Issue published January 25, 2022

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A genotype-phenotype correlation matrix for ABCA4 disease based on long-term prognostic outcomes

Lee et al. report a genotype-phenotype correlation matrix that provides quantifiable probabilities of long-term disease outcomes associated with specific ABCA4 genotypes from a large, age-restricted patient cohort.

Research Articles
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 antiinflammatory and antifibrotic agent that is approved by the FDA 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 (e.g., 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–KO 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.

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

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Abstract

Programmed death-1 homolog (PD-1H) is a coinhibitory molecule that negatively regulates T cell–mediated immune responses. In this study, we determined whether ablation of T cell–associated PD-1H could enhance adoptive T cell therapy in experimental tumor models. The expression of PD-1H is upregulated in activated and tumor-infiltrating CD8+ T cells. Activated CD8+ T cells from PD-1H–deficient (PD-1H–KO) mice exhibited increased cell proliferation, cytokine production, and antitumor activity in vitro. Adoptive transfer of PD-1H–KO CD8+ T cells resulted in the regression of established syngeneic mouse tumors. Similar results were obtained when PD-1H was ablated in T cells by CRISPR/Cas9-mediated gene silencing. Furthermore, ablation of PD-1H in CAR-T cells significantly improved their antitumor activity against human xenografts in vivo. Our results indicate that T cell–associated PD-1H could suppress immunity in the tumor microenvironment and that targeting PD-1H may improve T cell adoptive immunotherapy.

Authors

Li Hu, Ling Chen, Zexiu Xiao, Xu Zheng, Yuangui Chen, Na Xian, Christina Cho, Liqun Luo, Gangxiong Huang, Lieping Chen

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Abstract

BACKGROUND While most children who contract COVID-19 experience mild disease, high-risk children with underlying conditions may develop severe disease, requiring interventions. Kinetics of antibodies transferred via COVID-19 convalescent plasma early in disease have not been characterized.METHODS In this study, high-risk children were prospectively enrolled to receive high-titer COVID-19 convalescent plasma (>1:320 anti-spike IgG; Euroimmun). Passive transfer of antibodies and endogenous antibody production were serially evaluated for up to 2 months after transfusion. Commercial and research ELISA assays, virus neutralization assays, high-throughput phage-display assay utilizing a coronavirus epitope library, and pharmacokinetic analyses were performed.RESULTS Fourteen high-risk children (median age, 7.5 years) received high-titer COVID-19 convalescent plasma, 9 children within 5 days (range, 2–7 days) of symptom onset and 5 children within 4 days (range, 3–5 days) after exposure to SARS-CoV-2. There were no serious adverse events related to transfusion. Antibodies against SARS-CoV-2 were transferred from the donor to the recipient, but antibody titers declined by 14–21 days, with a 15.1-day half-life for spike protein IgG. Donor plasma had significant neutralization capacity, which was transferred to the recipient. However, as early as 30 minutes after transfusion, recipient plasma neutralization titers were 6.2% (range, 5.9%–6.7%) of donor titers.CONCLUSION Convalescent plasma transfused to high-risk children appears to be safe, with expected antibody kinetics, regardless of weight or age. However, current use of convalescent plasma in high-risk children achieves neutralizing capacity, which may protect against severe disease but is unlikely to provide lasting protection.Trial registration ClinicalTrials.gov NCT04377672.Funding The state of Maryland, Bloomberg Philanthropies, and the NIH (grants R01-AI153349, R01-AI145435-A1, K08-AI139371-A1, and T32-AI052071).

Authors

Oren Gordon, Mary Katherine Brosnan, Steve Yoon, Dawoon Jung, Kirsten Littlefield, Abhinaya Ganesan, Christopher A. Caputo, Maggie Li, William R. Morgenlander, Stephanie N. Henson, Alvaro A. Ordonez, Patricia De Jesus, Elizabeth W. Tucker, Nadine Peart Akindele, Zexu Ma, Jo Wilson, Camilo A. Ruiz-Bedoya, M. Elizabeth M. Younger, Evan M. Bloch, Shmuel Shoham, David Sullivan, Aaron A.R. Tobian, Kenneth R. Cooke, Ben Larman, Jogarao V.S. Gobburu, Arturo Casadevall, Andrew Pekosz, Howard M. Lederman, Sabra L. Klein, Sanjay K. Jain

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Abstract

We performed next-generation sequencing in patients with familial steroid-sensitive nephrotic syndrome (SSNS) and identified a homozygous segregating variant (p.H310Y) in the gene encoding clavesin-1 (CLVS1) in a consanguineous family with 3 affected individuals. Knockdown of the clavesin gene in zebrafish (clvs2) produced edema phenotypes due to disruption of podocyte structure and loss of glomerular filtration barrier integrity that could be rescued by WT CLVS1 but not the p.H310Y variant. Analysis of cultured human podocytes with CRISPR/Cas9-mediated CLVS1 knockout or homozygous H310Y knockin revealed deficits in clathrin-mediated endocytosis and increased susceptibility to apoptosis that could be rescued with corticosteroid treatment, mimicking the steroid responsiveness observed in patients with SSNS. The p.H310Y variant also disrupted binding of clavesin-1 to α-tocopherol transfer protein, resulting in increased reactive oxygen species (ROS) accumulation in CLVS1-deficient podocytes. Treatment of CLVS1-knockout or homozygous H310Y-knockin podocytes with pharmacological ROS inhibitors restored viability to control levels. Taken together, these data identify CLVS1 as a candidate gene for SSNS, provide insight into therapeutic effects of corticosteroids on podocyte cellular dynamics, and add to the growing evidence of the importance of endocytosis and oxidative stress regulation to podocyte function.

