In search of new prognostic markers, many mutation analyses of the HBV genome were performed. However, the Kozak sequence preceding precore was covered only infrequently in these analyses. In this study, HBV core promoter/precore region was sequenced in serum samples of European inactive HBV carriers (n=560). Quadruple mutation GCAC1809-1812TTCT was found with a high prevalence of 42% in the Kozak sequence preceding precore among all HBV genotypes. GCAC1809-1812TTCT was strongly associated with coexistence of basal core promoter (BCP) double mutation A1762T/G1764A and lower HBV DNA levels (p<0.0001). In vitro GCAC1809-1812TTCT leads to drastically diminished synthesis of pregenomic(pg)RNA, precore mRNA, core, HBsAg and HBeAg. Calculation of the pgRNA secondary structure suggests a destabilization of the pgRNA structure by A1762T/G1764A that is compensated by GCAC1809-1812TTCT. In 125 patients with HBV-related cirrhosis, GCAC1809-1812TTCT was not detected. While a strong association of GCAC1809-1812TTCT with inactive carrier status (p<0.0001) was observed, BCP double mutation was strongly correlated with cirrhosis (p<0.0001), but this was only observed in absence of GCAC1809-1812TTCT. In conclusion, our data reveal that GCAC1809-1812TTCT is highly prevalent in inactive carriers, and acts as a compensatory mutation for BCP double mutation. GCAC1809-1812TTCT seems to be a biomarker of good prognosis in HBV infection.
Kai-Henrik Peiffer, Catrina Spengler, Michael Basic, Bingfu Jiang, Lisa Kuhnhenn, Wiebke Obermann, Tobias Zahn, Mirco Glitscher, Alessandro Loglio, Floriana Facchetti, Gert Carra, Alica Kubesch, Johannes Vermehren, Viola Knop, Christiana Graf, Julia Dietz, Fabian Finkelmeier, Eva Herrmann, Jonel Trebicka, Arnold Grünweller, Stefan Zeuzem, Christoph Sarrazin, Pietro Lampertico, Eberhard Hildt
Heart failure is often accompanied by titin-dependent myocardial stiffness. Phosphorylation of titin by cGMP-dependent protein kinase (PKG)I increases cardiomyocyte distensibility. The upstream pathways stimulating PKGI-mediated titin phosphorylation are unclear. We studied whether C-type natriuretic peptide (CNP), via its guanylyl cyclase-B (GC-B) receptor and cGMP/PKGI signalling, modulates titin-based ventricular compliance. To dissect GC-B-mediated effects of endogenous CNP in cardiomyocytes, we generated mice with cardiomyocyte (CM)-restricted GC-B deletion. The impact on heart morphology and function, myocyte passive tension and titin isoform expression and phosphorylation was studied at baseline and after increased afterload induced by transverse aortic constriction (TAC).Pressure-overload increased left ventricular, especially endothelial CNP expression, with an early peak after 3 days. Concomitantly, titin phosphorylation at Ser4080, the site phosphorylated by PKGI, was augmented. Notably, in CM GC-B KO mice this titin response was abolished. TAC-induced hypertrophy and fibrosis were not different between genotypes. However, the KO mice presented mild systolic and diastolic dysfunction together with myocyte stiffness, which were not observed in control littermates. In vitro, recombinant PKGI rescued reduced Titin-Ser4080 phosphorylation and reverted passive stiffness of GC-B-deficient cardiomyocytes. CNP-induced activation of GC-B/cGMP/PKGI signalling in cardiomyocytes provides a protecting regulatory circuit preventing titin-based myocyte stiffening during early phases of pressure-overload.
