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Abstract
Despite decades of research there is no specific therapy for Acute Pancreatitis (AP). In the current study, we have evaluated the efficacy of pirfenidone, an anti-inflammatory and anti-fibrotic agent which is FDA-approved for treatment of idiopathic pulmonary fibrosis (IPF), in ameliorating local and systemic injury in AP. Our results suggest that treatment with pirfenidone in therapeutic settings (i.e. after initiation of injury), even when administered at the peak of injury, reduces severity of local and systemic injury and inflammation in multiple models of AP. In-vitro evaluation suggests that pirfenidone decreases cytokine release from acini and macrophages and disrupts acinar-macrophage crosstalk. Therapeutic pirfenidone treatment increases IL-10 secretion from macrophages preceding changes in histology and modulates the immune phenotype of inflammatory cells with decreased levels of inflammatory cytokines. Antibody-mediated IL-10 depletion, use of IL-10 Knock Out mice, and macrophage depletion experiments confirmed the role of IL-10 and macrophages in its mechanism of action, as pirfenidone was unable to reduce severity of AP in these scenarios. Since pirfenidone is FDA approved for IPF, a trial evaluating the efficacy of pirfenidone in patients with moderate to severe AP can be initiated expeditiously. Key Words: Acute Pancreatitis, Pirfenidone, Interleukin-10, L-arginine pancreatitis, Systemic inflammation, lung injury
Authors
Ejas Palathingal Bava, John George, Mohammad Tarique, Srikanth Iyer, Preeti Sahay, Beatriz Gomez Aguilar, Dujon B. Edwards, Bhuwan Giri, Vrishketan Sethi, Tejeshwar Jain, Prateek Sharma, Utpreksha Vaish, Harrys K. C. Jacob, Anthony Ferrantella, Craig L. Maynard, Ashok K. Saluja, Rajinder K. Dawra, Vikas Dudeja
Abstract
Identification and analysis of fungal communities commonly rely on internal transcribed spacer (ITS)-based amplicon sequencing. There is no gold standard to infer and classify fungal constituents since methodologies have been adapted from analyses of bacterial communities. To achieve high resolution inference of fungal constituents, we customized a DADA2-based pipeline using a mix of eleven medically relevant fungi. While DADA2 allowed the discrimination of ITS1 sequences differing by single nucleotides, quality filtering, sequencing bias, and database selection were identified as key variables determining the accuracy of sample inference. Due to species-specific differences in sequencing quality, default filtering settings removed most reads that originated from Aspergillus species, Saccharomyces cerevisiae, and Candida glabrata. By fine-tuning the quality filtering process, we achieved an improved representation of the fungal communities. By adapting a wobble nucleotide in the ITS1 forward primer region, we further increased the yield of S. saccharomyces and C. glabrata sequences. Finally, we showed that a BLAST-based algorithm based on the UNITE+INSD or the NCBI NT database achieved a higher reliability in species-level taxonomic annotation than the naïve Bayesian classifier implemented in DADA2. These steps optimized a robust fungal ITS1 sequencing pipeline that, in most instances, enabled species level-assignment of community members.
Authors
Thierry Rolling, Bing Zhai, John Frame, Tobias M. Hohl, Ying Taur
Abstract
While current thinking posits that insulin signaling to GLUT4 exocytic translocation and glucose uptake in skeletal muscle and adipocytes is controlled by phosphorylation-based signaling, many proteins in this pathway are acetylated on lysine residues. However, the importance of acetylation and lysine acetyltransferases to insulin-stimulated glucose uptake is incompletely defined. Here, we demonstrate that combined loss of the acetyltransferases E1A binding protein p300 (p300) and cAMP response element binding protein binding protein (CBP) in mouse skeletal muscle causes a complete loss of insulin-stimulated glucose uptake. Similarly, brief (i.e. 1 h) pharmacological inhibition of p300/CBP acetyltransferase activity recapitulates this phenotype in human and rodent myotubes, 3T3-L1 adipocytes, and mouse muscle. Mechanistically, these effects are due to p300/CBP-mediated regulation of GLUT4 exocytic translocation and occurs downstream of Akt signaling. Taken together, we highlight a fundamental role for acetylation and p300/CBP in the direct regulation of insulin-stimulated glucose transport in skeletal muscle and adipocytes.