Authors

Brandon M. Lane, Megan Chryst-Stangl, Guanghong Wu, Mohamed Shalaby, Sherif El Desoky, Claire C. Middleton, Kinsie Huggins, Amika Sood, Alejandro Ochoa, Andrew F. Malone, Ricardo Vancini, Sara E. Miller, Gentzon Hall, So Young Kim, David N. Howell, Jameela A. Kari, Rasheed Gbadegesin

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Abstract

Tetralogy of Fallot (TOF) is the most common cyanotic heart defect, yet the underlying genetic mechanisms remain poorly understood. Here, we performed whole-genome sequencing analysis on 146 nonsyndromic TOF parent-offspring trios of Chinese ethnicity. Comparison of de novo variants and recessive genotypes of this data set with data from a European cohort identified both overlapping and potentially novel gene loci and revealed differential functional enrichment between cohorts. To assess the impact of these mutations on early cardiac development, we integrated single-cell and spatial transcriptomics of early human heart development with our genetic findings. We discovered that the candidate gene expression was enriched in the myogenic progenitors of the cardiac outflow tract. Moreover, subsets of the candidate genes were found in specific gene coexpression modules along the cardiomyocyte differentiation trajectory. These integrative functional analyses help dissect the pathogenesis of TOF, revealing cellular hotspots in early heart development resulting in cardiac malformations.

Authors

Clara Sze Man Tang, Mimmi Mononen, Wai-Yee Lam, Sheng Chih Jin, Xuehan Zhuang, Maria-Mercè Garcia-Barcelo, Qiongfen Lin, Yujia Yang, Makoto Sahara, Elif Eroglu, Kenneth R. Chien, Haifa Hong, Paul Kwong Hang Tam, Peter J. Gruber

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Abstract

Acute respiratory distress syndrome (ARDS) is a life-threatening syndrome, constituted by respiratory failure and diffuse alveolar damage that results from dysregulated local and systemic immune activation, causing pulmonary vascular, parenchymal, and alveolar damage. SARS-CoV-2 infection has become the dominant cause of ARDS worldwide, and emerging evidence implicates neutrophils and their cytotoxic arsenal of effector functions as central drivers of immune-mediated lung injury in COVID-19 ARDS. However, key outstanding questions are whether COVID-19 drives a unique program of neutrophil activation or effector functions that contribute to the severe pathogenesis of this pandemic illness and whether this unique neutrophil response can be targeted to attenuate disease. Using a combination of high-dimensional single-cell analysis and ex vivo functional assays of neutrophils from patients with COVID-19 ARDS, compared with those with non-COVID ARDS (caused by bacterial pneumonia), we identified a functionally distinct landscape of neutrophil activation in COVID-19 ARDS that was intrinsically programmed during SARS-CoV-2 infection. Furthermore, neutrophils in COVID-19 ARDS were functionally primed to produce high amounts of neutrophil extracellular traps. Surprisingly, this unique pathological program of neutrophil priming escaped conventional therapy with dexamethasone, thereby revealing a promising target for adjunctive immunotherapy in severe COVID-19.

Authors

Rachita Panda, Fernanda V.S. Castanheira, Jared M. Schlechte, Bas G.J. Surewaard, Hanjoo Brian Shim, Amanda Z. Zucoloto, Zdenka Slavikova, Bryan G. Yipp, Paul Kubes, Braedon McDonald

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Abstract

Besides promoting inflammation by mobilizing lipid mediators, group IIA secreted phospholipase A2 (sPLA2-IIA) prevents bacterial infection by degrading bacterial membranes. Here, we show that, despite the restricted intestinal expression of sPLA2-IIA in BALB/c mice, its genetic deletion leads to amelioration of cancer and exacerbation of psoriasis in distal skin. Intestinal expression of sPLA2-IIA is reduced after treatment with antibiotics or under germ-free conditions, suggesting its upregulation by gut microbiota. Metagenome, transcriptome, and metabolome analyses have revealed that sPLA2-IIA deficiency alters the gut microbiota, accompanied by notable changes in the intestinal expression of genes related to immunity and metabolism, as well as in the levels of various blood metabolites and fecal bacterial lipids, suggesting that sPLA2-IIA contributes to shaping of the gut microbiota. The skin phenotypes in Pla2g2a–/– mice are lost (a) when they are cohoused with littermate WT mice, resulting in the mixing of the microbiota between the genotypes, or (b) when they are housed in a more stringent pathogen-free facility, where Pla2g2a expression in WT mice is low and the gut microbial compositions in both genotypes are nearly identical. Thus, our results highlight a potentially new aspect of sPLA2-IIA as a modulator of gut microbiota, perturbation of which affects distal skin responses.

