Abstract
In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture–conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/μg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements.
Authors
Sara Elgamal, Emanuele Cocucci, Ellen J. Sass, Xiaokui M. Mo, Angela R. Blissett, Edward P. Calomeni, Kerry A. Rogers, Jennifer A. Woyach, Seema A. Bhat, Natarajan Muthusamy, Amy J. Johnson, Karilyn T. Larkin, John C. Byrd
Abstract
Energy balance is controlled by interconnected brain regions in the hypothalamus, brain stem, cortex and limbic system. Gene expression signatures of these regions can help elucidate the pathophysiology underlying obesity. RNA sequencing was conducted on P56 C57BL/6NTac male mice and E14.5 C57BL/6NTac embryos punch-biopsies in 16 obesity-relevant brain regions. The expression of 190 known obesity-associated genes (monogenic, rare and low-frequency coding variants, genome-wide association studies (GWAS), syndromic) were analyzed in each anatomical region. Genes associated with these genetic categories of obesity had localized expression patterns across brain regions. Known monogenic obesity causal genes were highly enriched in the arcuate nucleus of the hypothalamus and developing hypothalamus. The obesity-associated genes clustered into distinct ‘modules’ of similar expression profile and these are distinct from expression ‘modules’ formed by similar analysis with genes known to be associated with other disease phenotypes (type 1 and type 2 diabetes, autism, breast cancer) in the same energy balance-relevant brain regions.
Authors
Maria Caterina De Rosa, Hannah J. Glover, George Stratigopoulos, Charles A. LeDuc, Qi Su, Yufeng Shen, Mark W. Sleeman, Wendy K. Chung, Rudolph L. Leibel, Judith Y. Altarejos, Claudia A. Doege
Abstract
The syndrome of spontaneous preterm birth (sPTB) presents a challenge to mechanistic understanding, effective risk stratification, and management. Individual associations between sPTB, ethnicity, vaginal microbiota, metabolome and innate immune response are known, but not fully understood and knowledge has yet to impact clinical practice. Here we use multi-data type integration and composite statistical models to gain insight into sPTB risk by exploring the cervicovaginal environment of an ethnically heterogenous pregnant population (n=346 women; n=60 sPTB <37 weeks’ gestation, including n=27 sPTB <34 weeks). Analysis of cervicovaginal samples (10-15+6 weeks) identified novel interactions between risk of sPTB and microbiota, metabolite, and maternal host defense molecules. Statistical modelling identified a composite of metabolites (leucine, tyrosine, aspartate, lactate, betaine, acetate and Ca2+) associated with risk of sPTB <37 weeks (Area Under the Curve - AUC 0.752). A combination of glucose, aspartate, Ca2+ and Lactobacillus crispatus and L. acidophilus relative abundance, identified risk of early sPTB <34 weeks, (AUC 0.758); improved by ethnicity stratification (AUC 0.835). Increased relative abundance of L. acidophilus appeared protective against sPTB <34 weeks. By using cervicovaginal fluid samples, we demonstrate the potential of multi-datatype integration for developing composite models towards understanding the contribution of the vaginal environment to risk of sPTB.
Authors
Flavia Flaviani, Natasha L. Hezelgrave, Tokuwa Kanno, Erica M. Prosdocimi, Evonne Chin-Smith, Alexandra E. Ridout, Djuna K. von Maydell, Vikash Mistry, William G. Wade, Andrew H. Shennan, Konstantina Dimitrakopoulou, Paul T. Seed, Andrew James Mason, Rachel M. Tribe
Abstract
Neutrophils are produced in the bone marrow (BM) in a process called granulopoiesis, in which progenitor cells sequentially develop into mature neutrophils. During the developmental process, which is finely regulated by distinct transcription factors, neutrophils acquire the ability to exit the BM, properly distribute throughout the body, and migrate to infection sites. Previous studies have demonstrated that CD40 ligand (CD40L) influences hematopoiesis and granulopoiesis. Here, we investigate the effect of CD40L on neutrophil development and trafficking by performing functional and transcriptome analyses. We found that CD40L signaling plays an essential role in the early stages of neutrophil generation and development in the BM. Moreover, CD40L modulates transcriptional signatures, indicating that this molecule enables neutrophils to traffic throughout the body and to migrate in response to inflammatory signals. Thus, our study provides new insights into the complex relationships between CD40L signaling and granulopoiesis and suggests a novel and non-redundant role of CD40L signaling in neutrophil development and function.
