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Abstract
BACKGROUND. The role of humoral immunity in the coronavirus disease 2019 (COVID-19) is not fully understood owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome. METHODS. Using SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution. RESULTS. We identified linear epitopes from the Spike (S) and Nucleocapsid (N) protein and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S(811-825), S(881-895) and N(156-170) epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes. CONCLUSIONS. Epitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, it may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern (VOC) in both the peptide array and latex agglutination formats. FUNDING. Ontario Research Fund (ORF)-COVID-19 Rapid Research Fund, the Toronto COVID-19 Action Fund, Western University, the Lawson Health Research Institute, the London Health Sciences Foundation, and the AMOSO Innovation Fund.
Authors
Courtney Voss, Sally Esmail, Xuguang Liu, Michael J. Knauer, Suzanne Ackloo, Tomonori Kaneko, Lori E. Lowes, Peter J. Stogios, Almagul Seitova, Ashley Hutchinson, Farhad Yusifov, Tatiana Skarina, Elena Evdokimova, Peter Loppnau, Pegah Ghiabi, Taraneh Hajian, Shanshan Zhong, Husam Abdoh, Benjamin D. Hedley, Vipin Bhayana, Claudio M. Martin, Marat Slessarev, Benjamin Chin-Yee, Douglas D. Fraser, Ian Chin-Yee, Shawn S.C. Li
Abstract
Exposure to maternal obesity may promote metabolic dysfunction in offspring. We use infant mesenchymal stem cells (MSC) to experimentally examine cellular mechanisms of intergenerational health transmission. Our earlier reports show MSCs collected from infants of mothers with obesity had a dichotomous distribution in metabolic efficiency; they were either efficient (Ef-Ob) or inefficient (In-Ob) with respect to fatty acid oxidation (FAO). Here, we sought to determine if this was due to a primary defect in FAO. Accordingly, we measured FAO in myogenic differentiating MSCs under three conditions: 1) myogenesis alone, 2) excess fatty acid exposure, and 3) excess fatty acid exposure plus a chemical uncoupler to increase metabolic rate. Compared to NW and Ef-Ob MSCs, In-Ob displayed lower FAO in myogenesis alone and after fatty acid plus uncoupler, indicating In-Ob were less metabolically flexible after increasing lipid availability and metabolic rate, demonstrating a primary deficit in FAO. MSC FAO was negatively associated with fasting maternal glucose and insulin, and positively associated with fasting HDL-cholesterol. MSC FAO was negatively associated with infant fat mass. These data indicate a less favorable maternal metabolic milieu, independent of maternal BMI, reduces intrinsic MSC FAO and is linked to higher infant adiposity as early as birth.
Authors
Melissa L. Erickson, Zachary W. Patinkin, Allison M. Duensing, Dana Dabelea, Leanne M. Redman, Kristen E. Boyle
Abstract
The main mechanisms underlying sexually dimorphic outcomes in neonatal lung injury are unknown. We tested the hypothesis that hormonal- or sex chromosome-mediated mechanisms interact with hyperoxia exposure to impact injury and repair in the neonatal lung. To distinguish sex differences caused by gonadal hormones versus sex chromosome complement (XX versus XY), we used the four core genotypes (FCG) mice and exposed them to hyperoxia (95% FiO2, PND1-4: saccular stage) or room air. This model generates XX and XY mice that each have either testes (with Sry, XXM or XYM) or ovaries (without Sry, XXF or XYF). Lung alveolarization and vascular development were more severely impacted in XYM and XYF compared to XXF and XXM mice. Cell cycle-related pathways were enriched in the gonadal or chromosomal females, while muscle-related pathways were enriched in the gonadal males, and immune-response related pathways were enriched in chromosomal males. Female gene signatures showed a negative correlation with human patients that developed BPD or needed oxygen therapy at 28 days. These results demonstrate that chromosomal sex and not gonadal sex impacted the response to neonatal hyperoxia exposure. The female sex chromosomal complement was protective and could mediate sex-specific differences in neonatal lung injury.
Authors
Sandra L. Grimm, Xiaoyu Dong, Yuhao Zhang, Alexandre F. Carisey, Arthur P. Arnold, Bhagavatula Moorthy, Cristian Coarfa, Krithika Lingappan
Abstract
The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) has resulted in an unprecedented pandemic that has been accompanied by a global health crisis. Although the lungs are the main organs involved in COVID-19, systemic disease with a wide range of clinical manifestations also develops in SARS-CoV-2-infected patients. One of the major systems affected by this virus is the cardiovascular system. The presence of pre-existing cardiovascular disease increases mortality in COVID-19 patients, and cardiovascular injuries, including myocarditis, cardiac rhythm abnormalities, endothelial cell injury, thrombotic events, and myocardial interstitial fibrosis, are observed in some COVID-19 patients. The underlying pathophysiology of COVID-19-associated cardiovascular complications is not fully understood, although direct viral infection of myocardium and cytokine storm have been suggested as possible mechanisms of myocarditis. In this review, we summarize available data on SARS-CoV-2-related cardiac damage and discuss potential mechanisms of cardiovascular implications of this rapidly spreading virus.