Authors

Yoshimi Miki, Yoshitaka Taketomi, Yuh Kidoguchi, Kei Yamamoto, Kazuaki Muramatsu, Yasumasa Nishito, Jonguk Park, Koji Hosomi, Kenji Mizuguchi, Jun Kunisawa, Tomoyoshi Soga, Eric Boilard, Siddabasave Gowda B. Gowda, Kazutaka Ikeda, Makoto Arita, Makoto Murakami

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Abstract

Secreted phospholipase A2-IIA (sPLA2-IIA) hydrolyzes phospholipids to liberate lysophospholipids and fatty acids. Given its poor activity toward eukaryotic cell membranes, its role in the generation of proinflammatory lipid mediators is unclear. Conversely, sPLA2-IIA efficiently hydrolyzes bacterial membranes. Here, we show that sPLA2-IIA affects the immune system by acting on the intestinal microbial flora. Using mice overexpressing transgene-driven human sPLA2-IIA, we found that the intestinal microbiota was critical for both induction of an immune phenotype and promotion of inflammatory arthritis. The expression of sPLA2-IIA led to alterations of the intestinal microbiota composition, but housing in a more stringent pathogen-free facility revealed that its expression could affect the immune system in the absence of changes to the composition of this flora. In contrast, untargeted lipidomic analysis focusing on bacteria-derived lipid mediators revealed that sPLA2-IIA could profoundly alter the fecal lipidome. The data suggest that a singular protein, sPLA2-IIA, produces systemic effects on the immune system through its activity on the microbiota and its lipidome.

Authors

Etienne Doré, Charles Joly-Beauparlant, Satoshi Morozumi, Alban Mathieu, Tania Lévesque, Isabelle Allaeys, Anne-Claire Duchez, Nathalie Cloutier, Mickaël Leclercq, Antoine Bodein, Christine Payré, Cyril Martin, Agnes Petit-Paitel, Michael H. Gelb, Manu Rangachari, Makoto Murakami, Laetitia Davidovic, Nicolas Flamand, Makoto Arita, Gérard Lambeau, Arnaud Droit, Eric Boilard

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Abstract

Cancer inflicts damage to surrounding normal tissues, which can culminate in fatal organ failure. Here, we demonstrate that cell death in organs affected by cancer can be detected by tissue-specific methylation patterns of circulating cell-free DNA (cfDNA). We detected elevated levels of hepatocyte-derived cfDNA in the plasma of patients with liver metastases originating from different primary tumors, compared with cancer patients without liver metastases. In addition, patients with localized pancreatic or colon cancer showed elevated hepatocyte cfDNA, suggesting liver damage inflicted by micrometastatic disease, by primary pancreatic tumor pressing the bile duct, or by a systemic response to the primary tumor. We also identified elevated neuron-, oligodendrocyte-, and astrocyte-derived cfDNA in a subpopulation of patients with brain metastases compared with cancer patients without brain metastasis. Cell type–specific cfDNA methylation markers enable the identification of collateral tissue damage in cancer, revealing the presence of metastases in specific locations and potentially assisting in early cancer detection.

Authors

Asael Lubotzky, Hai Zemmour, Daniel Neiman, Marc Gotkine, Netanel Loyfer, Sheina Piyanzin, Bracha-Lea Ochana, Roni Lehmann-Werman, Daniel Cohen, Joshua Moss, Judith Magenheim, Maureen F. Loftus, Lauren Brais, Kimmie Ng, Raul Mostoslavsky, Brian M. Wolpin, Aviad Zick, Myriam Maoz, Albert Grinshpun, Anatoli Kustanovich, Chen Makranz, Jonathan E. Cohen, Tamar Peretz, Ayala Hubert, Mark Temper, Azzam Salah, Shani Avniel-Polak, Simona Grozinsky-Glasberg, Kirsty L. Spalding, Ariel Rokach, Tommy Kaplan, Benjamin Glaser, Ruth Shemer, Yuval Dor

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Abstract

Approximately 80% of pancreatic cancer patients suffer from cachexia, and one-third die due to cachexia-related complications such as respiratory failure and cardiac arrest. Although there has been considerable research into cachexia mechanisms and interventions, there are, to date, no FDA-approved therapies. A major contributing factor for the lack of therapy options could be the failure of animal models to accurately recapitulate the human condition. In this study, we generated an aged model of pancreatic cancer cachexia to compare cachexia progression in young versus aged tumor-bearing mice. Comparative skeletal muscle transcriptome analyses identified 3-methyladenine (3-MA) as a candidate antiwasting compound. In vitro analyses confirmed antiwasting capacity, while in vivo analysis revealed potent antitumor effects. Transcriptome analyses of 3-MA–treated tumor cells implicated Perp as a 3-MA target gene. We subsequently (a) observed significantly higher expression of Perp in cancer cell lines compared with control cells, (b) noted a survival disadvantage associated with elevated Perp, and (c) found that 3-MA–associated Perp reduction inhibited tumor cell growth. Finally, we have provided in vivo evidence that survival benefits conferred by 3-MA administration are independent of its effect on tumor progression. Taken together, we report a mechanism linking 3-MA to Perp inhibition, and we further implicate Perp as a tumor-promoting factor in pancreatic cancer.