Authors
Vitor F. Martins, Samuel A. LaBarge, Alexandra Stanley, Kristoffer Svensson, Chao-Wei Hung, Omer Keinan, Theodore P. Ciaraldi, Dion Banoian, Ji E. Park, Christina Ha, Byron Hetrick, Gretchen A. Meyer, Andrew Philp, Larry L. David, Robert R. Henry, Joseph E. Aslan, Alan R. Saltiel, Carrie E. McCurdy, Simon Schenk
Abstract
Sangivamycin is a nucleoside analog that is well-tolerated by humans and broadly active against phylogenetically distinct viruses, including arenaviruses, filoviruses, and orthopoxviruses. Here, we show that sangivamycin is a potent antiviral against multiple variants of replicative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with half-maximal inhibitory concentration (IC50) in the nanomolar range in several cell types. Sangivamycin suppressed SARS-CoV-2 replication with greater efficacy than remdesivir (another broad-spectrum nucleoside analog). When we investigated sangivamycin’s potential for clinical administration, pharmacokinetic, ADME (absorption, distribution, metabolism, and excretion), and toxicity properties were found to be favorable. When tested in combination with remdesivir, efficacy was additive rather than competitive against SARS-CoV-2. The proven safety in humans, long half-life, potent antiviral activity (compared to remdesivir), and combinatorial potential suggest that sangivamycin is likely to be efficacious alone or in combination therapy to suppress viremia in patients. Sangivamycin may also have the ability to help combat drug-resistant or vaccine-escaping SARS-CoV-2 variants since it is antivirally active against several tested variants. Our results support the pursuit of sangivamycin for further preclinical and clinical development as a potential coronavirus disease 2019 (COVID-19) therapeutic.
Authors
Ryan P. Bennett, Elena N. Postnikova, Brett P. Eaton, Yingyun Cai, Shuiqing Yu, Charles O. Smith, Janie Liang, Huanying Zhou, Gregory A. Kocher, Michael J. Murphy, Harold C. Smith, Jens H. Kuhn
Abstract
The clinical utility of histone/protein deacetylase (HDAC) inhibitors (HDACi’s) in combinatorial regimens with proteasome inhibitors for patients with relapsed and refractory multiple myeloma (MM) is limited often by excessive toxicity due to HDACi promiscuity with multiple HDACs. Therefore, more selective inhibition minimizing off-target toxicity may increase the clinical effectiveness of HDACi’s. We demonstrate that both plasma cell (PC) development and survival are dependent upon HDAC11, suggesting this enzyme is a promising therapeutic target in MM. Mice lacking HDAC11 exhibited markedly decreased PC numbers. Accordingly, in vitro PC differentiation was arrested in B cells lacking functional HDAC11. Mechanistically, we show that HDAC11 is involved in the deacetylation of IRF4 at lysine103. Further, targeting HDAC11 led to IRF4 hyperacetylation resulting in impaired IRF4 nuclear localization and target promoter binding. Importantly, transient HDAC11 knockdown or treatment with elevenostat, an HDAC11-selective inhibitor, induced cell death in MM cell lines. Elevenostat produced similar anti-MM activity in vivo, improving survival among mice inoculated with 5TGM1 MM cells. Elevenostat demonstrated nanomolar ex vivo activity in 34 MM patient specimens and synergistic activity when combined with bortezomib. Collectively, our data indicate that HDAC11 is an emerging therapeutic vulnerability in MM by targeting an essential pathway in PC biology.
