Abstract
The anatomical routes for the clearance of cerebrospinal fluid (CSF) remain incompletely understood. However, recent evidence has given strong support for routes leading to lymphatic vessels. A current debate centers upon the routes through which CSF can access lymphatics, with evidence emerging for either direct routes to meningeal lymphatics or along cranial nerves to reach lymphatics outside the skull. Here, a method was established to infuse contrast agent into the ventricles using indwelling cannulae during imaging of mice at 2 and 12 months of age by magnetic resonance imaging. As expected, a significant decline in overall CSF turnover was found with aging. Quantifications demonstrated that the bulk of the contrast agent flowed from the ventricles to the subarachnoid space in the basal cisterns. Comparatively little contrast agent signal was found at the dorsal aspect of the skull. The imaging dynamics from the two cohorts revealed that the contrast agent cleared from the cranium through the cribriform plate to the nasopharyngeal lymphatics. On decalcified sections, we confirmed that fluorescentlylabeled ovalbumin drains through the cribriform plate and can be found within lymphatics surrounding the nasopharynx. In conclusion, routes leading to nasopharyngeal lymphatics appear to be a major efflux pathway for cranial CSF.
Authors
Yann Decker, Jonas Krämer, Li Xin, Andreas Müller, Anja Scheller, Klaus Fassbender, Steven T. Proulx
Abstract
Identification and analysis of fungal communities commonly rely on internal transcribed spacer (ITS)-based amplicon sequencing. There is no gold standard to infer and classify fungal constituents since methodologies have been adapted from analyses of bacterial communities. To achieve high resolution inference of fungal constituents, we customized a DADA2-based pipeline using a mix of eleven medically relevant fungi. While DADA2 allowed the discrimination of ITS1 sequences differing by single nucleotides, quality filtering, sequencing bias, and database selection were identified as key variables determining the accuracy of sample inference. Due to species-specific differences in sequencing quality, default filtering settings removed most reads that originated from Aspergillus species, Saccharomyces cerevisiae, and Candida glabrata. By fine-tuning the quality filtering process, we achieved an improved representation of the fungal communities. By adapting a wobble nucleotide in the ITS1 forward primer region, we further increased the yield of S. saccharomyces and C. glabrata sequences. Finally, we showed that a BLAST-based algorithm based on the UNITE+INSD or the NCBI NT database achieved a higher reliability in species-level taxonomic annotation than the naïve Bayesian classifier implemented in DADA2. These steps optimized a robust fungal ITS1 sequencing pipeline that, in most instances, enabled species level-assignment of community members.
Authors
Thierry Rolling, Bing Zhai, John Frame, Tobias M. Hohl, Ying Taur
Abstract
Human islet antigen reactive CD4+ memory T cells (IAR T cells) play a key role in the pathogenesis of autoimmune type 1 diabetes (T1D). Using single-cell RNA sequencing (scRNA-Seq) to identify T cell receptors (TCRs) in IAR T cells, we have identified a class of TCRs that share TCRα chains between individuals (“public” chains). We isolated IAR T cells from blood of healthy, new-onset T1D and established T1D donors using multiplexed CD154 enrichment and identified paired TCRαβ sequences from 2767 individual cells. More than a quarter of cells shared TCR junctions between 2 or more cells (“expanded”), and 29/47 (~62%) of expanded TCRs tested showed specificity for islet antigen epitopes. Public TCRs sharing TCRα junctions were most prominent in new-onset T1D. Public TCR sequences were more germline like than expanded unique, or “private,” TCRs, and had shorter junction sequences, suggestive of fewer random nucleotide insertions. Public TCRα junctions were often paired with mismatched TCRβ junctions in TCRs; remarkably, a subset of these TCRs exhibited cross-reactivity toward distinct islet antigen peptides. Our findings demonstrate a prevalent population of IAR T cells with diverse specificities determined by TCRs with restricted TCRα junctions and germline-constrained antigen recognition properties. Since these “innate-like” TCRs differ from previously described immunodominant TCRβ chains in autoimmunity, they have implications for fundamental studies of disease mechanisms. Self-reactive restricted TCRα chains and their associated epitopes should be considered in fundamental and translational investigations of TCRs in T1D.
