mouse hemochromatosis models, Smad158;Alb-Cre+ developed liver injury and fibrosis at 8 weeks. Liver injury and fibrosis were prevented in Smad158;Alb-Cre+ mice by a low-iron diet and were minimal in iron-loaded Cre- mice. Conclusion: Hepatocyte Smad1/5/8 knockout mice are a model of hemochromatosis that encompasses liver injury and fibrosis seen in human disease. These mice reveal the redundant but critical role of SMAD8 in hepcidin and iron homeostasis regulation, establish a requirement for SMAD1/5/8 in hepcidin regulation by testosterone and EGF but not inflammation, and suggest a pathogenic role for both iron loading and SMAD1/5/8 deficiency in liver injury and fibrosis.

The farnesoid X receptor agonist EDP-305 reduces interstitial renal fibrosis in a mouse model of unilateral ureteral obstruction.

Farnesoid X receptor (FXR) is a nuclear receptor that has emerged as a key regulator in the maintenance of hepatic steatosis, inflammation, and fibrosis. However, the role of FXR in renal fibrosis remains to be established. Here, we investigate the effects of the FXR agonist EDP-305 in a mouse model of tubulointerstitial fibrosis via unilateral ureteral obstruction (UUO). Male C57Bl/6 mice received a UUO on their left kidney. On postoperative d 4, mice received daily treatment by oral gavage with either vehicle control (0.5% methylcellulose) or 10 or 30 mg/kg EDP-305. All animals were euthanized on postoperative d 12. EDP-305 dose-dependently decreased macrophage infiltration as measured by the F4/80 staining area and proinflammatory cytokine gene expression. EDP-305 also dose-dependently reduced interstitial fibrosis as assessed by morphometric quantification of the collagen proportional area and kidney hydroxyproline levels. Finally, yes-associated protein (YAP) activation, a major driver of fibrosis, increased after UUO injury and was diminished by EDP-305 treatment. Consistently, EDP-305 decreased TGF-ß1-induced YAP nuclear localization in human kidney 2 cells by increasing inhibitory YAP phosphorylation. YAP inhibition may be a novel antifibrotic mechanism of FXR agonism, and EDP-305 could be used to treat renal fibrosis.-Li, S., Ghoshal, S., Sojoodi, M., Arora, G., Masia, R., Erstad, D. J., Ferriera, D. S., Li, Y., Wang, G., Lanuti, M., Caravan, P., Or, Y. S., Jiang, L.-J., Tanabe, K. K., Fuchs, B. C. The farnesoid X receptor agonist EDP-305 reduces interstitial renal fibrosis in a mouse model of unilateral ureteral obstruction.

Daily Aspirin Use Associated With Reduced Risk For Fibrosis Progression In Patients With Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: There are few data from prospective studies on the effects of aspirin on fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). METHODS: We performed a prospective cohort study of 361 adults with biopsy-confirmed NAFLD, from 2006 through 2015, examined every 3-12 months for incident advanced fibrosis defined using serial measurements of validated indices (the Fibrosis-4, NAFLD fibrosis score, and aspartate aminotransferase to platelet ratio indices). Histologic analyses of liver biopsies collected at baseline were performed by a blinded pathologist. Information collected at baseline and at each examination included frequency and duration of aspirin and nonsteroidal anti-inflammatory drug (NSAID) use. Using multivariable-adjusted logistic regression, we estimated the association of aspirin use with prevalent steatohepatitis (NASH) and fibrosis. Using multivariable-adjusted Cox proportional hazards modeling, we estimated the association between aspirin use and risk for fibrosis progression. RESULTS: At enrollment, 151 subjects used aspirin daily. Compared with non-regular use, daily aspirin use was associated with significantly lower odds of NASH (adjusted odds ratio, 0.68; 95% CI, 0.37-0.89) and fibrosis (adjusted odds ratio, 0.54; 95% CI, 0.31-0.82). Among individuals with baseline F0-F2 fibrosis (n = 317), 86 developed advanced fibrosis over 3692 person-years. Daily aspirin users had significantly lower risk for developing incident advanced fibrosis vs non-regular users (adjusted hazard ratio [aHR], 0.63; 95% CI, 0.43-0.85). This relationship appeared to be duration dependent (adjusted P trend=.026), with the greatest benefit found with at least 4 years or more of aspirin use (aHR, 0.50; 95% CI, 0.35-0.73). Conversely, use of nonaspirin NSAIDs was not associated with risk for advanced fibrosis (aHR, 0.93; 95% CI, 0.81-1.05). CONCLUSIONS: In a prospective study of patients with biopsy-proven NAFLD, daily aspirin use was associated with less severe histologic features of NAFLD and NASH, and lower risk for progression to advanced fibrosis with time.