Konstanze Michel, Melissa Herwig, Franziska Werner, Katarina Špiranec Spes, Marco Abeßer, Kai Schuh, Swati Dabral, Andreas Mügge, Hideo A. Baba, Boris V. Skryabin, Nazha Hamdani, Michaela Kuhn
The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass-spectrometry (dMS) followed by targeted analysis to facilitate this discovery. We validated this approach by analyzing Merkel cell carcinoma (MCC), an aggressive human neoplasm, in which ~80% of cases are caused by the human Merkel cell polyomavirus (MCV). Approximately 20% of MCC have a high mutational burden and are negative for MCV, but are microscopically indistinguishable from virus positive cases. Using 23 (12 MCV positive, 11 MCV negative) formalin-fixed MCC, DPS identified both viral and human biomarkers (MCV Large T antigen, CDKN2AIP, SERPINB5 and TRIM29) that discriminates MCV positive and negative MCC. Statistical analysis of 498,131 dMS features not matching the human proteome by DPS revealed 562 (0.11%) to be up-regulated in virus-infected samples. Remarkably, four (20%) of the top 20 candidate MS spectra originated from MCV T oncoprotein peptides and confirmed by reverse translation degenerate oligonucleotide sequencing. DPS is a robust proteomic approach to identify novel viral sequences in infectious tumors when nucleic acid-based methods are not feasible.
Tuna Toptan, Pamela S. Cantrell, Xuemei Zeng, Yang Liu, Mai Sun, Nathan A. Yates, Yuan Chang, Patrick S. Moore
Infections caused by multi-drug resistant Staphylococcus aureus, especially MRSA, are responsible for high mortality and morbidity worldwide. Resistant lineages were previously confined to hospitals, but are now also causing infections among healthy individuals in the community. It is therefore imperative to explore therapeutic avenues that are less prone to raise drug resistance compared to today’s antibiotics. An opportunity to achieve this ambitious goal could be provided by targeted antimicrobial photodynamic therapy (aPDT), which relies on the combination of a bacteria-specific targeting agent and light-induced generation of reactive oxygen species by an appropriate photosensitizer. Here we conjugated the near-infrared photosensitizer IRDye700DX to a fully human monoclonal antibody, specific for the invariantly expressed staphylococcal antigen IsaA. The resulting immunoconjugate 1D9-700DX was characterized biochemically and in preclinical infection models. As demonstrated in vitro, in vivo, and in a human post-mortem orthopedic implant infection model, targeted aPDT with 1D9-700DX is highly effective. Importantly, combined with the non-toxic aPDT-enhancing agent potassium iodide, 1D9-700DX overcomes the antioxidant properties of human plasma and fully eradicates high titers of MRSA. We show that the developed immunoconjugate 1D9-700DX targets MRSA and kills it upon illumination with red light, without causing collateral damage to human cells.
Mafalda Bispo, Andrea Anaya-Sanchez, Sabrina Suhani, Elisa J.M. Raineri, Marina López-Álvarez, Marjolein Heuker, Wiktor Szymański, Francisco Romero Pastrana, Girbe Buist, Alexander R. Horswill, Kevin P. Francis, Gooitzen M. van Dam, Marleen van Oosten, Jan Maarten van Dijl
Intratumoral immune infiltrate was recently reported in Gastrointestinal Stromal Tumors (GIST). However, what tumor-intrinsic factors dictate GIST immunogenicity is still largely undefined. To shed light on this issue a large cohort (82 samples) of primary untreated GIST, representative of major clinicopathological variables, was investigated by an integrated immunohistochemical, transcriptomic and computational approach. Our results indicate that tumor genotype, location and malignant potential concur to shape the immunogenicity of primary naïve GIST. Immune infiltration was greater in overt GIST than in lesions with limited malignant potential (miniGIST), in KIT/PDGFRA mutated than in KIT/PDGFRA wild-type tumors and in PDGFRA versus KIT mutated GIST. Within the KIT mutated subset, a higher degree of immune colonization was detected in the intestine. Immune hot tumors showed expression patterns compatible with a potentially proficient but curbed antigen-specific immunity, hinting at sensitivity to immunomodulatory treatments. Poorly infiltrated GIST, primarily KIT/PDGFRA wild-type intestinal tumors, showed activation of Hedgehog and WNT/β-catenin immune excluding pathways. This finding discloses a potential therapeutic vulnerability, as the targeting of these pathways might prove effective by both inhibiting pro-oncogenic signals and fostering anti-tumor immune responses. Finally, an intriguing anticorrelation between immune infiltration and ANO1/DOG1 expression was observed, suggesting an immunomodulatory activity for anoctamin-1.
Daniela Gasparotto, Marta Sbaraglia, Sabrina Rossi, Davide Baldazzi, Monica Brenca, Alessia Mondello, Federica Nardi, Dominga Racanelli, Matilde Cacciatore, Angelo Paolo Dei Tos, Roberta Maestro
Plasma antimalarial antibody can mediate anti-parasite immunity but has not previously been characterized at the molecular level. Here, we develop an innovative strategy to characterize humoral responses by integrating profiles of plasma immunoglobulins (IG) or antibodies with those expressed on B cells as part of BcR. We applied this strategy to define plasma IG and determine variable V gene usage after vaccination with the Plasmodium falciparum zygote antigen Pfs25. First, using proteomic tools coupled with bulk immunosequencing data, we determined human F(ab′)2 peptide sequences from plasma IG of adults who received four doses of Pfs25-EPA/Alhydrogel. Specifically, Pfs25 antigen-specific F(ab′)2 peptides (Pfs25-IG) were aligned to cDNA sequences of IGH complementarity determining region 3 (CDR3) from a dataset generated by total peripheral B cell immunosequencing of the entire vaccinated population. IGHV4 was the most commonly identified IGHV subgroup of Pfs25-IG, a pattern that was corroborated by VH/VL sequencing of Pfs25-specific single B cells from five vaccinees and by matching plasma Pfs25-IG peptides and V-(D)-J sequences of Pfs25-specific single B cells from the same donor. Among 13 recombinant human mAbs generated from IG sequences of Pfs25-specific single B cells, a single IGHV4 mAb displayed strong neutralizing activity, reducing the number of P. falciparum oocysts in infected mosquitoes by more than 80% at 100 μg/mL. Our approach characterizes the human plasma antibody repertoire in response to the Pfs25-EPA/Alhydrogel vaccine and will be useful to study circulating antibodies in response to other vaccines as well as those induced during infections or autoimmune disorders.
Camila H. Coelho, Steven T. Nadakal, Patricia A. Gonzales Hurtado, Robert Morrison, Jacob D. Galson, Jillian Neal, Yimin Wu, C. Richter King, Virginia Price, Kazutoyo Miura, Sharon Wong-Madden, Justin Y.A. Dortichamou, David L. Narum, Nicholas J. MacDonald, Maryonne Snow-Smith, Marissa Vignali, Justin J. Taylor, Marie-Paule Lefranc, Johannes Trück, Carole A. Long, Issaka Sagara, Michal Fried, Patrick E. Duffy
WNK1 is an atypical kinase protein ubiquitously expressed in humans and mice. A mutation in its encoding gene causes hypertension in humans which is associated with abnormal ion homeostasis. WNK1 is critical for in vitro decidualization in human endometrial stromal cells, thereby demonstrating its importance in female reproduction. Using a mouse model, WNK1 was ablated in the female reproductive tract to define its in vivo role in uterine biology. Loss of WNK1 altered uterine morphology, causing endometrial epithelial hyperplasia, adenomyotic features and a delay in embryo implantation, ultimately resulting in compromised fertility. Combining transcriptomic, proteomic and interactomic analyses revealed a novel regulatory pathway whereby WNK1 represses AKT phosphorylation through the phosphatase PP2A in endometrial cells from both humans and mice. We show that WNK1 interacts with PPP2R1A, the alpha isoform of the PP2A scaffold subunit. This maintains the levels of PP2A subunits and stabilizes its activity, which then dephosphorylates AKT. Therefore, loss of WNK1 reduced PP2A activity, causing AKT hypersignaling. Using FOXO1 as a readout of AKT activity, we demonstrate that there was escalated FOXO1 phosphorylation and nuclear exclusion, leading to a disruption in the expression of genes that are crucial for embryo implantation.