Authors
Peter S. Linsley, Fariba Barahmand-pour-Whitman, Elisa Balmas, Hannah A. DeBerg, Kaitlin J. Flynn, Alex K. Hu, Mario G. Rosasco, Janice Chen, Colin O’Rourke, Elisavet Serti, Vivian H. Gersuk, Keshav Motwani, Howard R. Seay, Todd M. Brusko, William W. Kwok, Cate Speake, Carla J. Greenbaum, Gerald T. Nepom, Karen Cerosaletti
Growth factors with valproic acid restore injury-impaired hearing by promoting neuronal regeneration
Abstract
Spiral ganglion neurons (SGNs) are primary auditory neurons in the spiral ganglion that transmit sound information from the inner ear to the brain and play an important role in hearing. Impairment of SGNs causes sensorineural hearing loss (SNHL), and it has been thought until now that SGNs cannot be regenerated once lost. Furthermore, no fundamental therapeutic strategy for SNHL has been established other than inserting devices such as hearing aids and cochlear implants. Here we show that the mouse spiral ganglion contains cells that are able to proliferate and indeed differentiate into neurons in response to injury. We suggest that SRY-box transcription factor 2/SRY-box transcription factor 10–double-positive (Sox2/Sox10–double-positive) Schwann cells sequentially started to proliferate, lost Sox10 expression, and became neurons, although the number of new neurons generated spontaneously was very small. To increase the abundance of new neurons, we treated mice with 2 growth factors in combination with valproic acid, which is known to promote neuronal differentiation and survival. This treatment resulted in a dramatic increase in the number of SGNs, accompanied by a partial recovery of the hearing loss induced by injury. Taken together, our findings offer a step toward developing strategies for treatment of SNHL.
Authors
Takahiro Wakizono, Hideyuki Nakashima, Tetsuro Yasui, Teppei Noda, Kei Aoyagi, Kanako Okada, Yasuhiro Yamada, Takashi Nakagawa, Kinichi Nakashima
Abstract
BACKGROUND. Although aberrant glycosylation is recognized as a hallmark of cancer, glycosylation in clinical breast cancer (BC) metastasis has not yet been studied. While preclinical studies show that the glycocalyx coating of cancer cells is involved in adhesion, migration, and metastasis, glycosylation changes from primary tumor (PT) to various metastatic sites remain unknown in patients. METHODS. We investigated N-glycosylation profiles in 17 metastatic BC patients from our rapid autopsy program. Primary breast tumor, lymph node metastases, multiple systemic metastases, and various normal tissue cores from each patient were arranged on unique single-patient tissue microarrays (TMAs). We performed mass spectrometry imaging (MSI) combined with extensive pathology annotation of these TMAs, which enabled spatially differentiated cell-based analysis of N-glycosylation patterns in metastatic BC. RESULTS. N-glycan abundance increased during metastatic progression independent of BC subtype and treatment regimen, with high-mannose glycans most frequently elevated in BC metastases, followed by fucosylated and complex glycans. Bone metastasis, however, displayed increased core-fucosylation and decreased high-mannose glycans. Consistently, N-glycosylated proteins and N-glycan biosynthesis genes were differentially expressed during metastatic BC progression, with reduced expression of EpCAM and mannose-trimming enzymes and elevated N-glycan branching and sialylation enzymes in BC metastases versus PT. CONCLUSION. We show for the first time in patients that N-glycosylation of breast cancer cells undergoing metastasis occurs in a metastatic site-specific manner, supporting the clinical importance of high-mannose, fucosylated, and complex N-glycans as future diagnostic markers and therapeutic targets in metastatic BC. FUNDING. United States National Institutes of Health grants NIH R01CA213428, R01CA213492, T32CA193145, