Triglyceride-derived fatty acids reduce autophagy in a model of retinal angiomatous proliferation
Heckel et al. report that excess dietary lipids sensed by free fatty acid receptor 1 restrain autophagy and energy metabolism in photoreceptors in mice, contributing to pathological neovascularization. The cover image shows confocal images of 3D reconstructed lectin-stained vessels of a very-low-density lipoprotein receptor–deficient mouse retina.
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Perspectives
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Abstract
MD-PhD trainees constitute an important source of physician-scientists. Persistence on this challenging path is facilitated by success in garnering independent (R grant) support from the NIH. Published research tracks academic appointments and global R01 success for MD-PhD trainees but has not included information on future funding success of individual MD-PhD predoctoral grant holders. Here, we used data from the NIH RePORTER database to identify and track the funding trajectory of physician-scientists who received predoctoral grant support through the F30 mechanism, which is specific for dual-degree candidates. Male and female F30 awardees did not differ in their success in garnering K (postdoctoral training) grants, but, among F30 grant awardees, men were 2.6 times more likely than women to receive R funding. These results underscore the need for analysis of factors that contribute to the disproportionate loss of NIH-supported female physician-scientists between the predoctoral F30 and the independent R grant–supported stages.
Authors
Shohini Ghosh-Choudhary, Neil Carleton, S. Mehdi Nouraie, Corrine R. Kliment, Richard A. Steinman
×Abstract
The average age when physician-scientists begin their career has been rising. Here, we focused on one contributor to this change: the increasingly common decision by candidates to postpone applying to MD-PhD programs until after college. This creates a time gap between college and medical school. Data were obtained from 3544 trainees in 73 programs, 72 program directors, and AAMC databases. From 2013 to 2020, the prevalence of gaps rose from 53% to 75%, with the time usually spent doing research. Gap prevalence for MD students also increased but not to the same extent and for different reasons. Differences by gender, underrepresented status, and program size were minimal. Most candidates who took a gap did so because they believed it would improve their chances of admission, but gaps were as common among those not accepted to MD-PhD programs as among those who were. Many program directors preferred candidates with gaps, believing without evidence that gaps reflects greater commitment. Although candidates with gaps were more likely to have a publication at the time of admission, gaps were not associated with a shorter time to degree nor have they been shown to improve outcomes. Together, these observations raise concerns that, by promoting gaps after college, current admissions practices have had unintended consequences without commensurate advantages.
Authors
Lawrence F. Brass, Reiko Maki Fitzsimonds, Myles H. Akabas
×Abstract
Postgraduate physician-scientist training programs (PSTPs) enhance the experiences of physician-scientist trainees following medical school graduation. PSTPs usually span residency and fellowship training, but this varies widely by institution. Applicant competitiveness for these programs would be enhanced, and unnecessary trainee anxiety relieved, by a clear understanding of what factors define a successful PSTP matriculant. Such information would also be invaluable to PSTP directors and would allow benchmarking of their admissions processes with peer programs. We conducted a survey of PSTP directors across the US to understand the importance they placed on components of PSTP applications. Of 41 survey respondents, most were from internal medicine and pediatrics residency programs. Of all components in the application, two elements were considered very important by a majority of PSTP directors: (a) having one or more first-author publications and (b) the thesis advisor’s letter. Less weight was consistently placed on factors often considered more relevant for non-physician-scientist postgraduate applicants — such as US Medical Licensing Examination scores, awards, and leadership activities. The data presented here highlight important metrics for PSTP applicants and directors and suggest that indicators of scientific productivity and commitment to research outweigh traditional quantitative measures of medical school performance.
Authors
Emily J. Gallagher, Don C. Rockey, Christopher D. Kontos, Jatin M. Vyas, Lawrence F. Brass, Patrick J. Hu, Carlos M. Isales, Olujimi A. Ajijola, W. Kimryn Rathmell, Paul R. Conlin, Robert A. Baiocchi, Barbara I. Kazmierczak, Myles H. Akabas, Christopher S. Williams
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Research Articles
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Abstract
The current strategy to detect acute injury of kidney tubular cells relies on changes in serum levels of creatinine. Yet serum creatinine (sCr) is a marker of both functional and pathological processes and does not adequately assay tubular injury. In addition, sCr may require days to reach diagnostic thresholds, yet tubular cells respond with programs of damage and repair within minutes or hours. To detect acute responses to clinically relevant stimuli, we created mice expressing Rosa26-floxed-stop uracil phosphoribosyltransferase (Uprt) and inoculated 4-thiouracil (4-TU) to tag nascent RNA at selected time points. Cre-driven 4-TU–tagged RNA was isolated from intact kidneys and demonstrated that volume depletion and ischemia induced different genetic programs in collecting ducts and intercalated cells. Even lineage-related cell types expressed different genes in response to the 2 stressors. TU tagging also demonstrated the transient nature of the responses. Because we placed Uprt in the ubiquitously active Rosa26 locus, nascent RNAs from many cell types can be tagged in vivo and their roles interrogated under various conditions. In short, 4-TU labeling identifies stimulus-specific, cell-specific, and time-dependent acute responses that are otherwise difficult to detect with other technologies and are entirely obscured when sCr is the sole metric of kidney damage.