Authors
AGM Mostofa, Allison Distler, Mark B. Meads, Eva Sahakian, John J. Powers, Alexandra Achille, David Noyes, Gabriela Wright, Bin Fang, Victoria Izumi, John Koomen, Rupal Rampakrishnan, Tuan P. Nguyen, Gabriel De Avila, Ariosto S. Silva, Praneeth Sudalagunta, Rafael Renatino Canevarolo, Maria D. Coelho Siqueira Silva, Raghunandan Reddy Alugubelli, Hongyue A. Dai, Amit Kulkarni, William S. Dalton, Oliver A. Hampton, Eric A. Welsh, Jamie K. Teer, Alexandre Tungesvik, Kenneth L. Wright, Javier Pinilla-Ibarz, Eduardo M. Sotomayor, Kenneth H. Shain, Jason Brayer
Abstract
Alport syndrome (AS) is a genetic disorder caused by mutations in type IV collagen that leads to defective glomerular basement membrane, glomerular filtration barrier (GFB) damage, and progressive chronic kidney disease. While the genetic basis of AS is well known, the molecular and cellular mechanistic details of disease pathogenesis have been elusive, hindering the development of mechanism-based therapies. Here we performed intravital multiphoton imaging of the local kidney tissue microenvironment in a X-linked AS mouse model to directly visualize the major drivers of AS pathology. Severely distended glomerular capillaries and aneurysms were found accompanied by numerous microthrombi, increased glomerular endothelial surface layer (glycocalyx) and immune cell homing, GFB albumin leakage, glomerulosclerosis and interstitial fibrosis by 5 months of age with an intermediate phenotype at 2 months. Renal histology in mouse or patient tissues largely failed to detect capillary aberrations. Treatment of AS mice with hyaluronidase or the ACE inhibitor enalapril reduced the excess glomerular endothelial glycocalyx and blocked immune cell homing, and GFB albumin leakage. This study identified central roles of glomerular mechanical forces and endothelial and immune cell activation early in AS, which could be therapeutically targeted to reduce mechanical strain and local tissue inflammation and improve kidney function.
Authors
Georgina Gyarmati, Urvi Nikhil Shroff, Audrey Izuhara, Xiaogang Hou, Stefano Da Sacco, Sargis Sedrakyan, Kevin V. Lemley, Kerstin Amann, Laura Perin, János Peti-Peterdi
Abstract
Neutrophils are recognized as important circulating effector cells in the pathophysiology of severe coronavirus disease 2019 (COVID-19). However, their role within the inflamed lungs is incompletely understood. Here, we collected broncho-alveolar lavage (BAL) fluids and parallel blood samples of critically ill COVID-19 patients requiring invasive mechanical ventilation and compared BAL fluid parameters with those of mechanically ventilated influenza patients, as a non-COVID-19 viral pneumonia cohort. Compared to influenza, BAL fluids of COVID-19 patients contained increased numbers of hyperactivated degranulating neutrophils and elevated concentrations of the cytokines IL-1β, IL-1RA, IL-17A, TNF-α and G-CSF, the chemokines CCL7, CXCL1, CXCL8, CXCL11 and CXCL12α, and the protease inhibitors elafin, secretory leukocyte protease inhibitor (SLPI) and tissue inhibitor of metalloproteinases 1 (TIMP-1). In contrast, α-1 antitrypsin levels and net proteolytic activity were comparable in COVID-19 and influenza BAL fluids. During antibiotics treatment for bacterial co-infections, increased BAL fluid levels of several activating and chemotactic factors for monocytes, lymphocytes and NK cells were detected in COVID-19 patients whereas concentrations tended to decrease in influenza patients, highlighting the persistent immunological response to co-infections in COVID-19. Finally, the high proteolytic activity in COVID-19 lungs suggests considering protease inhibitors as a treatment option.
Authors
Seppe Cambier, Mieke Metzemaekers, Ana C. Carvalho, Amber Nooyens, Cato Jacobs, Lore Vanderbeke, Bert Malengier-Devlies, Mieke Gouwy, Elisabeth Heylen, Philippe Meersseman, Greet Hermans, Els Wauters, Alexander Wilmer, Contagious Consortium, Dominique Schols, Patrick Matthys, Ghislain Opdenakker, Rafael Elias Marques, Joost Wauters, Jennifer Vandooren, Paul Proost
Abstract
BACKGROUND. Red blood cell (RBC) transfusion effectiveness varies due to donor, component, and recipient factors. Prior studies identified characteristics associated with variation in hemoglobin increments following transfusion. We extended these observations, examining donor genetic and non-genetic factors affecting transfusion effectiveness. METHODS. This is a multicenter retrospective study of 46,705 patients, and 102,043 evaluable RBC transfusions from 2013-2016 across 12 hospitals. Transfusion effectiveness was defined as hemoglobin, bilirubin, or creatinine increments following single RBC unit transfusion. Models incorporated a subset of donors with data on single nucleotide polymorphisms associated with osmotic and oxidative hemolysis in vitro. Mixed modelling accounting for repeated transfusion episodes identified predictors of transfusion effectiveness. RESULTS. Blood donor (sex, Rh status, fingerstick hemoglobin, smoking), component (storage duration, gamma irradiation, leukoreduction, apheresis collection, storage solution), and recipient (sex, body mass index, race, age) characteristics were associated with hemoglobin and bilirubin but not creatinine increments following RBC transfusions. Increased storage duration was associated with increased bilirubin and decreased hemoglobin increments, suggestive of in vivo hemolysis following transfusion. Donor G6PD-deficiency and polymorphisms in SEC14L4, HBA2, and MYO9B genes were associated with decreased hemoglobin increments. Donor G6PD-deficiency and polymorphisms in SEC14L4 were associated with increased transfusion requirements in the subsequent 48 hours. CONCLUSIONS. Donor genetic and other factors, such as RBC storage duration, affect transfusion effectiveness as defined by decreased hemoglobin or increased bilirubin increments. Addressing these factors will provide a precision medicine approach to improve patient outcomes, particularly for chronically-transfused RBC recipients, who would most benefit from more effective transfusion products.