Macrophage Activation Marker Soluble CD163 Is a Dynamic Marker of Liver Fibrogenesis in Human Immunodeficiency Virus/Hepatitis C Virus Coinfection.

Background: Coinfection with human immunodeficiency virus (HIV) accelerates hepatitis C virus (HCV)-related liver fibrosis. Macrophages are triggered during both viral infections and are critical in liver inflammation/fibrogenesis. Liver fibrosis strongly associates with serum soluble CD163 (sCD163, a macrophage activation marker); comprehensive evaluation in HIV/HCV coinfection is lacking. Methods: We retrospectively analyzed sCD163 (enzyme-linked immunosorbent assay) and hepatic CD163 (immunofluorescent CD163/CD68 costaining) in patients infected with HIV/HCV, HCV, or HIV, pre- and post-antiviral therapy. Results: sCD163 was significantly higher in HIV/HCV compared to either monoinfection, and decreased following successful antiviral therapy, although did not fully normalize. In HIV/HCV, sCD163 was associated with necroinflammation, Ishak fibrosis scores, and noninvasive fibrosis scores. We observed a novel trend whereby sCD163 levels progressively increase with increasing Ishak fibrosis score, peaking at stage 4, above which levels plateaued. Periportal CD163+ macrophage frequency was also higher with increasing fibrosis score. When stratified by fibrosis stage, sCD163 levels were higher in HIV/HCV than HCV but only in individuals with mild to moderate fibrosis. Conclusions: In HIV/HCV, increasing sCD163 levels accompanied periportal CD163+ macrophage enrichment in mild to moderate fibrosis, but not in established cirrhosis, suggesting that sCD163 is a dynamic biomarker of fibrogenesis rather than accumulated fibrosis. Our findings implicate HIV-related macrophage activation in accelerated fibrosis progression in HIV/HCV coinfection.

Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD+ ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD+ ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD+ ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD+ ratio, and this increase is mitigated by the presence of NAD+-generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD+ ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD+ ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real-time measurements of the ratio in living, functioning liver cells, overcoming many limitations of previous methods for measuring this important redox parameter. The feasibility of using Peredox in liver slices is particularly attractive because slices allow preservation of hepatic microanatomy and metabolic zonation of hepatocytes.

CM-101: Type I Collagen-targeted MR Imaging Probe for Detection of Liver Fibrosis.

Purpose To evaluate the biodistribution, metabolism, and pharmacokinetics of a new type I collagen-targeted magnetic resonance (MR) probe, CM-101, and to assess its ability to help quantify liver fibrosis in animal models. Materials and Methods Biodistribution, pharmacokinetics, and stability of CM-101 in rats were measured with mass spectrometry. Bile duct-ligated (BDL) and sham-treated rats were imaged 19 days after the procedure by using a 1.5-T clinical MR imaging unit. Mice were treated with carbon tetrachloride (CCl4) or with vehicle two times a week for 10 weeks and were imaged with a 7.0-T preclinical MR imaging unit at baseline and 1 week after the last CCl4 treatment. Animals were imaged before and after injection of 10 µmol/kg CM-101. Change in contrast-to-noise ratio (ΔCNR) between liver and muscle tissue after CM-101 injection was used to quantify liver fibrosis. Liver tissue was analyzed for Sirius Red staining and hydroxyproline content. The institutional subcommittee for research animal care approved all in vivo procedures. Results CM-101 demonstrated rapid blood clearance (half-life = 6.8 minutes ± 2.4) and predominately renal elimination in rats. Biodistribution showed low tissue gadolinium levels at 24 hours (<3.9% injected dose [ID]/g ± 0.6) and 10-fold lower levels at 14 days (<0.33% ID/g ± 12) after CM-101 injection with negligible accumulation in bone (0.07% ID/g ± 0.02 and 0.010% ID/g ± 0.004 at 1 and 14 days, respectively). ΔCNR was significantly (P < .001) higher in BDL rats (13.6 ± 3.2) than in sham-treated rats (5.7 ± 4.2) and in the CCl4-treated mice (18.3 ± 6.5) compared with baseline values (5.2 ± 1.0). Conclusion CM-101 demonstrated fast blood clearance and whole-body elimination, negligible accumulation of gadolinium in bone or tissue, and robust detection of fibrosis in rat BDL and mouse CCl4 models of liver fibrosis. © RSNA, 2017 Online supplemental material is available for this article.

