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Abstract
Understanding viral rebound in pediatric HIV-1 infection may inform the development of alternatives to lifelong antiretroviral therapy (ART) to achieve viral remission. We thus investigated viral rebound after analytical treatment interruption (ATI) in 10 infant macaques orally infected with SHIV.C.CH505 and treated with long-term ART. Rebound viremia was detected within 7-35 days of ATI in 9/10 animals, with post-treatment control of viremia seen in 5/5 Mamu-A*01+ macaques. Single-genome sequencing revealed initial rebound virus was similar to viral DNA present in CD4+ T cells from blood, rectum, and lymph nodes before ATI. We assessed the earliest sites of viral reactivation immediately following ATI using ImmunoPET imaging. The largest increase in signal that preceded detectable viral RNA in plasma was found in the gastrointestinal (GI) tract, a site with relatively high SHIV RNA/DNA ratios in CD4+ T cells prior to ATI. Thus, the GI tract may be an initial source of rebound virus but as ATI progresses, viral reactivation in other tissues likely contributes to the composition of plasma virus. Our study provides novel insight into the features of viral rebound in pediatric infection and highlights the application of a non-invasive technique to monitor areas of HIV-1 expression in children.
Authors
Veronica Obregon-Perko, Katherine M. Bricker, Gloria Mensah, Ferzan Uddin, Laura Rotolo, Daryll Vanover, Yesha Desai, Philip J. Santangelo, Sherrie Jean, Jennifer S. Wood, Fawn C. Connor-Stroud, Stephanie Ehnert, Stella J. Berendam, Shan Liang, Thomas H. Vanderford, Katharine J. Bar, George M. Shaw, Guido Silvestri, Amit Kumar, Genevieve G. Fouda, Sallie R. Permar, Ann Chahroudi
Abstract
Point mutations within sarcomeric proteins have been associated with altered function and cardiomyopathy development. Difficulties remain, however, in establishing the pathogenic potential of individual mutations, often limiting the use of genotype in management of affected families. To directly address this challenge, we utilized our all-atom computational model of the human full cardiac thin filament (CTF) to predict how sequence substitutions in CTF proteins might affect structure and dynamics on an atomistic level.Utilizing molecular dynamics (MD) calculations, we simulated 21 well-defined genetic pathogenic cardiac troponin T and tropomyosin variants to establish a baseline of pathogenic changes induced in computational observables. Computational results were verified via differential scanning calorimetry on a subset of variants to develop an experimental correlation. Calculations were performed on 9 independent variants of unknown significance (VUS) and results were compared to pathogenic variants to identify high resolution pathogenic signatures.Results for VUS were compared to the baseline set to determine induced structural and dynamic changes and potential variant reclassifications were proposed. This unbiased, high- resolution computational methodology can provide unique structural and dynamic information that can be incorporated into existing analyses to facilitate classification both for de novo variants and those where established approaches have provided conflicting information.
Authors
Allison B. Mason, Melissa L. Lynn, Anthony P. Baldo, Andrea E. Deranek, Jil C. Tardiff, Steven D. Schwartz
Abstract
Chronic inflammation and localized alterations in immune cell function are suspected to contribute to the progression of endometriosis and its associated symptoms. In particular, the alarmin, Interleukin (IL)-33 is elevated in the plasma, peritoneal fluid, and endometriotic lesions from endometriosis patients; however, the exact role of IL-33 in the pathophysiology of endometriosis is not well understood. In this study, we demonstrate, in both human patients and a murine model, that IL-33 contributes to the expansion of the novel group 2 innate lymphoid cells (ILC2s) and this IL-33 induced ILC2 expansion modulates the endometriosis lesion microenvironment. Importantly, we show that IL-33 drives hallmarks of severe endometriosis including elevated inflammation, lesion proliferation, and fibrosis and that this IL-33 induced aggravation is mediated by ILC2s. Finally, we demonstrate the functionality of IL-33 neutralization as a promising and novel therapeutic avenue for treating the debilitating symptoms of endometriosis.