Authors
Nareg H. Roubinian, Sarah E. Reese, Hannah Qiao, Colleen Plimier, Fang Fang, Grier P. Page, Ritchard G. Cable, Brian Custer, Mark T. Gladwin, Ruchika Goel, Bob Harris, Jeanne E. Hendrickson, Tamir Kanias, Steve Kleinman, Alan E. Mast, Steven R. Sloan, Bryan R. Spencer, Steven L. Spitalnik, Michael P. Busch, Eldad A. Hod
Abstract
The biosynthetic routes leading to de novo Nicotinamine Adenine Dinucleotide (NAD+) production are involved in acute kidney injury (AKI) with a critical role for Quinolinate Phosphoribosyl Transferase (QPRT), a bottleneck enzyme of de novo NAD+ biosynthesis. However, the molecular mechanisms determining reduced QPRT in AKI, and the role of impaired NAD+ biosynthesis in the progression to chronic kidney disease (CKD) are unknown. We demonstrate that a high urinary quinolinate to tryptophan ratio, an indirect indicator of impaired QPRT activity and reduced de novo NAD+ biosynthesis in the kidney, is a clinically applicable early marker of AKI after cardiopulmonary bypass, and is predictive of progression to chronic kidney disease (CKD) in kidney transplant recipients. We also provide evidence that the Endoplasmic Reticulum (ER) stress response impairs de novo NAD+ biosynthesis by repressing QPRT transcription. In conclusion, NAD+ biosynthesis impairment is an early event in AKI embedded with the ER stress response, and persistent reduction of QPRT expression is associated with AKI to CKD progression. This defines non-invasive metabolic biomarkers of kidney injury with prognostic and therapeutic implications.
Authors
Yohan Bignon, Anna Rinaldi, Zahia Nadour, Virginie Poindessous, Ivan Nemazanyy, Olivia Lenoir, Baptiste Fohlen, Pierre Weill-Raynal, Alexandre Hertig, Alexandre Karras, Pierre Galichon, Maarten Naesens, Dany Anglicheau, Pietro E. Cippà, Nicolas Pallet
Abstract
Colorectal cancers (CRCs) exhibit differences in incidence, pathogenesis, molecular pathways and outcome depending on the location of the tumor. The transcriptomes of 27,927 single human CRC cells, from three left-sided and three right-sided CRC patients were profiled by scRNA-seq. Right-sided CRC harbors a significant proportion of exhausted CD8 T cells of a highly migratory nature. One cluster of cells from left-sided CRC exhibiting states preceding exhaustion and a high ratio of “pre-exhausted” to exhausted T cells were favorable prognostic markers. Notably, we identified a novel RBP4+ NTS+ subpopulation of cancer cells that exclusively expands in left-sided CRC. Tregs from left-sided CRC showed higher levels of immunotherapy-related genes than those from right-sided CRC, indicating that left-sided CRC may have increased responsiveness to immunotherapy. Antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) induced by M2-like macrophages were more pronounced in left-sided CRC and correlated with a good prognosis in CRC.
Authors
Wei Guo, Cuiyu Zhang, Xia Wang, Dandan Dou, Dawei Chen, Jingxin Li
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