Background: Control of the tuberculosis (TB) pandemic remains hindered, in part, by a lack of simple and accurate measures of treatment efficacy. Current gold standard markers rely on sputum-based assays that are slow and challenging to implement. Previous work identified urinary N1, N12-diacetylspermine (DiAcSpm), neopterin, hydroxykynurenine, N-acetylhexosamine, ureidopropionic acid, sialic acid, and m/z 241.0903 as potential biomarkers of active pulmonary TB (ATB). Here, we evaluated their ability to serve as biomarkers of TB treatment response and mycobacterial load. Methods: We analyzed urine samples prospectively collected from two cohorts with ATB: 34 participants from African countries treated with first line TB therapy (HRZE) and followed for one year, and 35 participants from Haiti treated with either HRZE or an experimental drug followed for 14 days. Blinded samples were analyzed by untargeted high-performance liquid chromatography-coupled-time of flight-mass spectrometry. Results: Urinary levels of all seven molecules exhibited significant decreases by week 26 of successful treatment (p=0.01-p<0.0001), and positive correlations with sputum mycobacterial load (p<0.0001). Urinary levels of DiAcSpm exhibited significant decreases in participants treated with HRZE as early as 14 days (p<0.0001) but were unchanged in participants receiving ineffective therapy (p=0.14). Conclusion: Reductions in urinary DiAcSpm, neopterin, hydroxykynurenine, N-acetylhexosamine, ureidopropionic acid, sialic acid, and m/z 241.0903 correlated with successful anti-TB treatment and sputum mycobacterial load. Levels of DiAcSpm exhibited reductions capable of differentiating treatment success from failure as early as two weeks after the initiation of chemotherapy, commending its further development as a potentially simple, non-invasive biomarker of treatment response and bacterial load. Funding: This work was supported by the Clinical and Translational Science Center at Weill Cornell College of Medicine (NIH/NCATS 1 UL1 TR002384-02 and KL2TR000458), the Department of Defense (PR170782), the National Institute of Allergy and Infectious Disease grants (NIAID T32AI007613-16, K24 AI098627 and K23 AI131913), the NIH Fogarty International Center grants (R24 TW007988 and TW010062), the National Institute of Health grant (R01 GM135926 ), the Abby and Howard P. Milstein Program in Chemical Biology and Translational Medicine, and the Tuberculosis Research Units Networks (TBRU-N, AI111143).
Qianjing Xia, Myung Hee Lee, Kathleen F. Walsh, Kathrine McAulay, James M. Bean, Daniel W. Fitzgerald, Kathryn M. Dupnik, Warren D. Johnson, Jean W. Pape, Kyu Y. Rhee, Flonza Isa
Evaluation of potential immunity against the novel severe acute respiratory syndrome (SARS) coronavirus that emerged in 2019 (SARS-CoV-2) is essential for health, as well as social and economic recovery. Generation of antibody response to SARS-CoV-2 (seroconversion) may inform on acquired immunity from prior exposure, and antibodies to the SARS-CoV-2 spike protein receptor binding domain (S-RBD) are speculated to neutralize virus infection. Some serology assays rely solely on SARS-CoV-2 nucleocapsid protein (N-protein) as the antibody detection antigen; however, whether such immune responses correlate with S-RBD response and COVID-19 immunity remains unknown. Here, we generated a quantitative serological enzyme-linked immunosorbent assay (ELISA) using recombinant S-RBD and N-protein for the detection of circulating antibodies in 138 serial serum samples from 30 RT-PCR confirmed SARS-CoV-2 hospitalized patients, as well as 464 healthy and non-COVID-19 serum samples that were collected between June 2017 and June 2020. Quantitative detection of IgG antibodies to the two different viral proteins showed a moderate correlation. Antibodies to N-protein were detected at a rate of 3.6% in healthy and non-COVID-19 sera collected during the pandemic in 2020, whereas 1.6% of these sera were positive for S-RBD. Approximately 86% of individuals positive for S-RBD binding antibodies exhibited neutralizing capacity, but only 74% of N-protein positive individuals exhibited neutralizing capacity. Collectively, our studies show that detection of N-protein binding antibodies does not always correlate with presence of S-RBD neutralizing antibodies, and cautions against the extensive use of N-protein based serology testing for determination of potential COVID-19 immunity.