Authors

Aneesha Dasgupta, Paige C. Arneson-Wissink, Rebecca E. Schmitt, Dong Seong Cho, Alexandra M. Ducharme, Tara L. Hogenson, Eugene W. Krueger, William R. Bamlet, Lizhi Zhang, Gina L. Razidlo, Martin E. Fernandez-Zapico, Jason D. Doles

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Abstract

Platelet-neutrophil aggregates (PNAs) facilitate neutrophil activation and migration and could underpin the recruitment of neutrophils to the pancreas during type 1 diabetes (T1D) pathogenesis. PNAs, measured by flow cytometry, were significantly elevated in the circulation of autoantibody-positive (Aab+) children and new-onset T1D children, as well as in pre-T1D (at 4 weeks and 10–12 weeks) and T1D-onset NOD mice, compared with relevant controls, and PNAs were characterized by activated P-selectin+ platelets. PNAs were similarly increased in pre-T1D and T1D-onset NOD isolated islets/insulitis, and immunofluorescence staining revealed increased islet-associated neutrophil extracellular trap (NET) products (myeloperoxidase [MPO] and citrullinated histones [CitH3]) in NOD pancreata. In vitro, cell-free histones and NETs induced islet cell damage, which was prevented by the small polyanionic drug methyl cellobiose sulfate (mCBS) that binds to histones and neutralizes their pathological effects. Elevated circulating PNAs could, therefore, act as an innate immune and pathogenic biomarker of T1D autoimmunity. Platelet hyperreactivity within PNAs appears to represent a previously unrecognized hematological abnormality that precedes T1D onset. In summary, PNAs could contribute to the pathogenesis of T1D and potentially function as a pre-T1D diagnostic.

Authors

Sarah K. Popp, Federica Vecchio, Debra J. Brown, Riho Fukuda, Yuri Suzuki, Yuma Takeda, Rikako Wakamatsu, Mahalakshmi A. Sarma, Jessica Garrett, Anna Giovenzana, Emanuele Bosi, Antony R.A. Lafferty, Karen J. Brown, Elizabeth E. Gardiner, Lucy A. Coupland, Helen E. Thomas, Beng H. Chong, Christopher R. Parish, Manuela Battaglia, Alessandra Petrelli, Charmaine J. Simeonovic

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Abstract

Acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure yet has few pharmacologic therapies, reflecting the mechanistic heterogeneity of lung injury. We hypothesized that damage to the alveolar epithelial glycocalyx, a layer of glycosaminoglycans interposed between the epithelium and surfactant, contributes to lung injury in patients with ARDS. Using mass spectrometry of airspace fluid noninvasively collected from mechanically ventilated patients, we found that airspace glycosaminoglycan shedding (an index of glycocalyx degradation) occurred predominantly in patients with direct lung injury and was associated with duration of mechanical ventilation. Male patients had increased shedding, which correlated with airspace concentrations of matrix metalloproteinases. Selective epithelial glycocalyx degradation in mice was sufficient to induce surfactant dysfunction, a key characteristic of ARDS, leading to microatelectasis and decreased lung compliance. Rapid colorimetric quantification of airspace glycosaminoglycans was feasible and could provide point-of-care prognostic information to clinicians and/or be used for predictive enrichment in clinical trials.

Authors

Alicia N. Rizzo, Sarah M. Haeger, Kaori Oshima, Yimu Yang, Alison M. Wallbank, Ying Jin, Marie Lettau, Lynda A. McCaig, Nancy E. Wickersham, J. Brennan McNeil, Igor Zakharevich, Sarah A. McMurtry, Christophe J. Langouët-Astrié, Katrina W. Kopf, Dennis R. Voelker, Kirk C. Hansen, Ciara M. Shaver, V. Eric Kerchberger, Ryan A. Peterson, Wolfgang M. Kuebler, Matthias Ochs, Ruud A.W. Veldhuizen, Bradford J. Smith, Lorraine B. Ware, Julie A. Bastarache, Eric P. Schmidt