Authors
Tábata T. França, Ashraf Al-Sbiei, Ghada Bashir, Yassir A. Mohamed, Ranieri C. Salgado, Lucila A. Barreiros, Sarah M. da Silva Napoleão, Cristina W. Weber, Janáira F.S. Ferreira, Carolina S. Aranda, Carolina Prando, Mayra B. de Barros Dorna, Igor Jurisica, Maria J. Fernandez-Cabezudo, Hans D. Ochs, Antonio Condino-Neto, Basel K. Al-Ramadi, Otavio Cabral-Marques
Abstract
Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) infects epithelial cells of the human gastrointestinal (GI) tract and causes related symptoms. HIV infection impairs gut homeostasis and is associated with an increased risk of COVID-19 fatality. To investigate the potential link between these observations, we analysed single cell transcriptional profiles and SARS-CoV-2 entry receptor expression across lymphoid and mucosal human tissue from chronically HIV infected individuals and uninfected controls. Absorptive gut enterocytes displayed the highest co-expression of SARS-CoV-2 receptors ACE2, TMPRSS2 and TMPRSS4, of which ACE2 expression was associated with canonical interferon response and antiviral genes. Chronic treated HIV infection was associated with a clear antiviral response in gut enterocytes and, unexpectedly, with a significant reduction of ACE2 and TMPRSS2 target cells. Gut tissue from SARS-CoV-2 infected individuals, however, showed abundant SARS-CoV-2 nucleocapsid protein in both the large and small intestine, including an HIV co-infected individual. Thus, upregulation of antiviral response genes and downregulation of ACE2 and TMPRSS2 in the GI tract of HIV infected individuals, does not prevent SARS-CoV-2 infection in this compartment. The impact of these HIV-associated intestinal mucosal changes on SARS-CoV-2 infection dynamics, disease severity and vaccine responses remains unclear and require further investigation.
Authors
Rabiah Fardoos, Osaretin E. Asowata, Nicholas Herbert, Sarah K. Nyquist, Yenzekile Zungu, Alveera Singh, Abigail Ngoepe, Ian M. Mbano, Ntombifuthi Mthabela, Dirhona Ramjit, Farina Karim, Warren Kuhn, Fusi G. Madela, Vukani T. Manzini, Frank Anderson, Bonnie Berger, Tune H. Pers, Alex K. Shalek, Alasdair Leslie, Henrik Kløverpris
Abstract
The SARS-CoV-2 Receptor Binding Domain (RBD) is both the principal target of neutralizing antibodies, and one of the most rapidly evolving domains, which can result in the emergence of immune escape mutations limiting the effectiveness of vaccines and antibody therapeutics. To facilitate surveillance, we developed a rapid, high-throughput, multiplex assay able to assess the inhibitory response of antibodies to 24 RBD natural variants simultaneously. We demonstrate how this assay can be implemented as a rapid surrogate assay for functional cell-based serological methods to measure the SARS-CoV-2 neutralising capacity of antibodies at the ACE2-RBD interface. We describe the enhanced affinity of RBD variants N439K, S477N, Q493L, S494P and N501Y to the ACE2 receptor, and demonstrate the ability of this assay to bridge a major gap for SARS-CoV-2 research; informing selection of complementary monoclonal antibody candidates and the rapid identification of immune escape to emerging RBD variants following vaccination or natural infection.
Authors
Ester Lopez, Ebene R. Haycroft, Amy Adair, Francesca L. Mordant, Matthew T. O'Neill, Phillip Pymm, Samuel J. Redmond, Wen Shi Lee, Nicholas A. Gherardin, Adam K. Wheatley, Jennifer A. Juno, Kevin John Selva, Samantha K. Davis, Samantha L. Grimley, Leigh Harty, Damian F.J. Purcell, Kanta Subbarao, Dale I. Godfrey, Stephen J. Kent, Wai-Hong Tham, Amy W. Chung
Abstract
Diagnosis of organ transplant rejection relies upon biopsy approaches to confirm alloreactive T cell infiltration in the graft. Immune molecular monitoring is under investigation to screen for rejection, though these techniques have suffered from low specificity and lack of spatial information. ImmunoPET utilizing antibodies conjugated to radioisotopes has the potential to improve early and accurate detection of graft rejection. ImmunoPET is capable of noninvasively visualizing the dynamic distribution of cells expressing specific immune markers in the entire body over time. In this work, we identify and characterize OX40 as a surrogate biomarker for alloreactive T cells in organ transplant rejection and monitor its expression by utilizing immunoPET. In a dual murine heart transplant model that has both syngeneic and allogeneic hearts engrafted in bilateral ear pinna on the recipients, OX40 immunoPET clearly depicted alloreactive T cells in the allograft and draining lymph node that were not observed in their respective isograft counterparts. OX40 immunoPET signals also reflected the subject’s immunosuppression level with tacrolimus in this study. OX40 immunoPET is a promising approach that may bridge molecular monitoring and morphological assessment for improved transplant rejection diagnosis.