Authors
Tian Huai Shen, Jacob Stauber, Katherine Xu, Alexandra Jacunski, Neal Paragas, Miriam Callahan, Run Banlengchit, Abraham D. Levitman, Beatriz Desanti De Oliveira, Andrew Beenken, Madeleine S. Grau, Edwin Mathieu, Qingyin Zhang, Yuanji Li, Tejashree Gopal, Nathaniel Askanase, Siddarth Arumugam, Sumit Mohan, Pamela I. Good, Jacob S. Stevens, Fangming Lin, Samuel K. Sia, Chyuan-Sheng Lin, Vivette D’Agati, Krzysztof Kiryluk, Nicholas P. Tatonetti, Jonathan Barasch
×Abstract
Ca2+ is critical for cardiac electrical conduction and contractility, and aberrant Ca2+ homeostasis causes arrhythmia and heart failure. Chromatin remodeling modulates gene expression involved in cardiac sarcomere assembly and postnatal heart function. However, the chromatin-remodeling regulatory mechanism of cardiac Ca2+ homeostasis is unknown. Here, we found that Znhit1, a core subunit of the SRCAP remodeling complex, was essential for heart function. Deletion of Znhit1 in postnatal hearts of mice resulted in arrhythmia, idiopathic vacuolar cardiomyopathy, rapid heart failure, and premature sudden death. In addition, the level of Casq1, a sarcoplasmic reticulum Ca2+ regulatory protein, was massively elevated while SERCA2a showed reduced protein level. Mechanistically, the Znhit1 modulated the expression of Casq1 and SERCA2a by depositing H2A.Z at their promoters. Deletion of Casq1 could substantially alleviate the vacuolar formation in Znhit1 Casq1 KO mice. These findings demonstrate that Znhit1 is required for postnatal heart function and maintains cardiac Ca2+ homeostasis and that accumulation of Casq1 might be a causative factor for vacuolar cardiomyopathy.
Authors
Yingchao Shi, Wenli Fan, Mingjie Xu, Xinhua Lin, Wukui Zhao, Zhongzhou Yang
×Abstract
The chloride channel dysfunction caused by deleterious cystic fibrosis transmembrane conductance regulator (CFTR) variants generally correlates with severity of cystic fibrosis (CF). However, 3 adults bearing the common severe variant p.Phe508del (legacy: F508del) and a deletion variant in an ivacaftor binding region of CFTR (p.Phe312del; legacy: F312del) manifested only elevated sweat chloride concentration (sw[Cl–]; 87–105 mEq/L). A database review of 25 individuals with F312del and a CF-causing variant revealed elevated sw[Cl–] (75–123 mEq/L) and variable CF features. F312del occurs at a higher-than-expected frequency in the general population, confirming that individuals with F312del and a CF-causing variant do not consistently develop overt CF features. In primary nasal cells, CFTR bearing F312del and F508del generated substantial chloride transport (66.0% ± 4.5% of WT-CFTR) but did not respond to ivacaftor. Single-channel analysis demonstrated that F312del did not affect current flow through CFTR, minimally altered gating, and ablated the ivacaftor response. When expressed stably in CF bronchial epithelial (CFBE41o–) cells, F312del-CFTR demonstrated residual function (50.9% ± 3.3% WT-CFTR) and a subtle decrease in forskolin response compared with WT-CFTR. F312del provides an exception to the established correlation between CFTR chloride transport and CF phenotype and informs our molecular understanding of ivacaftor response.
Authors
Karen S. Raraigh, Kathleen C. Paul, Jennifer L. Goralski, Erin N. Worthington, Anna V. Faino, Stanley Sciortino, Yiting Wang, Melis A. Aksit, Hua Ling, Derek L. Osorio, Frankline M. Onchiri, Shivani U. Patel, Christian A. Merlo, Kristina Montemayor, Ronald L. Gibson, Natalie E. West, Amita Thakerar, Robert J. Bridges, David N. Sheppard, Neeraj Sharma, Garry R. Cutting
×Abstract
Mucosal healing is a key treatment goal for inflammatory bowel disease, and adequate epithelial regeneration is required for an intact gut epithelium. However, the underlying mechanism for mucosal healing is unclear. Long noncoding RNAs (lncRNAs) have been reported to be involved in the development of inflammatory bowel disease. Here, we report that a lncRNA named Gm31629 decreased in intestinal epithelial cells in response to inflammatory stimulation. Gm31629 deficiency led to exacerbated intestinal inflammation and delayed epithelial regeneration in dextran sulfate sodium–induced (DSS-induced) colitis model. Mechanistically, Gm31629 promoted E2F pathways and cell proliferation by stabilizing Y-box protein 1 (YB-1), thus facilitating epithelial regeneration. Genetic overexpression of Gm31629 protected against DSS-induced colitis in vivo. Theaflavin 3-gallate, a natural compound mimicking Gm31629, alleviated DSS-induced epithelial inflammation and mucosal damage. These results demonstrate an essential role of lncRNA Gm31629 in linking intestinal inflammation and epithelial cell proliferation, providing a potential therapeutic approach to inflammatory bowel disease.