Ru-pin Alicia Chi, Tianyuan Wang, Chou-Long Huang, San-Pin Wu, Steven Young, John Lydon, Francesco DeMayo
Rapid and specific antibody testing is crucial for improved understanding, control, and treatment of COVID-19 pathogenesis. Herein, we describe and apply a rapid, sensitive, and accurate virus neutralization assay for SARS-CoV-2 antibodies. The new assay is based on an HIV-1 lentiviral vector that contains a secreted intron Gaussia luciferase or secreted Nano-luciferase reporter cassette, pseudotyped with the SARS-CoV-2 spike (S) glycoprotein, and is validated with a plaque reduction assay using an authentic, infectious SARS-CoV-2 strain. The new assay was used to evaluate SARS-CoV-2 antibodies in serum from individuals with a broad range of COVID-19 symptoms, including intensive care unit (ICU) patients, health care workers (HCWs), and convalescent plasma donors. The highest neutralizing antibody titers were observed among ICU patients, followed by general hospitalized patients, HCWs and convalescent plasma donors. Our study highlights a wide phenotypic variation in human antibody responses against SARS-CoV-2, and demonstrates the efficacy of a novel lentivirus pseudotype assay for high-throughput serological surveys of neutralizing antibody titers in large cohorts.
Cong Zeng, John P. Evans, Rebecca Pearson, Panke Qu, Yi-Min Zheng, Richard T. Robinson, Luanne Hall-Stoodley, Jacob S. Yount, Sonal Pannu, Rama K. Mallampalli, Linda Saif, Eugene Oltz, Gerard Lozanski, Shan-Lu Liu
While the RV144 HIV vaccine trial lead to moderately reduced risk of HIV acquisition, emerging data from the repeat failure of the HVTN702 trial point to the critical need to re-examine the relationships between previously identified correlates of reduced risk of protection in the RV144 study. Specifically, the induction of V2-binding, non-IgA, IgG3 antibody responses with non-neutralizing functions were linked to reduced risk of infection in RV144 vaccinees. While each of these features was individually linked to reduced risk of infection, the relationships and interactions between these humoral immune signatures remain unclear. Thus, here we comprehensively profiled the humoral immune response in 300 RV144 vaccinees to specifically decipher the relationships between humoral biomarkers of protection and susceptibility. Here, we found that vaccine-specific IgG1, IgG3, and IgA were highly correlated. However, ratios of IgG1:IgG3:IgA provided new insights into subclass/isotype polyclonal functional regulation. For instance, in the absence of high IgG1 levels, IgG3 antibodies exhibited limited functional activity, pointing to IgG3 as a critical contributor, but not sole driver, of more effective antiviral humoral immunity. Moreover, in contrast to previous findings, higher IgA levels were linked to enhanced antibody effector function, including neutrophil phagocytosis (ADNP), complement deposition (ADCD) and NK degranulation (CD107a). Several IgA-associated functions were increased in infected vaccinees in a case:control dataset, suggesting that rather than blocking, IgA may have driven deleterious functions, thereby compromising immunity. These data highlight the interplay between IgG1, IgG3 and IgA, pointing to the critical need to deeply profile the relationships between subclass/isotype selection.
Stephanie Fischinger, Sepideh Dolatshahi, Madeleine F. Jennewein, Supachai Rerks-Ngarm, Punnee Pitisuttithum, Sorachai Nitayaphan, Nelson L. Michael, Sandhya Vasan, Margaret E Ackerman, Hendrik Streeck, Galit Alter
Long non-coding RNAs (lncRNAs) play important roles in regulating diverse cellular processes in the vessel wall, including atherosclerosis. RNAseq profiling of intimal lesions revealed a lncRNA, VINAS (Vascular INfllammation and Atherosclerosis lncRNA Sequence), that is enriched in the aortic intima and regulates vascular inflammation. Aortic intimal expression of VINAS fell with atherosclerotic progression and rose with regression. VINAS knockdown reduced atherosclerotic lesion formation by 55% in LDLR-/- mice, independent of effects on circulating lipids, by decreasing inflammation in the vessel wall. Loss- and gain-of-function studies in vitro demonstrated that VINAS serves as a critical regulator of inflammation by modulating NF-κB and MAPK signaling pathways. VINAS knockdown decreased the expression of key inflammatory markers, such as MCP-1, TNF-α, IL-1β , COX-2, in endothelial (EC), vascular smooth muscle cells, and bone marrow-derived macrophages. Moreover, VINAS silencing decreased expression of leukocyte adhesion molecules VCAM-1, E-selectin, and ICAM-1 and reduced monocyte adhesion to ECs. DEPDC4, an evolutionary conserved human ortholog of VINAS with ~74% homology, shows similar regulation in human and pig atherosclerotic specimens. DEPDC4 knockdown replicated VINAS’ anti-inflammatory effects in human ECs. These findings reveal a novel lncRNA that regulates vascular inflammation, with broad implications for vascular diseases.