Authors
Jessica E. Miller, Harshavardhan Lingegowda, Lindsey K. Symons, Olga Bougie, Steven L. Young, Bruce A. Lessey, Madhuri Koti, Chandrakant Tayade
Abstract
In response to liver injury, hepatic stellate cells activate and acquire proliferative and contractile features. The regression of liver fibrosis appears to involve the clearance of activated hepatic stellate cells, either by apoptosis or by reversion towards a quiescent-like state, a process denominated deactivation. Thus, deactivation of active hepatic stellate cells has emerged as a novel and promising therapeutic approach for liver fibrosis. However, our knowledge of the master regulators involved in the de/activation of fibrotic hepatic stellate cells is still limited. The transcription factor GATA4 has been previously shown to play an important role in embryonic hepatic stellate cells quiescence. In this work, we show that lack of GATA4 in adult mice causes hepatic stellate cell activation and consequently, liver fibrosis. During regression of liver fibrosis, Gata4 is reexpressed in deactivated hepatic stellate cells. Overexpression of Gata4 in hepatic stellate cells promotes liver fibrosis regression in CCl4-treated mice. GATA4 induces changes in the expression of fibrogenic and antifibrogenic genes promoting hepatic stellate cell deactivation. Finally, we show that GATA4 directly represses EPAS1 transcription in hepatic stellate cells and that stabilization of the HIF2α protein in hepatic stellate cells leads to liver fibrosis.
Authors
Noelia Arroyo, Laura Villamayor, Irene Díaz, Rita Carmona, Mireia Ramos-Rodríguez, Ramon Muñoz-Chapuli, Lorenzo Pasquali, Miguel G. Toscano, Franz Martin, David A. Cano, Anabel Rojas
Abstract
Glucagon-like peptide-1 receptor agonists (GLP-1RA) are used to treat diabetes and obesity and reduce rates of major cardiovascular events such as stroke and myocardial infarction. Nevertheless, the identity of GLP-1R-expressing cell types mediating the cardiovascular benefits of GLP-1RA remains incompletely characterized. Herein, we investigated the importance of murine Glp1r expression within endothelial and hematopoietic cells. Mice with targeted inactivation of the Glp1r in Tie2+ cells exhibited reduced levels of Glp1r mRNA transcripts in aorta, liver, spleen, blood and gut. Glp1r expression in bone marrow cells was very low, and not further reduced in Glp1rTie2-/- mice. The GLP-1RA semaglutide reduced the development of atherosclerosis induced by viral PCSK9 expression in both Glp1rTie2+/+ and Glp1rTie2-/- mice. Hepatic Glp1r mRNA transcripts were reduced in Glp1rTie2-/- mice and liver Glp1r expression was localized to γδ T cells. Moreover, semaglutide reduced hepatic Tnf, Abcg1, Tgfb1, Cd3g, Ccl2, and Il2 expression, triglyceride content and collagen accumulation in high fat high cholesterol (HFHC) diet-fed Glp1rTie2+/+ but not Glp1rTie2-/- mice. Collectively, these findings demonstrate that Tie2+ endothelial or hematopoietic cell GLP-1Rs are dispensable for the anti-atherogenic actions of GLP-1RA, whereas Tie2-targeted GLP-1R+ cells are required for a subset of the anti-inflammatory actions of semaglutide in the liver.