Authors
Xu Feng, Ye Xiao, Jian He, Mi Yang, Qi Guo, Tian Su, Yan Huang, Jun Yi, Chang-Jun Li, Xiang-Hang Luo, Xiao-Wei Liu, Hai-Yan Zhou
×Abstract
Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as low-flow sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation—the physical removal of cilia from the cell surface—is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress would manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro, and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer cilia-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared with healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (ARL13b) on their surface after adhesion to brain ECs. Brain ECs postinteraction with sickle RBCs showed high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreased cilia protein levels on RBCs and rescued ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered-flow conditions, which could serve as a surrogate biomarker of the damaged endothelium.
Authors
Ankan Gupta, Karthikeyan Thirugnanam, Madhan Thamilarasan, Ashraf M. Mohieldin, Hadeel T. Zedan, Shubhangi Prabhudesai, Meghan R. Griffin, Andrew D. Spearman, Amy Pan, Sean P. Palecek, Huseyin C. Yalcin, Surya M. Nauli, Kevin R. Rarick, Rahima Zennadi, Ramani Ramchandran
×CFTR-mediated monocyte/macrophage dysfunction revealed by cystic fibrosis proband-parent comparisons
Abstract
Cystic fibrosis (CF) is an inherited disorder caused by biallelic mutations of the CF transmembrane conductance regulator (CFTR) gene. Converging evidence suggests that CF carriers with only 1 defective CFTR copy are at increased risk for CF-related conditions and pulmonary infections, but the molecular mechanisms underpinning this effect remain unknown. We performed transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) of CF child-parent trios (proband, father, and mother) and healthy control (HC) PBMCs or THP-1 cells incubated with the plasma of these participants. Transcriptomic analyses revealed suppression of cytokine-enriched immune-related genes (IL-1β, CXCL8, CREM), implicating lipopolysaccharide tolerance in innate immune cells (monocytes) of CF probands and their parents. These data suggest that a homozygous as well as a heterozygous CFTR mutation can modulate the immune/inflammatory system. This conclusion is further supported by the finding of lower numbers of circulating monocytes in CF probands and their parents, compared with HCs, and the abundance of mononuclear phagocyte subsets, which correlated with Pseudomonas aeruginosa infection, lung disease severity, and CF progression in the probands. This study provides insight into demonstrated CFTR-related innate immune dysfunction in individuals with CF and carriers of a CFTR mutation that may serve as a target for personalized therapy.
Authors
Xi Zhang, Camille M. Moore, Laura D. Harmacek, Joanne Domenico, Vittobai Rashika Rangaraj, Justin E. Ideozu, Jennifer R. Knapp, Katherine J. Woods, Stephanie Jump, Shuang Jia, Jeremy W. Prokop, Russell Bowler, Martin J. Hessner, Erwin W. Gelfand, Hara Levy
×Abstract
CIC-DUX4 rearrangements define an aggressive and chemotherapy-insensitive subset of undifferentiated sarcomas. The CIC-DUX4 fusion drives oncogenesis through direct transcriptional upregulation of cell cycle and DNA replication genes. Notably, CIC-DUX4–mediated CCNE1 upregulation compromises the G1/S transition to confer a dependence on the G2/M cell cycle checkpoint. Through an integrative transcriptional and kinase activity screen using patient-derived specimens, we now show that CIC-DUX4 sarcomas depend on the G2/M checkpoint regulator WEE1 as part of an adaptive survival mechanism. Specifically, CIC-DUX4 sarcomas depended on WEE1 activity to limit DNA damage and unscheduled mitotic entry. Consequently, genetic or pharmacologic WEE1 inhibition in vitro and in vivo led to rapid DNA damage–associated apoptotic induction of patient-derived CIC-DUX4 sarcomas. Thus, we identified WEE1 as a vulnerability targetable by therapeutic intervention in CIC-DUX4 sarcomas.