Kathleen M. McAndrews, Dara P. Dowlatshahi, Jianli Dai, Lisa M. Becker, Janine Hensel, Laura M. Snowden, Jennifer M. Leveille, Michael R. Brunner, Kylie Holden, Nikolas S. Hopkins, Alexandria Harris, Jerusha J. Kumpati, Michael A. Whitt, J. Jack Lee, Luis Ostrosky-Zeichner, Ramesha Papanna, Valerie LeBleu, James Allison, Raghu Kalluri
Angiogenesis is essential for cardiac functional recovery after myocardial infarction (MI). HSPA12B is predominately expressed in endothelial cells and required for angiogenesis. Yes-associated protein (YAP) plays an important role in tumor angiogenesis. This study investigated the cooperative role of HSPA12B and YAP in angiogenesis post-MI. Silencing of either HSPA12B or YAP impairs hypoxia-promoted endothelial cell proliferation and angiogenesis. Deficiency of HSPA12B suppresses YAP expression and nuclear translocation following hypoxia. Knockdown of YAP attenuates hypoxia-stimulated HSPA12B nuclear translocation and abrogates HSPA12B-promoted endothelial cell angiogenesis. Mechanistically, hypoxia induced an interaction between endothelial HSPA12B and YAP. ChIP assay shows that HSPA12B is a target gene of YAP/transcriptional enhanced associated domain4 (TEAD4) and a co-activator in YAP-associated angiogenesis. In vivo studies using the MI model show that endothelial specific deficiency of HSPA12B (eHspa12b-/-) or YAP (eYap-/-) impairs angiogenesis and exacerbates cardiac dysfunction, when compared with wild type (WT) mice. MI increased YAP expression and nuclear translocation in WT hearts, but not in eHspa12b-/- hearts. HSPA12B expression and nuclear translocation were up-regulated in WT MI hearts, but not in eYap-/- MI myocardium. Our data demonstrated that endothelial HSPA12B is a novel target and co-activator for YAP/TEAD4 and cooperates with YAP to regulate endothelial angiogenesis post-MI.
Min Fan, Kun Yang, Xiaohui Wang, Yana Wang, Fei Tu, Tuanzhu Ha, Li Liu, David L. Williams, Chuanfu Li
Age-associated systemic, chronic inflammation is partially attributed to increased self (auto)-reactivity, resulting from disruption of central tolerance in the aged, involuted thymus. This involution causally results from gradually decreased expression of the transcription factor FOXN1 in thymic epithelial cells (TECs), while exogenous FOXN1 in TECs can partially rescue age-related thymic involution. Given the findings that TECs induced from FOXN1-overexpressing embryonic fibroblasts can generate an ectopic de novo thymus under the kidney capsule and intra-thymically injected naturally young TECs can lead to middle-aged thymus regrowth, we attempted to extend these two findings by combining them as a novel thymic rejuvenation strategy with two types of promoter-driven (Rosa26CreERT and FoxN1Cre) Cre-mediated FOXN1-reprogrammed embryonic fibroblasts (FREFs). We engrafted these two-types of FREFs directly into the aged murine thymus. We found significant regrowth of the native aged thymus with rejuvenated architecture and function in both males and females, exhibiting increased thymopoiesis and reinforced thymocyte negative selection, along with reduced senescent T cells and auto-reactive T cell-mediated inflammation in old mice. Therefore, this strategy has preclinical significance and presents a strategy to potentially rescue decreased thymopoiesis and perturbed negative selection to significantly, albeit partially, restore defective central tolerance and reduce subclinical autoimmune symptoms in the elderly.