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Abstract

Acute cardiac injury is prevalent in critical COVID-19 and associated with increased mortality. Its etiology remains debated, as initially presumed causes — myocarditis and cardiac necrosis — have proved uncommon. To elucidate the pathophysiology of COVID-19–associated cardiac injury, we conducted a prospective study of the first 69 consecutive COVID-19 decedents at CUIMC in New York City. Of 6 acute cardiac histopathologic features, presence of microthrombi was the most commonly detected among our cohort. We tested associations of cardiac microthrombi with biomarkers of inflammation, cardiac injury, and fibrinolysis and with in-hospital antiplatelet therapy, therapeutic anticoagulation, and corticosteroid treatment, while adjusting for multiple clinical factors, including COVID-19 therapies. Higher peak erythrocyte sedimentation rate and C-reactive protein were independently associated with increased odds of microthrombi, supporting an immunothrombotic etiology. Using single-nuclei RNA-sequencing analysis on 3 patients with and 4 patients without cardiac microthrombi, we discovered an enrichment of prothrombotic/antifibrinolytic, extracellular matrix remodeling, and immune-potentiating signaling among cardiac fibroblasts in microthrombi-positive, relative to microthrombi-negative, COVID-19 hearts. Non–COVID-19, nonfailing hearts were used as reference controls. Our study identifies a specific transcriptomic signature in cardiac fibroblasts as a salient feature of microthrombi-positive COVID-19 hearts. Our findings warrant further mechanistic study as cardiac fibroblasts may represent a potential therapeutic target for COVID-19–associated cardiac microthrombi.

Authors

Michael I. Brener, Michelle L. Hulke, Nobuaki Fukuma, Stephanie Golob, Robert S. Zilinyi, Zhipeng Zhou, Christos Tzimas, Ilaria Russo, Claire McGroder, Ryan D. Pfeiffer, Alexander Chong, Geping Zhang, Daniel Burkhoff, Martin B. Leon, Mathew S. Maurer, Jeffrey W. Moses, Anne-Catrin Uhlemann, Hanina Hibshoosh, Nir Uriel, Matthias J. Szabolcs, Björn Redfors, Charles C. Marboe, Matthew R. Baldwin, Nathan R. Tucker, Emily J. Tsai

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Abstract

Metabolomics has been used to explore the molecular mechanism and screen biomarkers. However, the critical metabolic signatures associated with benzene-induced hematotoxicity remain elusive. Here, we performed a plasma metabolomics study in 86 benzene-exposed workers and 76 healthy controls, followed by a validation analysis in mice, to investigate the dynamical change of the metabolic profile. We found that 8 fatty acids were significantly altered in both benzene-exposed worker and benzene-exposed animal models. These metabolites were significantly associated with S-phenylmercapturic acid and WBC, and they mediated the benzene-induced WBC decline. Furthermore, in vivo results confirm that fatty acid levels were dynamically altered, characterized by a decrease at 15 days and then sharp increases at 30 and 45 days. Following these identified fatty acids, the potential metabolic pathways were investigated. Fatty acids, as precursors for fatty acid oxidation, may disturb the balance of fatty acid biosynthesis and degradation. Our results reveal that fatty acid metabolism was strongly reprogrammed after benzene exposure. This abnormal change of fatty acids might be the key metabolic signature associated with benzene-induced hematotoxicity.

Authors

Xiaoli Guo, Lei Zhang, Jingyu Wang, Wei Zhang, Jing Ren, Yujiao Chen, Yanlin Zhang, Ai Gao

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Abstract

BACKGROUND Outcome measures sensitive to disease progression are needed for ATP-binding cassette, sub-family A, member 4–associated (ABCA4-associated) retinopathy. We aimed to quantify ellipsoid zone (EZ) loss and photoreceptor degeneration beyond EZ-loss in ABCA4-associated retinopathy and investigate associations between photoreceptor degeneration, genotype, and age.METHODS We analyzed 132 eyes from 66 patients (of 67 enrolled) with molecularly confirmed ABCA4-associated retinopathy from a prospective natural history study with a median [IQR] follow-up of 4.2 years [3.1, 5.1]. Longitudinal spectral-domain optical coherence tomography volume scans (37 B-scans, 30° × 15°) were segmented using a deep learning (DL) approach. For genotype-phenotype analysis, a model of ABCA4 variants was applied with the age of criterion EZ-loss (6.25 mm2) as the dependent variable.RESULTS Patients exhibited an average (square-root-transformed) EZ-loss progression rate of [95% CI] 0.09 mm/y [0.06, 0.11]. Outer nuclear layer (ONL) thinning extended beyond the area of EZ-loss. The average distance from the EZ-loss boundary to normalization of ONL thickness (to ±2 z score units) was 3.20° [2.53, 3.87]. Inner segment (IS) and outer segment (OS) thinning was less pronounced, with an average distance from the EZ-loss boundary to layer thickness normalization of 1.20° [0.91, 1.48] for the IS and 0.60° [0.49, 0.72] for the OS. An additive model of allele severity explained 52.7% of variability in the age of criterion EZ-loss.CONCLUSION Patients with ABCA4-associated retinopathy exhibited significant alterations of photoreceptors outside of EZ-loss. DL-based analysis of photoreceptor laminae may help monitor disease progression and estimate the severity of ABCA4 variants.TRIAL REGISTRATION ClinicalTrials.gov identifier: NCT01736293.FUNDING National Eye Institute Intramural Research Program and German Research Foundation grant PF950/1-1.