Authors
Rovingaile Kriska M. Ponce, Nicholas J. Thomas, Nam Q. Bui, Tadashi Kondo, Ross A. Okimoto
×Abstract
BACKGROUND Pathophysiology of type 1 diabetes (T1D) is illustrated by pancreatic islet infiltration of inflammatory lymphocytes, including CD8+ T cells; however, the molecular factors mediating their recruitment remain unknown. We hypothesized that single-cell RNA-sequencing (scRNA-Seq) analysis of immune cell populations isolated from islets of NOD mice captured gene expression dynamics providing critical insight into autoimmune diabetes pathogenesis.METHODS Pancreatic sections from human donors were investigated, including individuals with T1D, autoantibody-positive (aAb+) individuals, and individuals without diabetes who served as controls. IHC was performed to assess islet hormones and both novel and canonical immune cell markers that were identified from unbiased, state-of-the-art workflows after reanalyzing murine scRNA-Seq data sets.RESULTS Computational workflows identified cell adhesion molecule 1–mediated (Cadm1-mediated) homotypic binding among the most important intercellular interactions among all cell clusters, as well as Cadm1 enrichment in macrophages and DCs from pancreata of NOD mice. Immunostaining of human pancreata revealed an increased number of CADM1+glucagon+ cells adjacent to CD8+ T cells in sections from T1D and aAb+ donors compared with individuals without diabetes. Numbers of CADM1+CD68+ peri-islet myeloid cells adjacent to CD8+ T cells were also increased in pancreatic sections from both T1D and aAb+ donors compared with individuals without diabetes.CONCLUSION Increased detection of CADM1+ cells adjacent to CD8+ T cells in pancreatic sections of individuals with T1D and those who were aAb+ validated workflows and indicated CADM1-mediated intercellular contact may facilitate islet infiltration of cytotoxic T lymphocytes and serve as a potential therapeutic target for preventing T1D pathogenesis.FUNDING The Johns Hopkins All Children’s Foundation Institutional Research Grant Program, the National Natural Science Foundation of China (grant 82071326), and the Deutsche Forschungsgemeinschaft (grants 431549029–SFB1451, EXC2030–390661388, and 411422114-GRK2550).
Authors
Chandan Sona, Yu-Te Yeh, Andreas Patsalos, Laszlo Halasz, Xin Yan, Natalia L. Kononenko, Laszlo Nagy, Matthew N. Poy
×Abstract
Sporozoite-based approaches currently represent the most effective vaccine strategies for induction of sterile protection against Plasmodium falciparum (Pf) malaria. Clinical development of subunit vaccines is almost exclusively centered on the circum-sporozoite protein (CSP), an abundantly expressed protein on the sporozoite membrane. Anti-CSP antibodies are able to block sporozoite invasion and development in human hepatocytes and subsequently prevent clinical malaria. Here, we have investigated whether sporozoite-induced human antibodies with specificities different from CSP can reduce Pf-liver stage development. IgG preparations were obtained from 12 volunteers inoculated with a protective immunization regime of whole sporozoites under chloroquine prophylaxis. These IgGs were depleted for CSP specificity by affinity chromatography. Recovered non-CSP antibodies were tested for sporozoite membrane binding and for functional inhibition of sporozoite invasion of a human hepatoma cell line and hepatocytes both in vitro and in vivo. Postimmunization IgGs depleted for CS specificity of 9 of 12 donors recognized sporozoite surface antigens. Samples from 5 of 12 donors functionally reduced parasite-liver cell invasion or development using the hepatoma cell line HC-04 and FRG-huHep mice containing human liver cells. The combined data provide clear evidence that non-CSP proteins, as yet undefined, do represent antibody targets for functional immunity against Pf parasites responsible for malaria.
Authors
Amanda Fabra-García, Annie S.P. Yang, Marije C. Behet, Zen Yap, Youri van Waardenburg, Swarnendu Kaviraj, Kjerstin Lanke, Geert-Jan van Gemert, Matthijs M. Jore, Teun Bousema, Robert W. Sauerwein
×Abstract
Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by myofibroblast accumulation and progressive lung scarring. To identify transcriptional gene programs driving persistent lung fibrosis in aging, we performed RNA-Seq on lung fibroblasts isolated from young and aged mice during the early resolution phase after bleomycin injury. We discovered that, relative to injured young fibroblasts, injured aged fibroblasts exhibited a profibrotic state characterized by elevated expression of genes implicated in inflammation, matrix remodeling, and cell survival. We identified the proviral integration site for Moloney murine leukemia virus 1 (PIM1) and its target nuclear factor of activated T cells-1 (NFATc1) as putative drivers of the sustained profibrotic gene signatures in injured aged fibroblasts. PIM1 and NFATc1 transcripts were enriched in a pathogenic fibroblast population recently discovered in IPF lungs, and their protein expression was abundant in fibroblastic foci. Overexpression of PIM1 in normal human lung fibroblasts potentiated their fibrogenic activation, and this effect was attenuated by NFATc1 inhibition. Pharmacological inhibition of PIM1 attenuated IPF fibroblast activation and sensitized them to apoptotic stimuli. Interruption of PIM1 signaling in IPF lung explants ex vivo inhibited prosurvival gene expression and collagen secretion, suggesting that targeting this pathway may represent a therapeutic strategy to block IPF progression.
Authors
Tho X. Pham, Jisu Lee, Jiazhen Guan, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Steven K. Huang, Daniel J. Tschumperlin, Giovanni Ligresti
×Abstract
Dyslipidemia and autophagy have been implicated in the pathogenesis of blinding neovascular age-related macular degeneration (NV-AMD). VLDL receptor (VLDLR), expressed in photoreceptors with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acids. Since fatty acid uptake is reduced in Vldlr–/– tissues, more remain in circulation, and the retina is fuel deficient, driving the formation in mice of neovascular lesions reminiscent of retinal angiomatous proliferation (RAP), a subtype of NV-AMD. Nutrient scarcity and energy failure are classically mitigated by increasing autophagy. We found that excess circulating lipids restrained retinal autophagy, which contributed to pathological angiogenesis in the Vldlr–/– RAP model. Triglyceride-derived fatty acid sensed by free fatty acid receptor 1 (FFAR1) restricted autophagy and oxidative metabolism in photoreceptors. FFAR1 suppressed transcription factor EB (TFEB), a master regulator of autophagy and lipid metabolism. Reduced TFEB, in turn, decreased sirtuin-3 expression and mitochondrial respiration. Metabolomic signatures of mouse RAP-like retinas were consistent with a role in promoting angiogenesis. This signature was also found in human NV-AMD vitreous. Restoring photoreceptor autophagy in Vldlr–/– retinas, either pharmacologically or by deleting Ffar1, enhanced metabolic efficiency and suppressed pathological angiogenesis. Dysregulated autophagy by circulating lipids might therefore contribute to the energy failure of photoreceptors driving neovascular eye diseases, and FFAR1 may be a target for intervention.