Molecular MR Imaging of Myeloperoxidase Distinguishes Steatosis from Steatohepatitis in Nonalcoholic Fatty Liver Disease.

Purpose To test whether MPO-Gd, an activatable molecular magnetic resonance (MR) imaging agent specific for myeloperoxidase (MPO) activity, could detect MPO activity in nonalcoholic steatohepatitis (NASH) mouse models and human liver biopsy samples. Materials and Methods In this study, 20 leptin receptor-deficient and three MPO knockout mice were injected with endotoxin (lipopolysaccharide) or fed a methionine and choline-deficient (MCD) diet to induce experimental NASH and underwent MR imaging with MPO-Gd. Saline-injected and control diet-fed leptin receptor-deficient mice were used as respective controls. MPO protein and activity measurements and histologic analyses were performed. Eleven human liver biopsy samples underwent MPO-Gd-enhanced MR imaging ex vivo and subsequent histologic evaluation. Results were compared with Student t test or Mann-Whitney U test. Results With endotoxin, a significantly increased contrast-to-noise ratio (CNR) was found compared with sham (mean CNR, 1.81 [95% confidence interval {CI}: 1.53, 2.10] vs 1.02 [95% CI: 0.89, 1.14]; P = .03) at MPO-Gd MR imaging. In the diet-induced NASH model, an increased CNR was also found compared with sham mice (mean CNR, 1.33 [95% CI: 1.27, 1.40] vs 0.98 [95% CI: 0.83, 1.12]; P = .008). Conversely, CNR remained at baseline in NASH mice imaged with gadopentetate dimeglumine and in MPO knockout NASH mice with MPO-Gd, which proves specificity of MPO-Gd. Ex vivo molecular MR imaging of liver biopsy samples from NASH and control patients confirmed results from animal studies (mean CNR for NASH vs control patients, 2.61 [95% CI: 1.48, 3.74] vs 1.29 [95% CI: 1.06, 1.52]; P = .004). Conclusion MPO-Gd showed elevated MPO activity in NAFLD mouse models and human liver biopsy samples. © RSNA, 2017 Online supplemental material is available for this article. An earlier incorrect version of this article appeared online. This article was corrected on April 6, 2017.

The inward rectifier potassium channel Kir2.1 is expressed in mouse neutrophils from bone marrow and liver.

Neutrophils are phagocytic cells that play a critical role in innate immunity by destroying bacterial pathogens. Channels belonging to the inward rectifier potassium channel subfamily 2 (Kir2 channels) have been described in other phagocytes (monocytes/macrophages and eosinophils) and in hematopoietic precursors of phagocytes. Their physiological function in these cells remains unclear, but some evidence suggests a role in growth factor-dependent proliferation and development. Expression of functional Kir2 channels has not been definitively demonstrated in mammalian neutrophils. Here, we show by RT-PCR that neutrophils from mouse bone marrow and liver express mRNA for the Kir2 subunit Kir2.1 but not for other subunits (Kir2.2, Kir2.3, and Kir2.4). In electrophysiological experiments, resting (unstimulated) neutrophils from mouse bone marrow and liver exhibit a constitutively active, external K(+)-dependent, strong inwardly rectifying current that constitutes the dominant current. The reversal potential is dependent on the external K(+) concentration in a Nernstian fashion, as expected for a K(+)-selective current. The current is not altered by changes in external or internal pH, and it is blocked by Ba(2+), Cs(+), and the Kir2-selective inhibitor ML133. The single-channel conductance is in agreement with previously reported values for Kir2.1 channels. These properties are characteristic of homomeric Kir2.1 channels. Current density in short-term cultures of bone marrow neutrophils is decreased in the absence of growth factors that are important for neutrophil proliferation [granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF)]. These results demonstrate that mouse neutrophils express functional Kir2.1 channels and suggest that these channels may be important for neutrophil function, possibly in a growth factor-dependent manner.

Domain organization of the ATP-sensitive potassium channel complex examined by fluorescence resonance energy transfer.

