Pain signals are relayed to the brain via a nociceptive system, and in rare situations, this nociceptive system contains genetic variants that can limit pain response. Here we questioned whether a human transient receptor potential vanilloid 1 (TRPV1) missense variant causes a resistance to noxious stimuli and further if we can target this region by a cell-permeable peptide as a pain therapeutic. Initially using a computational approach, we identified a human K710N TRPV1 missense variant in an otherwise highly conserved region of mammalian TRPV1. After generating a TRPV1K710N knock-in mouse using CRISPR/Cas9, we discovered the K710N variant reduced capsaicin-induced calcium influx in dorsal root ganglion neurons. The TRPV1K710N rodents also had less acute behavioral response to chemical noxious stimuli and less hypersensitivity to nerve injury-induced pain, while leaving the response to noxious heat intact. Furthermore, blocking this K710 region in wild-type rodents by a cell-penetrating peptide limited acute behavioral responses to noxious stimuli and rescued pain hypersensitivity induced by nerve injury back to baseline. These findings identify K710 TRPV1 as a discrete site crucial for the control of nociception and provides new insights into how to leverage rare genetic variants in humans to uncover fresh strategies for developing pain therapeutics.
Shufang He, Vanessa O. Zambelli, Pritam Sinharoy, Laura Brabenec, Yang Bian, Freeborn Rwere, Rafaela C.R. Hell, Beatriz Stein Neto, Barbara Hung, Xuan Yu, Meng Zhao, Zhaofei Luo, Chao Wu, Lijun Xu, Katrin J. Svensson, Stacy L. McAllister, Creed M. Stary, Nana-Maria Wagner, Ye Zhang, Eric R. Gross
There is an unmet need for monoclonal antibodies (mAbs) for prevention or as adjunctive treatment of herpes simplex virus (HSV) disease. Most vaccine and mAb efforts focus on neutralizing antibodies, but for HSV this strategy has proven ineffective. Preclinical studies with a candidate HSV vaccine strain, ΔgD-2, demonstrated that non-neutralizing antibodies that activate Fc-gamma receptors (FcɣRs) to mediate antibody-dependent cellular cytotoxicity (ADCC) provide active and passive protection against HSV-1 and HSV-2. We hypothesized that this vaccine provides a tool to identify and characterize protective mAbs. We isolated HSV-specific mAbs from germinal center and memory B cells and bone marrow plasmacytes of ΔgD-2 vaccinated mice and evaluated these mAbs for binding, neutralizing and FcɣR-activating activity and for protective efficacy in mice. The most potent protective mAb, BMPC-23, was not neutralizing but activated murine FcɣRIV, a biomarker of ADCC. The cryo-EM structure of the Fab-glycoprotein B (gB) assembly identified domain IV of gB as the epitope. A single dose of BMPC-23 administered 24 hours before or after viral challenge provided significant protection when configured as mouse IgG2c and protected mice expressing human FcɣRIII when engineered as a human IgG1. These results highlight the importance of FcɣR-activating antibodies in protecting against HSV.
Masayuki Kuraoka, Clare Burn Aschner, Ian W. Windsor, Aakash Mahant Mahant, Scott J. Garforth, Susan Luozheng Kong, Jacqueline M. Achkar, Steven C. Almo, Garnett Kelsoe, Betsy C. Herold
BACKGROUND. The presence and reactivation of chronic viral infections such as Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human immunodeficiency virus (HIV) have been proposed as potential contributors to Long COVID (LC), but studies in well-characterized post-acute cohorts of individuals with COVID-19 over a longer time course consistent with current case definitions of LC are limited. METHODS. In a cohort of 280 adults with prior SARS-CoV-2 infection, we assessed the presence and types of LC symptoms and prior medical history (including COVID-19 history and HIV status), and performed serological testing for EBV and CMV using a commercial laboratory. We used covariate-adjusted binary logistic regression models to identify independent associations between variables and LC symptoms. RESULTS. We observed that LC symptoms such as fatigue and neurocognitive dysfunction at a median of 4months following initial diagnosis were independently associated with serological evidence suggesting recent EBV reactivation (early antigen-D [EA-D] IgG positivity) or high nuclear antigen (EBNA) IgG levels, but not with ongoing EBV viremia. Serological evidence suggesting recent EBV reactivation (EA-D IgG) was most strongly associated with fatigue (OR 2.12). Underlying HIV infection was also independently associated with neurocognitive LC (OR 2.5). Interestingly, participants who had serologic evidence of prior CMV infection were less likely to develop neurocognitive LC (OR 0.52). CONCLUSION. Overall, these findings suggest differential effects of chronic viral co-infections on the likelihood of developing LC and predicted distinct syndromic patterns. Further assessment during the acute phase of COVID-19 is warranted. TRIAL REGISTRATION. Long-term Impact of Infection with Novel Coronavirus (LIINC); NCT04362150 FUNDING. This work was supported by the National Institute of Allergy and Infectious Diseases NIH/NIAID 3R01AI141003-03S1 to TJ Henrich, R01AI158013 to M Gandhi and M Spinelli, K24AI145806 to P Hunt, and by the Zuckerberg San Francisco Hospital Department of Medicine and Division of HIV, Infectious Diseases, and Global Medicine. MJP is supported on K23 A137522 and received support from the UCSFBay Area Center for AIDS Research (P30-AI027763).