Authors
Emilie Heckel, Gael Cagnone, Tapan Agnihotri, Bertan Cakir, Ashim Das, Jin Sung Kim, Nicholas Kim, Geneviève Lavoie, Anu Situ, Sheetal Pundir, Ye Sun, Florian Wünnemann, Kerry A. Pierce, Courtney Dennis, Grant A. Mitchell, Sylvain Chemtob, Flavio A. Rezende, Gregor Andelfinger, Clary B. Clish, Philippe P. Roux, Przemyslaw Sapieha, Lois E.H. Smith, Jean-Sébastien Joyal
×Abstract
Cytomegalovirus (CMV) is a globally ubiquitous pathogen with a seroprevalence of approximately 50% in the United Kingdom. CMV infection induces expansion of immunosenescent T cell and NK cell populations, with these cells demonstrating lower responsiveness to activation and reduced functionality upon infection and vaccination. In this study, we found that CMV+ participants had normal T cell responses after a single-dose or homologous vaccination with the viral vector chimpanzee adenovirus developed by the University of Oxford (ChAdOx1). CMV seropositivity was associated with reduced induction of IFN-γ–secreting T cells in a ChAd-Modified Vaccinia Ankara (ChAd-MVA) viral vector vaccination trial. Analysis of participants receiving a single dose of ChAdOx1 demonstrated that T cells from CMV+ donors had a more terminally differentiated profile of CD57+PD1+CD4+ T cells and CD8+ T cells expressing less IL-2Rα (CD25) and fewer polyfunctional CD4+ T cells 14 days after vaccination. NK cells from CMV-seropositive individuals also had a reduced activation profile. Overall, our data suggest that although CMV infection enhances immunosenescence of T and NK populations, it does not affect antigen-specific T cell IFN-γ secretion or antibody IgG production after vaccination with the current ChAdOx1 nCoV-19 vaccination regimen, which has important implications given the widespread use of this vaccine, particularly in low- and middle-income countries with high CMV seroprevalence.
Authors
Hannah R. Sharpe, Nicholas M. Provine, Georgina S. Bowyer, Pedro Moreira Folegatti, Sandra Belij-Rammerstorfer, Amy Flaxman, Rebecca Makinson, Adrian V.S. Hill, Katie J. Ewer, Andrew J. Pollard, Paul Klenerman, Sarah Gilbert, Teresa Lambe
×Abstract
BACKGROUND Paclitaxel chemotherapy frequently induces dose-limiting sensory axonal polyneuropathy. Given that sensory symptoms are challenging to assess objectively in clinical practice, an easily accessible biomarker for chemotherapy-induced polyneuropathy (CIPN) holds the potential to improve early diagnosis. Here, we describe neurofilament light chain (NFL), a marker for neuroaxonal damage, as a translational surrogate marker for CIPN.METHODS NFL concentrations were measured in an in vitro model of CIPN, exposing induced pluripotent stem cell–derived sensory neurons (iPSC-DSNs) to paclitaxel. Patients with breast or ovarian cancer undergoing paclitaxel chemotherapy, breast cancer control patients without chemotherapy, and healthy controls were recruited in a cohort study and examined before chemotherapy (V1) and after 28 weeks (V2, after chemotherapy). CIPN was assessed by the validated Total Neuropathy Score reduced (TNSr), which combines patient-reported symptoms with data from clinical examinations. Serum NFL (NFLs) concentrations were measured at both visits with single-molecule array technology.RESULTS NFL was released from iPSC-DSNs upon paclitaxel incubation in a dose- and time-dependent manner and was inversely correlated with iPSC-DSN viability. NFLs strongly increased in paclitaxel-treated patients with CIPN, but not in patients receiving chemotherapy without CIPN or controls, resulting in an 86% sensitivity and 87% specificity. An NFLs increase of +36 pg/mL from baseline was associated with a predicted CIPN probability of more than 0.5.CONCLUSION NFLs was correlated with CIPN development and severity, which may guide neurotoxic chemotherapy in the future.TRIAL REGISTRATION ClinicalTrials.gov NCT02753036.FUNDING Deutsche Forschungsgemeinschaft (EXC 257 NeuroCure), BMBF (Center for Stroke Research Berlin, 01 EO 0801), Animalfree Research, EU Horizon 2020 Innovative Medicines Initiative 2 Joint Undertaking (TransBioLine, 821283), Charité 3R — Replace — Reduce — Refine.