Jiyoung Oh, Weikan Wang, Rachel Thomas, Dong-Ming Su
Abstract: S-Nitroso-L-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase (sGC)-independent effects are stereoselective - that is, not recapitulated by S-nitroso-D-cysteine (D-CSNO) – and are inhibited by chemical cogeners. However, candidate L-CSNO receptors have never been identified. Here, we have used two complementary affinity chromatography assays - followed by unbiased proteomic analysis - to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO-Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy, hydrogen deuterium exchange and, in Kv1.1/Kv1.2/Kvβ2 overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv 1.1 prevented this effect. In intact animals, L-CSNO injection at level of the carotid body (CB) dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO cogener S-methyl-L-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.
Benjamin Gaston, Laura A. Smith, Jürgen Bosch, James M. Seckler, Diana L. Kunze, Janna Kiselar, Nadzeya Marozkina, Craig Hodges, Patrick Wintrode, Kellen McGee, Tatiana Morozkina, Spencer T. Burton, Tristan Lewis, Timothy Strassmaier, Paulina Getsy, James N. Bates, Stephen J. Lewis
New strategies are needed to enhance the efficacy of anti-programmed cell death protein (PD-1) antibody (Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of CQ derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in melanoma. The combination resulted tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen primed T cells to macrophage conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma specific killing. Genetic or chemical PPT1 inhibition resulted an M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells (MDSCs) in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), tank-binding kinase 1 (TBK1) pathway activation and the secretion of interferon β (IFN-β) in macrophages which was a key component for augmented T cell-mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for a melanoma clinical trial testing this new immunotherapy combination and may also be effective in other cancers.
Gaurav Sharma, Rani Ojha, Estela Noguera-Ortega, Vito W. Rebecca, John Attanasio, Shujing Liu, Shengfu Piao, Jennifer J. Lee, Michael C. Nicastri, Sandra L. Harper, Amruta Ronghe, Vaibhav Jain, Jeffrey D. Winkler, David W. Speicher, Jerome Mastio, Phyllis A Gimotty, Xiaowei Xu, E. John Wherry, Dmitry I. Gabrilovich, Ravi K. Amaravadi
Tumor-associated macrophages (TAMs) affect cancer progression and therapy. Ovarian carcinoma often metastasizes to the peritoneal cavity. Here, we found two peritoneal macrophage subsets in mice bearing ID8 ovarian cancer based on the Tim-4 (T-cell immunoglobulin and mucin domain containing 4) expression. Tim-4+ TAMs were embryonically originated and locally sustained while Tim-4- TAMs were replenished from circulating monocytes. Tim-4+ TAMs, but not Tim-4- TAMs, promoted tumor growth in vivo. Relative to Tim-4- TAMs, Tim-4+ TAMs manifested high oxidative phosphorylation and adapted mitophagy to alleviate oxidative stress. High levels of arginase-1 in Tim-4+ TAMs contributed to potent mitophagy activities via weakened mTORC1 activation due to low arginine resultant from arginase-1-mediated metabolism. Furthermore, genetic deficiency of autophagy element FIP200 resulted in Tim-4+ TAM loss via ROS-mediated apoptosis, and elevated T cell-immunity and ID8 tumor inhibition in vivo. Moreover, human ovarian cancer-associated CRIg (complement receptor of the Immunoglobulin superfamily) positive macrophages were transcriptionally, metabolically, and functionally similar to murine Tim-4+ TAMs. Thus, targeting CRIg+ (Tim-4+) TAMs may potentially treat ovarian cancer patients with peritoneal metastasis.