Michael J. Peluso, Tyler-Marie Deveau, Sadie E. Munter, Dylan M. Ryder, Amanda M. Buck, Gabriele Beck-Engeser, Fay Chan, Scott Lu, Sarah A. Goldberg, Rebecca Hoh, Viva Tai, Leonel Torres, Nikita S. Iyer, Monika Deswal, Lynn H. Ngo, Melissa Buitrago, Antonio E. Rodriguez, Jessica Y. Chen, Brandon C. Yee, Ahmed Chenna, John W. Winslow, Christos J. Petropoulos, Amelia N. Deitchman, Joanna Hellmuth, Matthew A. Spinelli, Matthew S. Durstenfeld, Priscilla Y. Hsue, John Daniel Kelly, Jeffrey N. Martin, Steven G. Deeks, Peter W. Hunt, Timothy J. Henrich
Comprehensive cis-regulatory landscapes are essential for accurate enhancer prediction and disease variant mapping. Although cis-regulatory element (CRE) resources exist for most tissues and organs, many rare – yet functionally important – cell types remain overlooked. Despite representing only a small fraction of the heart’s cellular biomass, the cardiac conduction system (CCS) unfailingly coordinates every life-sustaining heartbeat. To globally profile the mouse CCS cis-regulatory landscape, we genetically tagged CCS component-specific nuclei for comprehensive ATAC-seq analysis. Thus, we established a global CCS-enriched CRE database (CCS-ATAC) as a key resource for studying CCS-wide and component-specific regulatory functions. Using transcription factor (TF) motifs to construct CCS component-specific gene regulatory networks (GRNs), we identify and independently confirm several novel TF sub-networks. Highlighting the functional importance of CCS-ATAC, we also validate numerous CCS-enriched enhancer elements and suggest gene targets based on CCS single-cell RNA-seq data. Furthermore, we leverage CCS-ATAC to improve annotation of existing human variants related to cardiac rhythm and nominate a potential enhancer-target pair dysregulated by a specific SNP. Collectively, our results establish a CCS regulatory compendium, identify novel CCS enhancer elements, and illuminate potential functional associations between human genomic variants and CCS component-specific CREs.
Samadrita Bhattacharyya, Rahul K. Kollipara, Gabriela Orquera-Tornakian, Sean Goetsch, Minzhe Zhang, Cameron Perry, Boxun Li, John M. Shelton, Minoti Bhakta, Jialei Duan, Yang Xie, Guanghua Xiao, Bret M. Evers, Gary C. Hon, Ralf Kittler, Nikhil V. Munshi
Chronic-pain is a debilitating illness that has become exceedingly widespread with currently limited treatments. Differences in the molecular signature of nociceptors, have been demonstrated between human and the commonly-used mouse model, suggesting functional differences in detection and transmission of noxious-stimuli. Therefore, direct understanding of pain-physiology in humans is required for pain treatment. This could be facilitated by studying humans carrying deleterious genetic mutations affecting pain sensation. The transient receptor potential vanilloid 1 (TRPV1) channel is associated with several body-functions, in particular, noxious-heat detection and inflammatory-pain. Reports of adverse effects in human trials have hinder the clinical development of TRPV1 antagonists as novel pain relievers. Hence, studies on the functional roles of TRPV1, which currently rely mainly on evidences obtained from rodents, should be extended to humans. Here, we examined humans carrying a unique missense mutation in TRPV1, rendering the channel non-functional. The affected individual demonstrated lack of aversion towards capsaicin and elevated heat-pain threshold. Surprisingly, he showed elevated cold-pain threshold and extensive neurogenic inflammatory flare and pain-responses following application of the TRPA1 channel-activator, mustard-oil. Our study provides the first direct evidence for pain-related functional-changes linked to TRPV1 in humans, which is a prime target in the development of novel pain-relievers.
Ben Katz, Rachel Zaguri, Simon Edvardson, Channa Maayan, Orly Elpeleg, Shaya Lev, Elyad Davidson, Maximilian Peters, Shlomit Kfir-Erenfeld, Esther Berger, Shifa Ghazalin, Alexander M. Binshtok, Baruch Minke
The emergence of the novel henipavirus, Langya virus, received global attention earlier this month after the virus sickened over three dozen people in China. There is heightened concern henipaviruses as respiratory pathogens could spark another pandemic, most notably the deadly Nipah virus (NiV). NiV causes near annual outbreaks in Bangladesh and India and induces a highly fatal respiratory disease and encephalitis in humans. No licensed countermeasures against this pathogen exist. An ideal NiV vaccine would confer both fast-acting and long-lived protection. Recently, we reported the generation of a recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing the NiV glycoprotein (rVSV-ΔG-NiVBG) that protected 100% of nonhuman primates from NiV-associated lethality within a week. Here, to evaluate the durability of rVSV-ΔG-NiVBG, we vaccinated African green monkeys (AGMs) one year prior to challenge with a uniformly lethal dose of NiV. The rVSV-ΔG-NiVBG vaccine induced stable and robust humoral responses, whereas cellular responses were modest. All immunized AGMs (whether receiving a single dose or prime-boosted) survived with no detectable clinical signs or NiV replication. Transcriptomic analyses indicated adaptive immune signatures correlated with vaccine-mediated protection. While vaccines for certain respiratory infections (e.g., COVID-19) have yet to provide durable protection, our results suggest rVSV-ΔG-NiVBG elicits long-lasting immunity.
Courtney Woolsey, Viktoriya Borisevich, Alyssa C. Fears, Krystle N. Agans, Daniel J. Deer, Abhishek N. Prasad, Rachel O'Toole, Stephanie L. Foster, Natalie S. Dobias, Joan B. Geisbert, Karla A. Fenton, Robert W. Cross, Thomas Geisbert
Absence of Interferon-γ Receptor (IFNGR) or Signal Transducer and Activator of Transcription 1 (STAT1) signaling in donor cells has been shown to result in reduced acute GVHD induction. In this study, we unexpectedly observed increased activation and expansion of donor lymphocytes in both lymphohematopoietic organs and GVHD target tissues of IFNGR/STAT1-deficient recipient mice, leading to rapid mortality following the induction of GVHD. Lipopolysaccharide (LPS)-matured bone marrow-derived Ifngr1-/-/Stat1-/- dendritic cells (BMDCs) were more potent allogeneic stimulators and expressed increased levels of MHC II and costimulatory molecules. Similar effects were observed in human APCs with knockdown of Stat1 by CRISPR/Cas9 and treatment with a JAK1/2 inhibitor. Furthermore, we demonstrated that the absence of IFNGR/STAT1 signaling in hematopoietic APCs impaired the presentation of exogenous antigens while promoting the presentation of endogenous antigens. In contrast, the indirect presentation of host antigens to donor lymphocytes was defective in IFNGR/STAT1-deficient donor-derived APCs in fully donor chimeric mice. The differential effects of IFNGR/STAT1 signaling on endogenous and exogenous antigen presentation could provide further insight into the roles of the IFN-γ/STAT1 signal pathway in the pathogenesis of GVHD, organ rejection, and autoimmune diseases.
Caisheng Lu, Huihui Ma, Liangsong Song, Hui Wang, Lily Wang, Shirong Li, Stephen M. Lagana, Antonia R. Sepulveda, Kasper Hoebe, Samuel S. Pan, Yong-Guang Yang, Suzanne Lentzsch, Markus Y. Mapara
KRAS is one of the most frequently activated oncogenes in human cancers. While the role of KRAS mutation in tumorigenesis and tumor maintenance has been extensively studied, the relationship between KRAS and the tumor immune microenvironment is not fully understood. Herein, we identified a novel role of KRAS in driving tumor evasion from innate immune surveillance. In lung adenocarcinoma patient samples and Kras-driven genetic mouse models of lung cancer, mutant KRAS activated the expression of cluster of differentiation 47 (CD47), an antiphagocytic signal in cancer cells, leading to decreased phagocytosis of cancer cells by macrophages. Mechanistically, mutant KRAS activated PI3K-STAT3 signaling, which restrained miR-34a expression and relieved the post-transcriptional repression of miR-34a on CD47. In three independent lung cancer patient cohorts, KRAS mutation status positively correlated with CD47 expression. Therapeutically, disruption of the KRAS-CD47 signaling axis with KRAS siRNA, the KRASG12C inhibitor AMG 510 or miR-34a mimic suppressed CD47 expression, enhanced the phagocytic capacity of macrophages and restored innate immune surveillance. Our results revealed a direct mechanistic link between active KRAS and innate immune evasion and identified CD47 as a major effector underlying KRAS-mediated immunosuppressive tumor microenvironment.