Authors
Farnaz Farshidfar, Navid Koleini, Hossein Ardehali
Abstract
The skin lesion erythema migrans (EM) is an initial sign of the Ixodes-tick transmitted Borreliella spirochetal infection known as Lyme disease. T cells and innate immune cells have previously been shown to predominate the EM lesion and promote the reaction. Despite the established importance of B cells and antibodies in preventing infection, the role of B cells in the skin immune response to Borreliella is unknown. Here, we used single-cell RNA-Seq in conjunction with B cell receptor (BCR) sequencing to immunophenotype EM lesions and their associated B cells and BCR repertoires. We found that B cells were more abundant in EM in comparison to autologous uninvolved skin; many were clonally expanded and had circulating relatives. EM-associated B cells upregulated expression of MHC class II genes and exhibited preferential IgM isotype usage. A subset also exhibited low levels of somatic hypermutation despite a gene expression profile consistent with memory B cells. Our study demonstrates that single-cell gene expression with paired BCR sequencing can be used to interrogate the sparse B cell populations in human skin and reveals that B cells in the skin infection site in early Lyme disease express a phenotype consistent with local antigen presentation and antibody production.
Authors
Ruoyi Jiang, Hailong Meng, Khadir Raddassi, Ira Fleming, Kenneth B. Hoehn, Kenneth R. Dardick, Alexia A. Belperron, Ruth R. Montgomery, Alex K. Shalek, David A. Hafler, Steven H. Kleinstein, Linda K. Bockenstedt
Abstract
Using genetically engineered mouse models we demonstrate that protein synthesis is essential for efficient urothelial cancer formation and growth but dispensable for bladder homeostasis. Through a candidate gene analysis for translation regulators implicated in this dependency, we discovered that phosphorylation of the translation initiation factor eIF4E at serine 209 is increased in both murine and human bladder cancer which corresponds with an increase in de novo protein synthesis. Employing an eIF4E serine 209 to alanine knock-in mutant mouse model we show that this single post-translational modification is critical for bladder cancer initiation and progression despite having no impact on normal bladder tissue maintenance. Using murine and human models of advanced bladder cancer, we demonstrate that only tumors with high levels of eIF4E phosphorylation are therapeutically vulnerable to eFT508, the first clinical grade inhibitor of MNK1 and MNK2, the upstream kinases of eIF4E. Together, our results show that phospho-eIF4E plays an important role in bladder cancer pathogenesis and targeting its upstream kinases could be an effective therapeutic option for bladder cancer patients with high levels of eIF4E phosphorylation.
Authors
Sujata Jana, Rucha Deo, Rowan P. Hough, Yuzhen Liu, Jessie L. Horn, Jonathan L. Wright, Hung-Ming Lam, Kevin R. Webster, Gary G. Chiang, Nahum Sonenberg, Andrew C. Hsieh
Abstract
Fetal growth restriction, or low birthweight is a strong determinant for eventual obesity and Type 2 diabetes. Clinical studies suggest placental mechanistic target of rapamycin (mTOR) signaling regulate fetal birthweight and the metabolic health trajectory of the offspring. In the current study, we used genetic model with loss of placental mTOR function (mTORKOPlacenta) to test the direct role of mTOR signaling on birthweight and the metabolic health in the adult offspring. mTORKOPlacenta animals displayed reduced placental area and total weight, as well as fetal bodyweight at embryonic day (e) 17.5. Birthweight and serum insulin levels were reduced; however, β-cell mass was normal in mTORKOPlacenta newborns. Adult mTORKOPlacenta offspring, under a metabolic high-fat challenge, displayed exacerbated obesity and metabolic dysfunction compared to littermate controls. Subsequently, we tested whether enhancing placental mTOR complex 1 (mTORC1) signaling, via genetic ablation of TSC2, in utero would improve glucose homeostasis in the offspring. Indeed, increased placental mTORC1 conferred protection from a diet-induced obesity in the offspring. In conclusion, placental mTORC1 serves as a mechanistic link between placental function and programming of obesity and insulin resistance in the adult offspring.