Viorel Simion, Haoyang Zhou, Jacob B. Pierce, Dafeng Yang, Stefan Haemmig, Yevgenia Tesmenitsky, Galina Sukhova, Peter H. Stone, Peter Libby, Mark W. Feinberg
Amyotrophic Lateral Sclerosis (ALS) and FrontoTemporal Lobar Degeneration (FTLD), two incurable neurodegenerative disorders, share the same pathological hallmark named TDP43 (TAR DNA binding protein 43) proteinopathy. This event is characterized by a consistent cytoplasmic mislocalization and aggregation of the protein TDP43 which loses its physiological properties leading neurons to death. Antibody-based approaches are now emerging interventions in the field of neurodegenerative disorders. Here we tested the target specificity, in vivo distribution and therapeutic efficacy of a monoclonal full-length antibody, named E6, in TDP43 related conditions. We observed that the antibody recognizes specifically the cytoplasmic fraction of TDP43. We demonstrated its ability in targeting large neurons in the spinal cord of mice and in reducing TDP43 mislocalization and NF-B activation. We also recognized the proteasome as well as the lysosome machineries as possible mechanisms used by the antibody to reduce TDP43 proteinopathy. To our knowledge this is the first report showing the therapeutic efficacy and feasibility of a full-length antibody against TDP43 in reducing TDP43 proteinopathy in spinal neurons of an ALS/FTLD mouse model.
Silvia Pozzi, Philippe Codron, Genevieve Soucy, Laurence Renaud, Pierre Cordeau, Kallol Dutta, Christine Bareil, Jean-Pierre Julien
Primary varicella-zoster virus (VZV) infection in adults is often complicated by severe pneumonia, which is difficult to treat and associated with high morbidity and mortality. Here, the simian varicella virus (SVV) nonhuman primate (NHP) model was used to investigate the pathogenesis of varicella pneumonia. SVV infection resulted in transient fever, viremia and robust virus replication in alveolar pneumocytes and bronchus-associated lymphoid tissue. Clearance of infectious virus from lungs coincided with robust innate immune responses, leading to recruitment of inflammatory cells, mainly neutrophils and lymphocytes, and finally severe acute lung injury. SVV infection caused neutrophil activation and formation of neutrophil extracellular traps (NETs) in vitro and in vivo. Notably, NETs were also detected in lung and blood specimens of varicella pneumonia patients. Lung pathology in the SVV NHP model was associated with dysregulated expression of alveolar epithelial cell tight junction proteins (claudin-2, claudin-10 and claudin-18) and alveolar endothelial adherens junction protein VE-cadherin. Importantly, factors released by activated neutrophils, including NETs, were sufficient to reduce claudin-18 and VE-cadherin expression in NHP lung slice cultures. Collectively, the data indicate that local inflammatory responses involving activated neutrophils contribute to impaired alveolar epithelial/endothelial barrier integrity in varicella pneumonia and possibly other virus-induced acute lung injuries.
Werner J.D. Ouwendijk, Henk Jan van den Ham, Mark W. Delany, Jeroen J.A. van Kampen, Gijsbert P. van Nierop, Tamana Mehraban, Fatiha Zaaraoui-Boutahar, Wilfred F.J. van IJcken, Judith M.A. van den Brand, Rory D. De Vries, Arno C. Andeweg, Georges M.G.M. Verjans
Symbiotic microbial colonization through the establishment of the intestinal microbiome is critical to many intestinal functions including nutrient metabolism, intestinal barrier integrity and immune regulation. Recent studies suggest that education of the intestinal immunity maybe ongoing in utero. However, the drivers of this process are unknown. The microbiome and its byproducts are one potential source. Whether a fetal intestinal microbiome exists is controversial and if microbially derived metabolites are present in utero is unknown. Here, we aimed to determine whether bacterial DNA and microbially-derived metabolites can be detected in second trimester human intestinal samples. Although, we were unable to amplify bacterial DNA from fetal intestines, we report a unique fetal metabolomic intestinal profile with an abundance of bacterially derived and host derived metabolites commonly produced in response to microbiota. Though we did not directly assess their source and function, we hypothesize that these microbial associated metabolites come either from the maternal microbiome and are vertically transmitted to the fetus to prime the fetal immune system and prepare the gastrointestinal tract for postnatal microbial encounters or are produced locally by bacteria that was below our detection threshold.