Authors
Brent McLean, Chi Kin Wong, Kiran Deep Kaur, Randy J. Seeley, Daniel J. Drucker
Abstract
Ischemic retinopathies including diabetic retinopathy are major causes of blindness. While neurons and Müller glia are recognized as important regulators of reparative and pathologic angiogenesis, the role of mononuclear phagocytes (MPs), such as microglia/macrophages, is unclear, particularly microglia, the resident retinal immune cells. Here we found microglial/macrophage activation in human diabetic retinopathy, especially in neovessels from human neovascular membranes in proliferative retinopathy, including TNF-α expression. There was similar activation in the mouse oxygen-induced retinopathy (OIR) model of ischemia-induced neovascularization. Glucagon-like peptide-1 receptor (GLP-1R) agonists are in clinical use for glycemic control in diabetes and are also known to modulate microglia. We investigated the effect of a long-acting GLP-1R agonist, NLY01. Following intravitreal administration, NLY01 selectively localized to MPs in OIR retina. NLY01 modulated MP but not retinal endothelial cell viability, apoptosis, and tube formation in vitro. In OIR, NLY01 treatment inhibited MP infiltration and activation, including microglia/macrophage expression of cytokines in vivo. NLY01 significantly suppressed global induction of retinal inflammatory cytokines, promoted reparative angiogenesis, and suppressed pathologic retinal neovascularization. Collectively, these findings indicate the important role of microglia/macrophages in regulation of retinal vascularization in ischemia and suggest modulation of MPs as a new treatment strategy for ischemic retinopathies.
Authors
Lingli Zhou, Zhenhua Xu, Yumin Oh, Rico Gamuyao, Grace Lee, Yangyiran Xie, Hongkwan Cho, Seulki Lee, Elia J. Duh
Abstract
Medulloblastoma (MB), one of the most malignant brain tumors of childhood, comprises distinct molecular subgroups, with p53 mutant sonic hedgehog (SHH)-activated MB patients having a very severe outcome that is associated with unfavorable histological large cell/anaplastic (LC/A) features. To identify the molecular underpinnings of this phenotype, we analyzed a large cohort of MBs developing in p53-deficient Ptch+/- SHH mice that, unexpectedly, showed LC/A traits that correlated with mechanistic Target Of Rapamycin Complex 1 (mTORC1) hyperactivation. Mechanistically, mTORC1 hyperactivation was mediated by a decrease in the p53-dependent expression of mTORC1 negative regulator Tsc2. Ectopic mTORC1 activation in mouse MB cancer stem cells (CSCs) promoted the in vivo acquisition of LC/A features and increased malignancy; accordingly, mTORC1 inhibition in p53-mutant Ptch+/- SHH MBs and CSC-derived MBs resulted in reduced tumor burden and aggressiveness. Most remarkably, mTORC1 hyperactivation was detected only in p53-mutant SHH MB patients’ samples and treatment with rapamycin of a human preclinical model phenocopying this subgroup decreased tumor growth and malignancy. Thus, mTORC1 may act as a specific druggable target for this subset of SHH MB, resulting in the implementation of a stringent risk stratification and in the potentially rapid translation of this precision medicine approach into the clinical setting.
Authors
Valentina Conti, Manuela Cominelli, Valentina Pieri, Alberto L. Gallotti, Ilaria Pagano, Matteo Zanella, Stefania Mazzoleni, Flavia Pivetta, Monica Patanè, Giulia M. Scotti, Ignazio S. Piras, Bianca Pollo, Andrea Falini, Alessio Zippo, Antonella Castellano, Roberta Maestro, Pietro L. Poliani, Rossella Galli
Abstract
Immune cells exhibit low-level, constitutive signaling at rest (tonic signaling). Such tonic signals are required for fundamental processes, including the survival of B lymphocytes, but when elevated by genetic or environmental causes can lead to autoimmunity. Events that control ongoing signal transduction are therefore tightly regulated by submembrane cytoskeletal polymers like filamentous (F)-actin. The actin-binding proteins that underpin the process, however, are poorly described. By investigating patients with ARPC1B-deficiency, we report that ARPC1B-containing ARP2/3 complexes are stimulated by Wiskott Aldrich Syndrome protein (WASP) to nucleate the branched actin networks that control tonic signaling from the B cell receptor (BCR). Despite an upregulation of ARPC1A, ARPC1B-deficient cells were not capable of WASP-mediated nucleation by ARP2/3 and this caused the loss of WASP-dependent structures including podosomes in macrophages and lamellipodia in B cells. In the B cell compartment, ARPC1B-deficiency also led to weakening of the cortical F-actin cytoskeleton that normally curtails the diffusion of B cell receptors and ultimately resulted in increased tonic lipid signaling, oscillatory calcium release from the endoplasmic reticulum (ER), and phosphorylated Akt. These events contributed to skewing the threshold for B cell activation in response to microbial associated molecular patterns (MAMPs). Thus, ARPC1B is critical for ARP2/3 complexes to control steady-state signaling of immune cells.