Huanhuan Hu, Rongjie Cheng, Yanbo Wang, Xiaojun Wang, Jianzhuang Wu, Yan Kong, Shoubin Zhan, Zhen Zhou, Hongyu Zhu, Ranran Yu, Gaoli Liang, Qingyan Wang, Xiaoju Zhu, Chen-Yu Zhang, Rong Yin, Chao Yan, Xi Chen
BACKGROUND. Sanfilippo type B is a mucopolysaccharidosis (MPS) with a major neuronopathic component characterized by heparan sulfate (HS) accumulation due to mutations in the NAGLU gene encoding for alfa-N-acetyl-glucosaminidase. Enzyme replacement therapy for neuronopathic MPS requires efficient enzyme delivery throughout the brain in order to normalize HS, prevent brain atrophy and potentially delay cognitive decline. METHODS. In this phase 1/2, open-label study, subjects (n=22) affected with MPS IIIB were treated with tralesinidase alfa administered intracerebroventricularly (ICV). Subjects were monitored for drug exposure, total HS and HS non-reducing end (HS-NRE) levels in both cerebrospinal fluid (CSF) and plasma, anti-drug antibody response, brain, spleen and liver volumes as measured by magnetic resonance imaging and cognitive development as measured by age-equivalent (AEq) scores. RESULTS. In the Part 1 dose escalation (30, 100, and 300 mg) phase, tralesinidase alfa 300 mg was necessary to achieve normalization of HS and HS-NRE in CSF and plasma. In Part 2, tralesinidase alfa 300 mg sustained HS and HS-NRE normalization in the CSF and stabilized cortical grey matter volume (CGMV) over 48 weeks of treatment. Resolution of hepatomegaly and reduction in spleen volume were observed in most subjects. Significant correlations were also established between change in cognitive AEq and plasma drug exposure, plasma HS-NRE level and change in CGMV. CONCLUSION. ICV administration of tralesinidase alfa effectively normalized HS and HS-NRE as a prerequisite for clinical efficacy. Peripheral drug exposure data suggests a role for the glymphatic system in altering tralesinidase alfa efficacy. TRIAL REGISTRATION. Clinicaltrials.gov: NCT02754076.
Nicole Muschol, Anja Koehn, Katharina von Cossel, Ilyas Okur, Fatih Ezgu, Paul Harmatz, Maria Jose de Castro Lopez, Maria Luz Couce, Shuan-Pei Lin, Spyros Batzios, Maureen Cleary, Martha Solano, Igor Nestrasil, Brian D. Kaufman, Adam J. Shaywitz, Stephen M. Maricich, Bernice Kuca, Joseph Kovalchin, Eric H. Zanelli
BACKGROUND. Studies assessing the efficacy of therapies for neovascular age-related macular degeneration (nvAMD) have demonstrated that aflibercept may have a longer treatment interval than its lesser-expensive alternative, bevacizumab. However, whether this benefit justifies the additional cost of aflibercept remains under debate. We have recently reported that a “treat-and-extend-pause/monitor” (TEP/M) approach can be used to successfully wean 31% of nvAMD patients off anti-VEGF therapy. Here we examine whether the choice of therapy influences the outcomes of this approach. METHODS. In this retrospective analysis, 122 eyes of 106 patients with nvAMD underwent 3 consecutive monthly injections with either aflibercept (n=70) or bevacizumab (n=52) followed by a treat-and-extend protocol in which the decision to extend the interval between treatments was based on visual acuity, clinical exam, and the presence or absence of fluid on optical coherence tomography (OCT). Eyes that remained stable 12 weeks from their prior treatment were given a 6-week trial of holding further treatment, followed by quarterly monitoring. Treatment was resumed for worsening vision, clinical exam, or OCT findings. RESULTS. At the end of one year, eyes receiving bevacizumab had similar vision but required more injections (8.7 ±0.3 vs. 7.2 ±0.3) compared to aflibercept. However, eyes treated with aflibercept were almost 3-times more likely to be weaned off treatment (43% vs. 15%) compared to eyes treated with bevacizumab at the end of one year. CONCLUSIONS. These observations expose a previously unappreciated advantage of aflibercept over bevacizumab and have important clinical implications for the selection of therapy for patients with nvAMD.
Xuan Cao, Jaron Castillo Sanchez, Tapan P. Patel, Zhiyong Yang, Chuanyu Guo, Danyal Malik, Anuoluwapo Sopeyin, Silvia Montaner, Akrit Sodhi
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