Yujia Li, Jessica M. Toothaker, Shira Ben-Simon, Lital Ozeri, Ron Schweitzer, Blake T. McCourt, Collin C. McCourt, Lael Werner, Scott B. Snapper, Dror S. Shouval, Soliman Khatib, Omry Koren, Sameer Agnihorti, George Tseng, Liza Konnikova
Mottled skin pigmentation and solar lentigines from chronic photodamage with aging involves complex interactions between keratinocytes and melanocytes. However, the precise signaling mechanisms that could serve as therapeutic targets are unclear. Herein, we report that expression of nuclear factor erythroid 2-related factor 2 (NRF2), which regulates reduction–oxidation reactions, is altered in solar lentigines and photodamaged skin. Moreover, mottled skin pigmentation in humans could be treated with topical application of the NRF2 inducer sulforaphane (SF). Similarly, ultraviolet (UV) light-induced pigmentation of wildtype mouse ear skin could be treated or prevented with SF treatment. Conversely, SF treatment was unable to reduce UV-induced ear skin pigmentation in mice deficient in NRF2 or in mice with keratinocyte-specific conditional deletion of IL-6Rα. Taken together, NRF2 and IL-6Rα signaling are involved in the pathogenesis of UV-induced skin pigmentation and specific enhancement of NRF2-signaling could represent a potential therapeutic target.
Michelle L. Kerns, Robert J. Miller, Momina Mazhar, Angel S. Byrd, Nathan K. Archer, Bret L. Pinsker, Lance S. Lew, Carly A. Dillen, Ruizhi Wang, Lloyd S. Miller, Anna L. Chien, Sewon Kang
Chronic kidney disease (CKD) induces the failure of arteriovenous fistulas (AVF) and promotes the differentiation of vascular adventitial GLI1+ mesenchymal stem cells (GMCs). However, the roles of GMCs in forming neointima in AVFs remains unknown. GMCs isolated from CKD mice showed increased potential capacity of differentiation into myofibroblast-like cells. Increased activation of expression of PDGFRA and hedgehog (HH) signaling were detected in adventitial cells of AVFs from ESRD patients and CKD mice. PDGFRA was translocated and accumulated in early endosome when hedgehog signaling stimulates was activated. In endosome, PDGFRA mediated activation of TGFB1/SMAD signaling promoting GMCs differentiation into myofibroblast, extracellular matrix deposition, and vascular fibrosis. These responses resulted in neointima formation and AVF failure. Knockout (KO) of Pdgfra or inhibition of HH signaling in GMCs suppressed the differentiation of GMCs into myofibroblasts. In vivo, specific KO of Pdgfra inhibited GMC activation and vascular fibrosis, resulting in suppression of neointima formation and improvement of AVF patency despite CKD. Our findings could yield strategies for maintaining AVF functions.