Authors
Gabriella Leung, Yuhuan Zhou, Philip Ostrowski, Sivakami Mylvaganam, Parastoo Boroumand, Daniel J. Mulder, Conghui Guo, Aleixo M. Muise, Spencer Freeman
Abstract
Angiogenesis, a hallmark of cancer, is induced by vascular endothelial growth factor-A (VEGF). As a result, anti-VEGF therapy is commonly employed for cancer treatment. Recent studies have found that VEGF expression is also associated with immune suppression in cancer patients. This connection has been investigated in preclinical and clinical studies by evaluating the therapeutic effect of combining anti-angiogenic reagents with immune therapy. However, the mechanisms of how anti-VEGF strategies enhance immune therapy are not fully understood. We and others have shown selective elevation of VEGFR2 expression on tumor-associated myeloid cells in tumor-bearing animals. Here we investigated the function of VEGFR2+ myeloid cells in regulating tumor immunity and found VEGF induces an immunosuppressive phenotype in VEGFR2+ myeloid cells including directly upregulating the expression of programmed cell death 1-ligand 1 (PD-L1). Moreover, we found that VEGF blockade inhibits the immunosuppressive phenotype of VEGFR2+ myeloid cells, increases T cell activation and enhances the efficacy of immune checkpoint blockade. This study highlights the function of VEGFR2 on myeloid cells and provides mechanistic insight on how VEGF inhibition potentiates immune checkpoint blockade.
Authors
Yuqing Zhang, Huocong Huang, Morgan Coleman, Arturas Ziemys, Purva Gopal, Syed M. Kazmi, Rolf A. Brekken
Abstract
Understanding the presence and durability of antibodies against SARS-CoV-2 in the airways is required to provide insights on the ability of individuals to neutralize the virus locally and prevent viral spread. Here, we longitudinally assessed both systemic and airway immune responses upon SARS-CoV-2 infection in a clinically well-characterized cohort of 147 infected individuals representing the full spectrum of COVID-19 severity; from asymptomatic infection to fatal disease. In addition, we evaluated how SARS-CoV-2 vaccination influenced the antibody responses in a subset of these individuals during convalescence as compared to naïve individuals. Not only systemic but also airway antibody responses correlated with the degree of COVID-19 disease severity. However, while systemic IgG levels were durable for up to 8 months, airway IgG and IgA had declined significantly within 3 months. After vaccination, there was an increase in both systemic and airway antibodies, in particular IgG, often exceeding the levels found during acute disease. In contrast, naïve individuals showed low airway antibodies after vaccination. In the former COVID-19 patients, airway antibody levels were significantly elevated after the boost vaccination, highlighting the importance of prime and boost vaccination also for previously infected individuals to obtain optimal mucosal protection.
Authors
Alberto Cagigi, Meng Yu, Björn Österberg, Julia Svensson, Sara Falck-Jones, Sindhu Vangeti, Eric Åhlberg, Lida Azizmohammadi, Anna Warnqvist, Ryan Falck-Jones, Pia C. Gubisch, Mert Ödemis, Farangies Ghafoor, Mona Eisele, Klara Lenart, Max Bell, Niclas Johansson, Jan Albert, Jörgen Sälde, Deleah D. Pettie, Michael P. Murphy, Lauren Carter, Neil P. King, Sebastian Ols, Johan Normark, Clas Ahlm, Mattias N. Forsell, Anna Färnert, Karin Loré, Anna Smed-Sörensen
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