Authors
Petra Huehnchen, Christian Schinke, Nikola Bangemann, Adam D. Dordevic, Johannes Kern, Smilla K. Maierhof, Lois Hew, Luca Nolte, Peter Körtvelyessy, Jens C. Göpfert, Klemens Ruprecht, Christopher J. Somps, Jens-Uwe Blohmer, Jalid Sehouli, Matthias Endres, Wolfgang Boehmerle
×Abstract
Malignant pleural effusion (MPE) is an incurable common manifestation of many malignancies. Its formation is orchestrated by complex interactions among tumor cells, inflammatory cells, and the vasculature. Tumor-associated macrophages present the dominant inflammatory population of MPE, and M2 macrophage numbers account for dismal prognosis. M2 polarization is known to be triggered by CSF1/CSF1 receptor (CSF1R) signaling. We hypothesized that CSF1R+ M2 macrophages favor MPE formation and could be therapeutically targeted to limit MPE. We generated mice with CSF1R-deficient macrophages and induced lung and colon adenocarcinoma–associated MPE. We also examined the therapeutic potential of a clinically relevant CSF1R inhibitor (BLZ945) in lung and colon adenocarcinoma–induced experimental MPE. We showed that CSF1R+ macrophages promoted pleural fluid accumulation by enhancing vascular permeability, destabilizing tumor vessels, and favoring immune suppression. We also showed that CSF1R inhibition limited MPE in vivo by reducing vascular permeability and neoangiogenesis and impeding tumor progression. This was because apart from macrophages, CSF1R signals in cancer-associated fibroblasts leading to macrophage inflammatory protein 2 secretion triggered the manifestation of suppressive and angiogenic properties in macrophages upon CXCR2 paracrine activation. Pharmacological targeting of the CSF1/CSF1R axis can therefore be a vital strategy for limiting MPE.
Authors
Chrysavgi N. Kosti, Photene C. Vaitsi, Apostolos G. Pappas, Marianthi P. Iliopoulou, Katherina K. Psarra, Sophia F. Magkouta, Ioannis T. Kalomenidis
×Abstract
In this investigation, a potentially novel signaling pathway in gentamicin-induced acute kidney injury—worsened by overexpression of proximal tubular enzyme, myo-inositol oxygenase (MIOX)—was elucidated. WT, MIOX-transgenic (MIOX-Tg), and MIOX-KO mice were used. Gentamicin was administered to induce tubular injury. MIOX-Tg mice had severe tubular lesions associated with increased serum creatinine and proteinuria. Lesions were relatively mild, with no rise in serum creatinine and no albuminuria in MIOX-KO mice. Transfection of HK-2 cells with MIOX-pcDNA led to increased gentamicin-induced reactive oxygen species (ROS). Marked increase of ROS-mediated lipid hydroperoxidation was noted in MIOX-Tg mice, as assessed by 4-HNE staining. This was associated with increased expression of arachidonate 12-lipoxygenase (ALOX-12) and generation of 12-hydroxyeicosatetraenoic acid (12-HETE). In addition, notable monocyte/macrophage influx, upregulation of NF-κB and inflammatory cytokines, and apoptosis was observed in MIOX-Tg mice. Treatment of cells with ALOX-12 siRNA abolished gentamicin-mediated induction of cytokines and 12-HETE generation. HETE-12 treatment promoted this effect, along with upregulation of various signaling kinases and activation of GPCR31. Similarly, treatment of cells or mice with the ALOX-12 inhibitor ML355 attenuated inflammatory response, kinase signaling cascade, and albuminuria. Collectively, these studies highlight a potentially novel mechanism (i.e., the ROS/ALOX-12/12-HETE/GPR31 signaling axis) relevant to gentamicin-induced nephrotoxicity modulated by MIOX.
Authors
Isha Sharma, Yingjun Liao, Xiaoping Zheng, Yashpal S. Kanwar
×Abstract
Type 2 alveolar epithelial cells (AT2s), facultative progenitor cells of the lung alveolus, play a vital role in the biology of the distal lung. In vitro model systems that incorporate human cells, recapitulate the biology of primary AT2s, and interface with the outside environment could serve as useful tools to elucidate functional characteristics of AT2s in homeostasis and disease. We and others recently adapted human induced pluripotent stem cell–derived AT2s (iAT2s) for air-liquid interface (ALI) culture. Here, we comprehensively characterize the effects of ALI culture on iAT2s and benchmark their transcriptional profile relative to both freshly sorted and cultured primary human fetal and adult AT2s. We find that iAT2s cultured at ALI maintain an AT2 phenotype while upregulating expression of transcripts associated with AT2 maturation. We then leverage this platform to assay the effects of exposure to clinically significant, inhaled toxicants including cigarette smoke and electronic cigarette vapor.