Ke Song, Ying Qing, Qunying Guo, Eric K. Peden, Changyi Chen, William E. Mitch, Luan Truong, Jizhong Cheng
Actin-associated nonmuscle myosin II (NM2) motor proteins play critical roles in a myriad of cellular functions including endocytosis and organelle transport pathways. Cell type-specific expression and unique subcellular localization of the NM2 proteins, encoded by the Myh9 and Myh10 genes, in the mouse kidney tubules led us to hypothesize that these proteins have specialized functional roles within the renal epithelium. Inducible, conditional knockout (cKO) of Myh9 and Myh10 in the renal tubules of adult mice resulted in progressive kidney disease. Prior to overt renal tubular injury, we observed intracellular accumulation of the GPI-anchored protein uromodulin and gradual loss of Na+ K+ 2Cl- cotransporter from the apical membrane of the thick ascending limb (TAL) epithelia. The UMOD accumulation coincided with expansion of endoplasmic reticulum (ER) tubules, activation of ER stress and unfolded protein response pathways in Myh9&10 cKO kidneys. We conclude that NM2 proteins are required for localization and transport of UMOD and loss of function results in accumulation of UMOD and ER stress mediated progressive renal tubulointerstitial disease. These observations establish cell type-specific role(s) for NM2 proteins in regulation of specialized renal epithelial transport pathways and reveal the possibility that human kidney disease associated with MYH9 mutations could be of renal epithelial origin..
Karla L. Otterpohl, Brook W. Busselman, Ishara Ratnayake, Ryan G. Hart, Kimberly Hart, Claire Evans, Carrie L. Phillips, Jordan R. Beach, Phil Ahrenkiel, Bruce Molitoris, Kameswaran Surendran, Indra Chandrasekar
Canagliflozin (Cana) is an inhibitor of the sodium glucose transporter 2 (SGLT2), and is thought to act by blocking renal reuptake and intestinal absorption of glucose. Cana is FDA-approved for treatment of diabetes, and affords protection from cardiovascular and kidney diseases. In the context of the mouse Interventions Testing Program, genetically heterogeneous mice were given chow containing 180 ppm Cana at 7 months of age until their death. Cana extended median survival of male mice by 14%, with p < 0.001 by log-rank test. Cana also increased by 9% the age for 90th percentile survival (p < 0.001 by Wang/Allison test), with parallel effects seen at each of three test sites. Cana did not alter the distribution of inferred cause of death, nor of incidental pathology findings at end-of-life necropsies. No benefits were seen in female mice. The lifespan benefit of Cana is likely to reflect blunting of peak glucose levels, because similar longevity effects are seen in mice given acarbose, a diabetes drug that blocks glucose surges through a distinct mechanism, i.e. slowing breakdown of carbohydrate in the intestine. Interventions that control daily peak glucose levels deserve attention as possible preventive medicines to protect from a wide range of late-life neoplastic and degenerative diseases.
Richard A. Miller, David E. Harrison, David B. Allison, Molly A. Bogue, Lucas K. Debarba, Vivian Diaz, Elizabeth Fernandez, Andrzej T. Galecki, W. Timothy Garvey, Hashan Jayarathne, Navasuja Kumar, Martin Javors, Warren Ladiges, Francesca Macchiarini, James F. Nelson, Peter C. Reifsnyder, Nadia Rosenthal, Marianna Sadagurski, Adam B. Salmon, Daniel L. Smith, Jr., Jessica M. Snyder, David B. Lombard, Randy Strong
Somatic KRAS mutations are highly prevalent in many human cancers. In addition, a distinct spectrum of germline KRAS mutations cause developmental disorders called RASopathies. The mutant proteins encoded by these germline KRAS mutations are less biochemically and functionally activated than the mutant proteins found in cancer. We generated mice harboring conditional KrasLSL-P34R and KrasLSL-T58I “knock in” alleles and characterized the consequences of each mutation in vivo. Embryonic expression of KrasT58I resulted in craniofacial abnormalities reminiscent of RASopathy disorders, and these mice also exhibited hyperplastic growth of multiple organs, modest alterations in cardiac valvulogenesis, myocardial hypertrophy, and myeloproliferation. By contrast, embryonic KrasP34R expression resulted in early perinatal lethality from respiratory failure due to defective lung sacculation, which was associated with aberrant ERK activity in lung epithelial cells. Somatic Mx1-Cre-mediated activation in the hematopoietic compartment showed that KrasP34R and KrasT58I expression had distinct signaling effects despite causing a similar spectrum of hematologic diseases. These novel mouse strains are robust models for investigating the consequences of endogenous hyperactive K-Ras signaling in different developing and adult tissues, for comparing how oncogenic and germline K-Ras proteins perturb signaling networks and cell fate decisions, and for performing preclinical therapeutic trials.