Houjun Xia, Shasha Li, Xiong Li, Weichao Wang, Yingjie Bian, Shuang Wei, Sara Grove, Weimin Wang, Linda Vatan, J. Rebecca Liu, Karen McLean, Ramandeep Rattan, Adnan R. Munkarah, Jun-Lin Guan, Ilona Kryczek, Weiping Zou
Electroconvulsive therapy is highly effective in neuropsychiatric disorders by unknown mechanisms. Microglial toxicity plays key role in neuroinflammatory and degenerative diseases, where there is critical shortage in therapies. This study examined the effects of electroconvulsive seizures (ECS) on chronic neuroinflammation and microglial neurotoxicity.Electric brain stimulation inducing full tonic-clonic seizures during chronic relapsing-progressive experimental autoimmune encephalomyelitis (EAE) reduced spinal immune cell infiltration, reduced myelin and axonal loss, and prevented clinical deterioration. Using the transfer EAE model we examined the effect of ECS on systemic immune response in donor mice versus ECS effect on CNS innate immune activity in recipient mice. ECS did not affect encephalitogenicity of systemic T cells, but targeted the CNS directly to inhibit T-cell induced neuroinflammation. In vivo and ex-vivo assays indicated that ECS suppressed microglial neurotoxicity, by reducing iNOS expression, nitric oxide and reactive oxygen species (ROS) production, and by reducing CNS oxidative stress. Microglia from ECS treated EAE mice expressed less T cell stimulatory and chemoattractant factors. Our finding indicate that Electroconvulsive therapy targets the CNS innate immune system to reduce neuroinflammation by attenuating microglial neurotoxicity. These findings signify a novel therapeutic approach for chronic neuroinflammatory, neuropsychiatric and neurodegenerative diseases.
Smadar Goldfarb, Nina Fainstein, Tamir Ben-Hur
Background Tuberculosis (TB) kills more people than any other infection and new diagnostic tests to identify active cases are urgently required. We aimed to discover and verify novel markers for TB in non-depleted plasma. Methods We applied an optimised quantitative proteomics discovery methodology based on multidimensional and orthogonal liquid chromatographic separation hyphenated with high-resolution mass spectrometry (q3D LC-MS) to study non-depleted plasma of 11 patients with active TB compared to 10 healthy control donors. Prioritised candidates were verified in an independent UK-based (n=118) and a South African cohorts (n=203). Results We generated the most comprehensive TB plasma proteome to date, profiling 5022 proteins spanning 11 orders-of-magnitude concentration range with diverse biochemical and molecular properties. We further analysed the predominantly low molecular weight sub-proteome; identifying 46 proteins with significantly increased and 90 with decreased abundance (peptide FDR ≤1%, q-value ≤0.05). Biological network analysis showed regulation of new pathways involving lipid and organophosphate ester transport. Verification was performed for novel candidate biomarkers (CFHR5, ILF2) in two independent cohorts. These proteins were elevated in both TB and other respiratory diseases (ORD). Receiver-operating-characteristics analyses using a 5-protein panel (CFHR5, LRG1, CRP, LBP and SAA1) exhibited discriminatory power in distinguishing between TB and ORD (AUC =0.81). Conclusions We report the most comprehensive TB plasma proteome to date, identifying numerous novel markers with verification in two independent cohorts, which led to a 5-protein biosignature with potential to improve TB diagnosis. With further development, these biomarkers have potential as a diagnostic triage test. Funding Colombia: Colciencias. UK: Medical Research Council, Innovate UK, National Institute for Health Research, Academy of Medical Sciences. Peru: Program for Advanced Research Capacities for AIDS. South Africa: Wellcome Centre for Infectious Diseases Research.
Diana J. Garay-Baquero, Cory H. White, Naomi F. Walker, Marc Tebruegge, Hannah F. Schiff, Cesar Ugarte-Gil, Stephen Morris-Jones, Ben G. Marshall, Antigoni Manousopoulou, John H. Adamson, Andres F. Vallejo, Magdalena K. Bielecka, Robert J. Wilkinson, Liku B. Tezera, Christopher H. Woelk, Spiros D. Garbis, Paul Elkington
Establishing the interactome of the cancer associated stress protein NUPR1 (NUclear PRotein 1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1 thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further reported that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system indicating NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation dependent functions of key proteins involved in the DDR.
Wenjun Lan, Patricia Santofimia-Castaño, Mirna Swayden, Yi Xia, Zhengwei Zhou, Stephane Audebert, Luc Camoin, Can Huang, Ling Peng, Ana Jiménez-Alesanco, Adrián Velázquez-Campoy, Olga Abian, Gwen Lomberk, Raul Urrutia, Bruno Rizzuti, Vincent Geli, Philippe Soubeyran, Jose Luis Neira, Juan L. Iovanna
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