Authors
Kristine M. Abo, Julio Sainz de Aja, Jonathan Lindstrom-Vautrin, Konstantinos-Dionysios Alysandratos, Alexsia Richards, Carolina Garcia-de-Alba, Jessie Huang, Olivia T. Hix, Rhiannon B. Werder, Esther Bullitt, Anne Hinds, Isaac Falconer, Carlos Villacorta-Martin, Rudolf Jaenisch, Carla F. Kim, Darrell N. Kotton, Andrew A. Wilson
×Abstract
Two HER2-specific mAbs, trastuzumab and pertuzumab (T+P), combined with chemotherapy comprise standard-of-care treatment for advanced HER2+ breast cancers (BC). While this antibody combination is highly effective, its synergistic mechanism-of-action (MOA) remains incompletely understood. Past studies have suggested that the synergy underlying this combination occurs through the different mechanisms elicited by these antibodies, with pertuzumab suppressing HER2 heterodimerization and trastuzumab inducing antitumor immunity. However, in vivo evidence for this synergy is lacking. In this study, we found that the therapeutic efficacy elicited by their combination occurs through their joint ability to activate the classical complement pathway, resulting in both complement-dependent cytotoxicity and complement-dependent cellular phagocytosis of HER2+ tumors. We also demonstrate that tumor C1q expression is positively associated with survival outcome in HER2+ BC patients and that complement regulators CD55 and CD59 were inversely correlated with outcome, suggesting the clinical importance of complement activity. Accordingly, inhibition of C1q in mice abolished the synergistic therapeutic activity of T+P therapy, whereas knockdown of CD55 and CD59 expression enhanced T+P efficacy. In summary, our study identifies classical complement activation as a significant antitumor MOA for T+P therapy that may be functionally enhanced to potentially augment clinical therapeutic efficacy.
Authors
Li-Chung Tsao, Erika J. Crosby, Timothy N. Trotter, Junping Wei, Tao Wang, Xiao Yang, Amanda N. Summers, Gangjun Lei, Christopher A. Rabiola, Lewis A. Chodosh, William J. Muller, Herbert Kim Lyerly, Zachary C. Hartman
×Abstract
Prion protein (PrP) concentration controls the kinetics of prion replication and is a genetically and pharmacologically validated therapeutic target for prion disease. In order to evaluate PrP concentration as a pharmacodynamic biomarker and assess its contribution to known prion disease risk factors, we developed and validated a plate-based immunoassay reactive for PrP across 6 species of interest and applicable to brain and cerebrospinal fluid (CSF). PrP concentration varied dramatically across different brain regions in mice, cynomolgus macaques, and humans. PrP expression did not appear to contribute to the known risk factors of age, sex, or common PRNP genetic variants. CSF PrP was lowered in the presence of rare pathogenic PRNP variants, with heterozygous carriers of P102L displaying 55%, and D178N just 31%, of the CSF PrP concentration of mutation-negative controls. In rodents, pharmacologic reduction of brain Prnp RNA was reflected in brain parenchyma PrP and, in turn in CSF PrP, validating CSF as a sampling compartment for the effect of PrP-lowering therapy. Our findings support the use of CSF PrP as a pharmacodynamic biomarker for PrP-lowering drugs and suggest that relative reduction from individual baseline CSF PrP concentration may be an appropriate marker for target engagement.
Authors
Meredith A. Mortberg, Hien T. Zhao, Andrew G. Reidenbach, Juliana E. Gentile, Eric Kuhn, Jill O’Moore, Patrick M. Dooley, Theresa R. Connors, Curt Mazur, Shona W. Allen, Bianca A. Trombetta, Alison McManus, Matthew R. Moore, Jiewu Liu, Deborah E. Cabin, Holly B. Kordasiewicz, Joel Mathews, Steven E. Arnold, Sonia M. Vallabh, Eric Vallabh Minikel
×A 4-week high-AGE diet does not impair glucose metabolism and vascular function in obese individuals
Abstract
BACKGROUND Accumulation of advanced glycation endproducts (AGEs) may contribute to the pathophysiology of type 2 diabetes and its vascular complications. AGEs are widely present in food, but whether restricting AGE intake improves risk factors for type 2 diabetes and vascular dysfunction is controversial.METHODS Abdominally obese but otherwise healthy individuals were randomly assigned to a specifically designed 4-week diet low or high in AGEs in a double-blind, parallel design. Insulin sensitivity, secretion, and clearance were assessed by a combined hyperinsulinemic-euglycemic and hyperglycemic clamp. Micro- and macrovascular function, inflammation, and lipid profiles were assessed by state-of-the-art in vivo measurements and biomarkers. Specific urinary and plasma AGEs Nε-(carboxymethyl)lysine (CML), Nε-(1-carboxyethyl)lysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) were assessed by mass spectrometry.RESULTS In 73 individuals (22 males, mean ± SD age and BMI 52 ± 14 years, 30.6 ± 4.0 kg/m2), intake of CML, CEL, and MG-H1 differed 2.7-, 5.3-, and 3.7-fold between the low- and high-AGE diets, leading to corresponding changes of these AGEs in urine and plasma. Despite this, there was no difference in insulin sensitivity, secretion, or clearance; micro- and macrovascular function; overall inflammation; or lipid profile between the low and high dietary AGE groups (for all treatment effects, P > 0.05).CONCLUSION This comprehensive RCT demonstrates very limited biological consequences of a 4-week diet low or high in AGEs in abdominally obese individuals.TRIAL REGISTRATION Clinicaltrials.gov, NCT03866343; trialregister.nl, NTR7594.FUNDING Diabetesfonds and ZonMw.