Authors

Maximilian Pfau, Catherine A. Cukras, Laryssa A. Huryn, Wadih M. Zein, Ehsan Ullah, Marisa P. Boyle, Amy Turriff, Michelle A. Chen, Aarti S. Hinduja, Hermann E.A. Siebel, Robert B. Hufnagel, Brett G. Jeffrey, Brian P. Brooks

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Abstract

Invariant NK T (iNKT) cells are implicated in viral clearance; however, their role in hepatitis C virus (HCV) infection remains controversial. Here, iNKT cells were studied during different stages of HCV infection. iNKT cells from patients with acute HCV infection and people who inject drugs (PWID) with chronic or spontaneously resolved HCV infection were characterized by flow cytometry. In a longitudinal analysis during acute HCV infection, frequencies of activated CD38+ iNKT cells reproducibly declined in spontaneously resolving patients, whereas they were persistently elevated in patients progressing to chronic infection. During the first year of infection, the frequency of activated CD38+ or CD69+ iNKT cells strongly correlated with alanine transaminase levels with particularly pronounced correlations in spontaneously resolving patients. Increased frequencies of activated iNKT cells in chronic HCV infection were confirmed in cross-sectional analyses of PWID with chronic or spontaneously resolved HCV infection; however, no apparent functional differences were observed with various stimulation protocols. Our data suggest that iNKT cells are activated during acute hepatitis C and that activation is sustained in chronic infection. The correlation between the frequency of activated iNKT cells and alanine transaminase may point toward a role of iNKT cells in liver damage.

Authors

Tina Senff, Christopher Menne, Christine Cosmovici, Lia Laura Lewis-Ximenez, Jasneet Aneja, Ruth Broering, Arthur Y. Kim, Astrid M. Westendorf, Ulf Dittmer, Norbert Scherbaum, Georg M. Lauer, Jörg Timm

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Abstract

Isolation guidelines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are largely derived from data collected prior to the emergence of the delta variant. We followed a cohort of ambulatory patients with postvaccination breakthrough SARS-CoV-2 infections with longitudinal collection of nasal swabs for SARS-CoV-2 viral load quantification, whole-genome sequencing, and viral culture. All delta variant infections in our cohort were symptomatic, compared with 64% of non-delta variant infections. Symptomatic delta variant breakthrough infections were characterized by higher initial viral load, longer duration of virologic shedding by PCR, greater likelihood of replication-competent virus at early stages of infection, and longer duration of culturable virus compared with non-delta variants. The duration of time since vaccination was also correlated with both duration of PCR positivity and duration of detection of replication-competent virus. Nonetheless, no individuals with symptomatic delta variant infections had replication-competent virus by day 10 after symptom onset or 24 hours after resolution of symptoms. These data support US CDC isolation guidelines as of November 2021, which recommend isolation for 10 days or until symptom resolution and reinforce the importance of prompt testing and isolation among symptomatic individuals with delta breakthrough infections. Additional data are needed to evaluate these relationships among asymptomatic and more severe delta variant breakthrough infections.

Authors

Mark J. Siedner, Julie Boucau, Rebecca F. Gilbert, Rockib Uddin, Jonathan Luu, Sebastien Haneuse, Tammy Vyas, Zahra Reynolds, Surabhi Iyer, Grace C. Chamberlin, Robert H. Goldstein, Crystal M. North, Chana A. Sacks, James Regan, James P. Flynn, Manish C. Choudhary, Jatin M. Vyas, Amy K. Barczak, Jacob E. Lemieux, Jonathan Z. Li

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Abstract

Cellular and molecular mechanisms driving morbidity following SARS-CoV-2 infection have not been well defined. The receptor for advanced glycation end products (RAGE) is a central mediator of tissue injury and contributes to SARS-CoV-2 disease pathogenesis. In this study, we temporally delineated key cell and molecular events leading to lung injury in mice following SARS-CoV-2 infection and assessed efficacy of therapeutically targeting RAGE to improve survival. Early following infection, SARS-CoV-2 replicated to high titers within the lungs and evaded triggering inflammation and cell death. However, a significant necrotic cell death event in CD45– populations, corresponding with peak viral loads, was observed on day 2 after infection. Metabolic reprogramming and inflammation were initiated following this cell death event and corresponded with increased lung interstitial pneumonia, perivascular inflammation, and endothelial hyperplasia together with decreased oxygen saturation. Therapeutic treatment with the RAGE antagonist FPS-ZM1 improved survival in infected mice and limited inflammation and associated perivascular pathology. Together, these results provide critical characterization of disease pathogenesis in the mouse model and implicate a role for RAGE signaling as a therapeutic target to improve outcomes following SARS-CoV-2 infection.