Jasmine C. Wong, Pedro A. Perez-Mancera, Tannie Q. Huang, Jangkyung Kim, Joaquim Grego-Bessa, Maria del pilar Alzamora, Scott C. Kogan, Amnon Sharir, Susan H. Keefe, Carolina E. Morales, Denny Schanze, Pau Castel, Kentaro Hirose, Guo N. Huang, Martin Zenker, Dean Sheppard, Ophir Klein, David Tuveson, Benjamin S. Braun, Kevin Shannon
With an expanding aging population burdened with comorbidities, there is considerable interest in treatments that optimize health in later life. Acarbose (ACA), a drug used clinically to treat Type 2 diabetes (T2DM) can extend mouse lifespan, with greater effect in males than in females. Utilizing a genetically heterogeneous mouse model, we tested the ability of ACA to ameliorate functional, pathological and biochemical changes that occur during aging, and determined which of the effects of age and drug were sex-dependent. In both sexes, ACA prevented age-dependent loss of body mass, in addition to improving balance/coordination on an accelerating rotarod, rotarod endurance, and grip strength. Age-related cardiac hypertrophy was seen only in male mice, and this male-specific aging effect was attenuated by ACA. ACA-sensitive cardiac changes were associated with reduced activation of cardiac growth promoting pathways and increased abundance of peroxisomal proteins involved in lipid metabolism. ACA further ameliorated age-associated changes in cardiac lipid species, particularly lysophospholipids – changes which have previously been associated with aging, cardiac dysfunction and cardiovascular disease in humans. In the liver, ACA had pronounced effects on lipid handling in both sexes, reducing hepatic lipidosis during aging and shifting the liver lipidome in adulthood, particularly favoring reduced triglyceride (TAG) accumulation. Our results demonstrate that ACA, already in clinical use for T2DM, has broad-ranging anti-aging effects in multiple tissues, and may have the potential to increase physical function and alter lipid biology to preserve or improve health at older ages.
Jonathan J Herrera, Sean Louzon, Kaitlyn Pifer, Danielle Leander, Gennifer E. Merrihew, Jea H. Park, Kate Szczesniak, Jeremy A. Whitson, John E. Wilkinson, Oliver Fiehn, Michael J. MacCoss, Sharlene M. Day, Richard A. Miller, Michael Garratt
Patient-derived organoid models are proving to be a powerful platform for both basic and translational studies. Here we conduct a methodical analysis of pancreatic ductal adenocarcinoma (PDAC) tumor organoid drug response in paired PDX and PDX-derived organoid (PXO) models grown under WNT-free culture conditions. We report a specific relationship between Area Under the Curve value of organoid drug dose-response and in vivo tumor growth, irrespective of the drug treatment. In addition, we analyzed the glycome of PDX and PXO models and demonstrate that PXOs recapitulate the in vivo glycan landscape. In addition, we identify a core set of 57 N-glycans detected in all 10 models that represent 50-94% of the relative abundance of all N-glycans detected in each of the model. Lastly, we developed a secreted biomarker discovery pipeline using media supernatant of organoid cultures and identified potentially new extracellular vesicles (EV) protein markers. We validated our findings using plasma samples from patients with PDAC, benign gastrointestinal diseases, and chronic pancreatitis, and discover that four EV proteins are potential circulating biomarkers for PDAC. Thus, we demonstrate the utility of organoid cultures to not only model in vivo drug responses but also serve as a powerful platform for discovering clinically-actionable serologic biomarkers.
Ling Huang, Bruno Bockorny, Indranil Paul, Dipikaa Akshinthala, Pierre-Olivier Frappart, Omar Gandarilla, Arindam Bose, Veronica Sanchez-Gonzalez, Emily Rouse, Sylvain Lehoux, Nicole Pandell, Christine Lim, John G. Clohessy, Joseph E. Grossman, Raul S. Gonzalez, Sofia Perea, George Daaboul, Mandeep Sawhney, Steven D. Freedman, Alexander Kleger, Richard D. Cummings, Andrew Emili, Lakshmi Muthuswamy, Manuel Hidalgo, Senthil Muthuswamy