Authors
Armand M.A. Linkens, Alfons J.H.M. Houben, Petra M. Niessen, Nicole E.G. Wijckmans, Erica E.C. de Goei, Mathias D.G. Van den Eynde, Jean L.J.M. Scheijen, Marjo P.H. van den Waarenburg, Andrea Mari, Tos T.J.M. Berendschot, Lukas Streese, Henner Hanssen, Martien C.J.M. van Dongen, Christel C.J.A.W. van Gool, Coen D.A. Stehouwer, Simone J.M.P. Eussen, Casper G. Schalkwijk
×Abstract
Kawasaki disease (KD) is the leading cause of noncongenital heart disease in children. Studies in mice and humans propound the NLRP3/IL-1β pathway as the principal driver of KD pathophysiology. Endoplasmic reticulum (ER) stress can activate the NLRP3 inflammasome, but the potential implication of ER stress in KD pathophysiology has not been investigated to our knowledge. We used human patient data and the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis to characterize the impact of ER stress on the development of cardiovascular lesions. KD patient transcriptomics and single-cell RNA sequencing of the abdominal aorta from LCWE-injected mice revealed changes in the expression of ER stress genes. Alleviating ER stress genetically, by conditional deletion of inositol-requiring enzyme 1 (IRE1) in myeloid cells, or pharmacologically, by inhibition of IRE1 endoribonuclease (RNase) activity, led to significant reduction of LCWE-induced cardiovascular lesion formation as well as reduced caspase-1 activity and IL-1β secretion. These results demonstrate the causal relationship of ER stress to KD pathogenesis and highlight IRE1 RNase activity as a potential new therapeutic target.
Authors
Stefanie Marek-Iannucci, Asli D. Yildirim, Syed M. Hamid, Asli B. Ozdemir, Angela C. Gomez, Begüm Kocatürk, Rebecca A. Porritt, Michael C. Fishbein, Takao Iwawaki, Magali Noval Rivas, Ebru Erbay, Moshe Arditi
×Abstract
Altered islet architecture is associated with β cell dysfunction and type 2 diabetes (T2D) progression, but molecular effectors of islet spatial organization remain mostly unknown. Although Notch signaling is known to regulate pancreatic development, we observed “reactivated” β cell Notch activity in obese mouse models. To test the repercussions and reversibility of Notch effects, we generated doxycycline-dependent, β cell–specific Notch gain-of-function mice. As predicted, we found that Notch activation in postnatal β cells impaired glucose-stimulated insulin secretion and glucose intolerance, but we observed a surprising remnant glucose intolerance after doxycycline withdrawal and cessation of Notch activity, associated with a marked disruption of normal islet architecture. Transcriptomic screening of Notch-active islets revealed increased Ephrin signaling. Commensurately, exposure to Ephrin ligands increased β cell repulsion and impaired murine and human pseudoislet formation. Consistent with our mouse data, Notch and Ephrin signaling were increased in metabolically inflexible β cells in patients with T2D. These studies suggest that β cell Notch/Ephrin signaling can permanently alter islet architecture during a morphogenetic window in early life.
Authors
Alberto Bartolomé, Nina Suda, Junjie Yu, Changyu Zhu, Jinsook Son, Hongxu Ding, Andrea Califano, Domenico Accili, Utpal B. Pajvani
×Abstract
SARS-CoV-2 provokes a robust T cell response. Peptide-based studies exclude antigen processing and presentation biology, which may influence T cell detection studies. To focus on responses to whole virus and complex antigens, we used intact SARS-CoV-2 and full-length proteins with DCs to activate CD8 and CD4 T cells from convalescent people. T cell receptor (TCR) sequencing showed partial repertoire preservation after expansion. Resultant CD8 T cells recognize SARS-CoV-2–infected respiratory tract cells, and CD4 T cells detect inactivated whole viral antigen. Specificity scans with proteome-covering protein/peptide arrays show that CD8 T cells are oligospecific per subject and that CD4 T cell breadth is higher. Some CD4 T cell lines enriched using SARS-CoV-2 cross-recognize whole seasonal coronavirus (sCoV) antigens, with protein, peptide, and HLA restriction validation. Conversely, recognition of some epitopes is eliminated for SARS-CoV-2 variants, including spike (S) epitopes in the Alpha, Beta, Gamma, and Delta variant lineages.
Authors
Lichen Jing, Xia Wu, Maxwell P. Krist, Tien-Ying Hsiang, Victoria L. Campbell, Christopher L. McClurkan, Sydney M. Favors, Lawrence A. Hemingway, Charmie Godornes, Denise Q. Tong, Stacy Selke, Angela C. LeClair, Chu-Woo Pyo, Daniel E. Geraghty, Kerry J. Laing, Anna Wald, Michael Gale Jr., David M. Koelle