Authors

Forrest Jessop, Benjamin Schwarz, Dana Scott, Lydia M. Roberts, Eric Bohrnsen, John R. Hoidal, Catharine M. Bosio

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Abstract

Background More than 1500 variants in the ATP-binding cassette, sub-family A, member 4 (ABCA4), locus underlie a heterogeneous spectrum of retinal disorders ranging from aggressive childhood-onset chorioretinopathy to milder late-onset macular disease. Genotype-phenotype correlation studies have been limited in clinical applicability as patient cohorts are typically small and seldom capture the full natural history of individual genotypes. To overcome these limitations, we constructed a genotype-phenotype correlation matrix that provides quantifiable probabilities of long-term disease outcomes associated with specific ABCA4 genotypes from a large, age-restricted patient cohort.Methods The study included 112 unrelated patients at least 50 years of age in whom 2 pathogenic variants were identified after sequencing of the ABCA4 locus. Clinical characterization was performed using the results of best corrected visual acuity, retinal imaging, and full-field electroretinogram testing.Results Four distinct prognostic groups were defined according to the spatial severity of disease features across the fundus. Recurring genotypes were observed in milder prognoses, including a newly defined class of rare hypomorphic alleles. PVS1 (predicted null) variants were enriched in the most severe prognoses; however, missense variants were present in a larger-than-expected fraction of these patients. Analysis of allele combinations and their respective prognostic severity showed that certain variants, such as p.(Gly1961Glu), and both rare and frequent hypomorphic alleles, were “clinically dominant” with respect to patient phenotypes irrespective of the allele in trans.Conclusion These results provide much-needed structure to the complex genetic and clinical landscape of ABCA4 disease and add a tool to the clinical repertoire to quantitatively assess individual genotype-specific prognoses in patients.FUNDING National Eye Institute, NIH, grants R01 EY028203, R01 EY028954, R01 EY029315, P30 19007 (Core Grant for Vision Research); the Foundation Fighting Blindness USA, grant no. PPA-1218-0751-COLU; and Research to Prevent Blindness.

Authors

Winston Lee, Jana Zernant, Pei-Yin Su, Takayuki Nagasaki, Stephen H. Tsang, Rando Allikmets

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Review
Abstract

In recent decades, immunotherapeutic strategies have been used to treat a wide range of pathologies, many of which were previously incurable, such as cancer and autoimmune disorders. Despite this unprecedented success, a considerable number of patients fail to respond to currently approved immunotherapies or develop resistance over time. Therefore, there is an urgent need to develop the next generation of immune-targeted therapies. Various members of the Ig superfamily play essential roles in regulating leukocyte functions. One such group, the leukocyte Ig-like receptors (LILRs), have been implicated in both innate and adaptive immune regulation. Human inhibitory LILRs (LILRBs) are primarily expressed on leukocytes and mediate their signaling through multiple cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. Engagement of LILRBs by endogenous and pathogenic ligands can markedly suppress immune responses, leading to tolerance or immunoevasion, whereas blocking these inhibitory receptors can potentiate immune responses. In this Review, we discuss the immunoregulatory functions of human LILRBs and the potential of targeting them to manipulate immune responses in various pathologies.

Authors

Calvin D. De Louche, Ali Roghanian

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Abstract

Commensal microbes critically regulate skeletal homeostasis, yet the impact of specific microbiota communities on osteoimmune response mechanisms is unknown. To discern osteoimmunomodulatory effects imparted by the commensal oral microbiota that are distinct from the systemic microbiota, osteoimmunology studies were performed in both alveolar bone and non-oral skeletal sites of specific-pathogen-free (SPF) vs. germ-free (GF) mice, and SPF mice subjected to saline vs. chlorhexidine oral rinses. SPF vs. GF mice had reduced cortical/trabecular bone and an enhanced pro-osteoclastic phenotype in alveolar bone. Toll-like receptor signaling and TH17 cells that have known pro-osteoclastic actions were increased in alveolar, but not long-bone marrow, of SPF vs. GF mice. MHC class-II antigen presentation genes, activated dendritic cells, and activated CD4+ T-cells were elevated in alveolar, but not long-bone marrow, of SPF vs. GF mice. These findings were substantiated by in vitro allostimulation studies demonstrating increased activated dendritic cells derived from alveolar, but not long-bone marrow, of SPF vs. GF mice. Chlorhexidine antiseptic rinse depleted the oral, but not gut, bacteriome in SPF mice. Findings from saline- vs. chlorhexidine-treated SPF mice corroborated outcomes from SPF vs. GF mice, which reveals that the commensal oral microbiota imparts osteoimmunomodulatory effects separate from the systemic microbiome.

Authors

Jessica D. Hathaway-Schrader, Johannes D. Aartun, Nicole A. Poulides, Megan B. Kuhn, Blakely E. McCormick, Michael E. Chew, Emily Huang, Richard P. Darveau, Caroline Westwater, Chad M. Novince

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Abstract

COPD is a debilitating chronic disease and the third cause of mortality worldwide. It is characterized by airway neutrophilia, promoting tissue injury through release of toxic mediators and proteases. Recently, it has been shown that neutrophil-derived extracellular vesicles (EVs) from COPD patient lungs can cause a neutrophil elastase (NE)-dependent COPD-like disease upon transfer to mouse airways. However in vivo preclinical models elucidating the impact of EVs on disease are lacking, delaying opportunities for therapeutic testing. Here, we developed an in vivo preclinical mouse model of lung EV-induced COPD. EVs from in vivo LPS activated mouse neutrophils induced COPD-like disease in naive recipients through an alpha-1 antitrypsin resistant, NE-dependent mechanism. Together, these results show a key pathogenic and mechanistic role for neutrophil-derived EVs in a new mouse model of COPD. Broadly, the in vivo model described herein could be leveraged to develop targeted therapies for severe lung disease.