K(ATP) channels link cell metabolism to excitability in many cells. They are formed as tetramers of Kir6.2 subunits, each associated with a SUR1 subunit. We used mutant GFP-based FRET to assess domain organization in channel complexes. Full-length Kir6.2 subunits were linked to YFP or cyan fluorescent protein (CFP) at N or C termini, and all such constructs, including double-tagged YFP-Kir6.2-CFP (Y6.2C), formed functional K(ATP) channels. In intact COSm6 cells, background emission of YFP excited by 430-nm light was ∼6%, but the Y6.2C construct expressed alone exhibited an apparent FRET efficiency of ∼25%, confirmed by trypsin digestion, with or without SUR1 co-expression. Similar FRET efficiency was detected in mixtures of CFP- and YFP-tagged full-length Kir6.2 subunits and transmembrane domain only constructs, when tagged at the C termini but not at the N termini. The FRET-reported Kir6.2 tetramer domain organization was qualitatively consistent with Kir channel crystal structures: C termini and M2 domains are centrally located relative to N termini and M1 domains, respectively. Additional FRET analyses were performed on cells in which tagged full-length Kir6.2 and tagged SUR1 constructs were co-expressed. These analyses further revealed that 1) NBD1 of SUR1 is closer to the C terminus of Kir6.2 than to the N terminus; 2) the Kir6.2 cytoplasmic domain is not essential for complexation with SUR1; and 3) the N-terminal half of SUR1 can complex with itself in the absence of either the C-terminal half or Kir6.2.

Cytomegalovirus Hepatitis in Allograft Livers May Show Histologic Features of Acute Cellular Rejection.

CONTEXT.­: Cytomegalovirus (CMV) hepatitis in allograft livers is an important infectious complication, with histology that historically has been described to overlap with that of acute cellular rejection (ACR), a diagnosis that compels a different treatment regimen. OBJECTIVE.­: To update the clinicopathologic features of CMV hepatitis and explore its clinical and histologic relationship with ACR. DESIGN.­: A retrospective analysis of 26 patients with a diagnosis of CMV hepatitis across 4 institutions was performed, including clinical, histologic, and immunohistochemical features. RESULTS.­: Patients were predominantly CMV donor positive/recipient negative (D+/R-; n = 9 of 15) and received a diagnosis of CMV hepatitis at a mean age of 52 years (SD, 17 years), at a mean interval of 184 days (SD, 165 days) from transplantation. Mean CMV viral load at diagnosis was 241 000 IU/mL (SD, 516 000 IU/mL), and liver biochemical enzymes were elevated (mean alanine aminotransferase, 212 U/L [SD, 180 U/L]; mean aspartate aminotransferase, 188 U/L [SD, 151 U/L]; mean alkaline phosphatase, 222 U/L [SD, 153 U/L]). Ten cases did not show histologic features of ACR, and 16 cases demonstrated features of ACR (including marked bile duct injury and endotheliitis). Viral cytopathic change was found in all cases. All patients were treated with a combination of antiviral therapy and CMV intravenous immunoglobulin, with near resolution of biochemical enzymes in all patients with undetectable serum CMV viral titers. CONCLUSIONS.­: CMV hepatitis and ACR are complex processes with interlinking mechanisms that are important to distinguish. A subset of transplantation patients with CMV hepatitis show histologic changes that mimic ACR but were treated successfully with antiviral therapy alone.

CD73 Inhibits cGAS-STING and Cooperates with CD39 to Promote Pancreatic Cancer.

The ectonucleotidases CD39 and CD73 catalyze extracellular ATP to immunosuppressive adenosine, and as such, represent potential cancer targets. We investigated biological impacts of CD39 and CD73 in pancreatic ductal adenocarcinoma (PDAC) by studying clinical samples and experimental mouse tumors. Stromal CD39 and tumoral CD73 expression significantly associated with worse survival in human PDAC samples and abolished the favorable prognostic impact associated with the presence of tumor-infiltrating CD8+ T cells. In mouse transplanted KPC tumors, both CD39 and CD73 on myeloid cells, as well as CD73 on tumor cells, promoted polarization of infiltrating myeloid cells towards an M2-like phenotype, which enhanced tumor growth. CD39 on tumor-specific CD8+ T cells and pancreatic stellate cells also suppressed IFNγ production by T cells. Although therapeutic inhibition of CD39 or CD73 alone significantly delayed tumor growth in vivo, targeting of both ectonucleotidases exhibited markedly superior antitumor activity. CD73 expression on human and mouse PDAC tumor cells also protected against DNA damage induced by gemcitabine and irradiation. Accordingly, large-scale pharmacogenomic analyses of human PDAC cell lines revealed significant associations between CD73 expression and gemcitabine chemoresistance. Strikingly, increased DNA damage in CD73-deficient tumor cells associated with activation of the cGAS-STING pathway. Moreover, cGAS expression in mouse KPC tumor cells was required for antitumor activity of the CD73 inhibitor AB680 in vivo. Our study, thus, illuminates molecular mechanisms whereby CD73 and CD39 seemingly cooperate to promote PDAC progression.