Authors
Brian Akhaphong, Daniel C. Baumann, Megan Beetch, Amber D. Lockridge, Seokwon Jo, Alicia Wong, Tate Zemanovic, Ramkumar Mohan, Danica L. Fondevilla, Michelle Sia, Maria R.B. Pineda-Cortel, Emilyn U. Alejandro
Abstract
The presence of an immunosuppressive tumor microenvironment is a major obstacle in the success of cancer immunotherapies. Because extracellular matrix components can shape the microenvironment, we investigated the role of matrix metalloproteinase 2 (MMP2) in melanoma tumorigenesis. Significantly, we found that MMP2 signals pro-inflammatory pathways on antigen presenting cells which requires both toll-like receptor (TLR) 2 and TLR4. B16 melanoma cells that express MMP2 at baseline have slower kinetics in Tlr2-/-Tlr4-/- mice, implicating MMP2 in promoting tumor growth. Indeed, Mmp2 overexpression in B16 cells potentiated rapid tumor growth which was accompanied by reduced intra-tumoral cytolytic cells and increased M2 macrophages. In contrast, knockdown of Mmp2 slowed tumor growth, and enhanced T cell proliferation and NK cell recruitment. Finally we found that these effects of MMP2 are mediated through dysfunctional dendritic cell (DC) - T cell cross-talk as they are lost in Batf3-/- and Rag2-/- mice, respectively. These findings provide insights into the detrimental role of endogenous alarmins like MMP2 in modulating immune responses in the tumor microenvironment.
Authors
Luciana R. Muniz-Bongers, Christopher B. McClain, Mansi Saxena, Gerold Bongers, Miriam Merad, Nina Bhardwaj
Abstract
ECSIT is a protein with roles in early development, activation of the transcription factor NFB and production of mitochondrial reactive oxygen species (mROS) that facilitates clearance of intracellular bacteria like Salmonella. ECSIT is also an important assembly factor for mitochondrial complex I. Unlike the murine form of Ecsit (mEcsit), we demonstrate here that human ECSIT (hECSIT) to be highly labile. In order to explore if the instability of hECSIT affects functions previously ascribed to its murine counterpart, we created a novel transgenic mouse in which the murine Ecsit gene is replaced by the human ECSIT gene. The humanised mouse has low levels of hECSIT protein in keeping with its intrinsic instability. Whereas low level expression of hECSIT was capable of fully compensating for mEcsit in its roles in early development and activation of the NFB pathway, macrophages from humanised mice showed impaired clearance of Salmonella that was associated with reduced production of mROS. Notably, severe cardiac hypertrophy manifested in ageing humanised mice leading to premature death. The cellular and molecular basis to this phenotype is delineated by showing that low levels of human ECSIT protein leads to marked reduction in assembly and activity of mitochondrial complex I with impaired oxidative phosphorylation and reduced production of ATP. Cardiac tissue from humanised hECSIT mice also shows reduced mitochondrial fusion and more fission but impaired clearance of fragmented mitochondria. A cardiomyocyte-intrinsic role for Ecsit in mitochondrial function and cardioprotection is also demonstrated. We also show that cardiac fibrosis and damage in humans correlates with low expression of human ECSIT. In summary, our findings identify a new role for ECSIT in cardioprotection whilst also generating a valuable new experimental model to study mitochondrial dysfunction and cardiac pathophysiology.
Authors
Linan Xu, Fiachra Humphries, Nezira Delagic, Bingwei Wang, Ashling Holland, Kevin S. Edgar, Jose R. Hombrebueno, Donna Beer Stolz, Ewa Oleszycka, Aoife M. Rodgers, Nadezhda Glezeva, Kenneth McDonald, Chris J. Watson, Mark T. Ledwidge, Rebecca J. Ingram, David J. Grieve, Paul N. Moynagh
Abstract
Recent advances in high-throughput T cell receptor (TCR) sequencing have allowed for new insights into the human TCR repertoire. However, methods for capturing antigen-specific repertoires remain an area of development. Here, we describe a potentially novel approach that utilizes both a biological and statistical enrichment to define putatively antigen-specific complementarity-determining region 3 (CDR3) repertoires in unselected individuals. The biological enrichment entails fluorescence-activated cell sorting of in vitro antigen-activated memory CD4+ T cells, followed by TCRβ sequencing. The resulting TCRβ sequences are then filtered by selecting those that are statistically enriched when compared to their frequency in the autologous resting T cell compartment. Applying this method to define putatively peanut protein-specific repertoires in 27 peanut-allergic individuals resulted in a library of 7345 unique CDR3β amino acid sequences that had similar characteristics to other validated antigen-specific repertoires in terms of homology and diversity. In-depth analysis of these CDR3βs revealed 36 public sequences that demonstrated high levels of convergent recombination. In a network analysis, the public CDR3βs unveiled themselves as core sequences with more edges than their private counterparts. This method has the potential to be applied to a wide range of T cell-mediated disorders, and to yield new biomarkers and biological insights.
Authors
Neal P. Smith, Bert Ruiter, Yamini V. Virkud, Ang A. Tu, Brinda Monian, James J. Moon, J. Christopher Love, Wayne G. Shreffler
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