Authors

Camilla Margaroli, Matthew C. Madison, Liliana Viera, Derek W. Russell, Amit Gaggar, Kristopher R. Genschmer, J. Edwin Blalock

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Abstract

Currently, the most effective strategy for dealing with Alzheimer’s disease (AD) is delaying the onset of dementia. Severe hypoglycemia is strongly associated with dementia; however, the effects of recurrent moderate hypoglycemia (RH) on progression of cognitive deficits in diabetic patients with genetic susceptibility to AD remain unclear. Here, we report that insulin-controlled hyperglycemia only slightly aggravated AD-type pathologies and cognitive impairment; however, RH significantly increased neuronal hyperactivity and accelerated the progression of cognitive deficits in streptozotocin(STZ)-induced diabetic APP/PS1 mice. GLUT3-mediated neuronal glucose uptake was not significantly altered under hyperglycemia, but was markedly reduced by RH, which induced excessive mitochondrial fission in the hippocampus. Overexpression of GLUT3 specifically in DG area of hippocampus enhanced mitochondrial function and improved cognitive deficits induced by RH. Activation of TRPC6 increased GLUT3-mediated glucose uptake in brain and alleviated RH-induced cognitive deficits, and inactivation of Ca2+/AMPK pathway was responsible for TRPC6-induced GLUT3 inhibition. Taken together, RH impairs brain GLUT3-mediated glucose uptake and further provokes neuronal mitochondrial dysfunction by inhibiting TRPC6 expression, which then accelerates the progression of cognitive deficits in diabetic APP/PS1 mice. Avoiding RH is essential for glycemic control in diabetic patients, and TRPC6/GLUT3 represent potent targets for delaying the onset of dementia in diabetic patients.

Authors

Chengkang He, Qiang Li, Yuanting Cui, Peng Gao, WenTao Shu, Qing Zhou, Lijuan Wang, Li Li, Zongshi Lu, Yu Zhao, Huan Ma, Xiaowei Chen, Hongbo Jia, Hongting Zheng, Gangyi Yang, Daoyan Liu, Martin Tepel, Zhiming Zhu

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Abstract

Lung alveolar type 2 (AT2) cells are progenitors for alveolar type 1 (AT1) cells. Although many factors regulate AT2 cell plasticity, the role of mitochondrial calcium (mCa2+) uptake in controlling AT2 cells remains unclear. We previously identified that the microRNA family, miR-302, supports lung epithelial progenitor cell proliferation and less differentiated phenotypes during development. Here we report that a sustained elevation of miR-302 in adult AT2 cells decreases AT2-to-AT1 cell differentiation during the Streptococcus pneumoniae induced lung injury repair. We identified that miR-302 targets and represses the expression of mitochondrial Ca2+ uptake 1 (MICU1), which regulates mCa2+ uptake through the mCa2+ uniporter channel by acting as a gatekeeper at low cytosolic Ca2+ levels. Our results reveal a marked increase in MICU1 protein expression and decreased mCa2+ uptake during AT2-to-AT1 cell differentiation in the adult lung. Deletion of Micu1 in AT2 cells reduces AT2-to-AT1 cell differentiation during steady-state tissue maintenance and alveolar epithelial regeneration following bacterial pneumonia. These studies indicate that mCa2+ uptake is extensively modulated during AT2-to-AT1 cell differentiation and that MICU1-dependent mCa2+ uniporter channel gating is a prominent mechanism modulating AT2-to-AT1 cell differentiation.

Authors

Mir Ali, Xiaoying Zhang, Ryan LaCanna, Dhanendra Tomar, John W. Elrod, Ying Tian

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Abstract

Symmetric, progressive, necrotizing lesions in the brainstem are a defining feature of Leigh syndrome (LS). A mechanistic understanding of the pathogenesis of these lesions has been elusive. Here, we report that leukocyte proliferation is causally involved in the pathogenesis of LS. Depleting leukocytes with a colony-stimulating factor 1 receptor inhibitor disrupts disease progression, including suppression of CNS lesion formation and a substantial extension of survival. Leukocyte depletion rescues diverse symptoms including seizures, respiratory center function, hyperlactemia, and neurologic sequelae. These data reveal a mechanistic explanation for the beneficial effects of mTOR inhibition. More importantly, these findings dramatically alter our understanding of the pathogenesis of LS, demonstrating that immune involvement is causal in disease. This work has significant implications for the mechanisms of mitochondrial disease and may lead to novel therapeutic strategies.

Authors

Julia C. Stokes, Rebecca L. Bornstein, Katerina James, Kyung Yeon Park, Kira A. Spencer, Katie Vo, John C. Snell, Brittany M. Johnson, Philip G. Morgan, Margaret M. Sedensky, Nathan A. Baertsch, Simon C. Johnson

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