Authors
Toshihito Hirai, Aaron T. Mayer, Tomomi W. Nobashi, Po-Yu Lin, Zunyu Xiao, Tomokatsu Udagawa, Kinya Seo, Federico Simonetta, Jeanette Baker, Alan G. Cheng, Robert S. Negrin, Sanjiv S. Gambhir
Abstract
The liver is the major source of glucose production during fasting under normal physiological conditions. However, the kidney may also contribute to maintaining glucose homeostasis in certain circumstances. To test the ability of the kidney to compensate for impaired hepatic glucose production in vivo, we developed a stable isotope approach to simultaneously quantify gluconeogenic and oxidative metabolic fluxes in the liver and kidney. Hepatic gluconeogenesis from phosphoenolpyruvate was disrupted via liver-specific knockout of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; KO). 2H/13C isotopes were infused in fasted KO and WT littermate mice, and fluxes were estimated from isotopic measurements of tissue and plasma metabolites using a multicompartment metabolic model. Hepatic gluconeogenesis and glucose production were reduced in KO mice, yet whole-body glucose production and arterial glucose were unaffected. Glucose homeostasis was maintained by a compensatory rise in renal glucose production and gluconeogenesis. Renal oxidative metabolic fluxes of KO mice increased to sustain the energetic and metabolic demands of elevated gluconeogenesis. These results show the reciprocity of the liver and kidney in maintaining glucose homeostasis by coordinated regulation of gluconeogenic flux through PEPCK-C. Combining stable isotopes with mathematical modeling provides a versatile platform to assess multitissue metabolism in various genetic, pathophysiological, physiological, and pharmacological settings.
Authors
Mohsin Rahim, Clinton M. Hasenour, Tomasz K. Bednarski, Curtis C. Hughey, David H. Wasserman, Jamey D. Young
Abstract
Neurodegeneration mediates neurological disability in inflammatory demyelinating diseases of the CNS. The role of innate immune cells in mediating this damage has remained controversial with evidence for destructive and protective effects. This has complicated efforts to develop treatment. The time sequence and dynamic evolution of the opposing functions are especially unclear. Given limits of in vivo monitoring in human diseases such as multiple sclerosis (MS), animal models are warranted to investigate the association and timing of innate immune activation with neurodegeneration. Using noninvasive in vivo retinal imaging of experimental autoimmune encephalitis (EAE) in CX3CR1GFP/+–knock-in mice followed by transcriptional profiling, we are able to show 2 distinct waves separated by a marked reduction in the number of innate immune cells and change in cell morphology. The first wave is characterized by an inflammatory phagocytic phenotype preceding the onset of EAE, whereas the second wave is characterized by a regulatory, antiinflammatory phenotype during the chronic stage. Additionally, the magnitude of the first wave is associated with neuronal loss. Two transcripts identified — growth arrest–specific protein 6 (GAS6) and suppressor of cytokine signaling 3 (SOCS3) — might be promising targets for enhancing protective effects of microglia in the chronic phase after initial injury.
Authors
Andrés Cruz-Herranz, Frederike C. Oertel, Kicheol Kim, Ester Cantó, Garrett Timmons, Jung H. Sin, Michael Devereux, Nicholas Baker, Brady Michel, Ryan D. Schubert, Lakshmisahithi Rani, Christian Cordano, Sergio E. Baranzini, Ari J. Green
Abstract
Existing patient-derived-xenograft (PDX) mouse models of solid tumors lack a fully tumor-donor matched, syngeneic, and functional immune system. We developed such a model by engrafting lymphopenic recipient mice with a fresh, undisrupted piece of solid tumor, whereby tumor-infiltrating lymphocytes (TILs) persisted in the recipient mice for several weeks. Successful tumor engraftment was achieved in eighty-three to eighty-nine percent of tumor-infiltrating-lymphocytes-PDX (TIL-PDX) mice, and these were seen to harbor exhausted immuno-effector as well as functional immuno-regulatory cells persisting for at least six months post-engraftment. Combined treatment with interleukin-15 (IL-15) stimulation and immune checkpoint inhibition (ICI) resulted in complete or partial tumor response in this model. Further, depletion of Cytotoxic T-lymphocytes (CTLs) and/or Natural Killer (NK) cells before combined immunotherapy revealed that both cell types were required for maximal tumor regression. Our novel TIL-PDX model provides a valuable resource for powerful mechanistic and therapeutic studies in solid tumors.
Authors
Duy T. Le, Tridu R. Huynh, Bryan M. Burt, George Van Buren, Shawn A. Abeynaike, Cristina Zalfa, Rana Nikzad, Farrah Kheradmand, John J. Tyner, Silke Paust
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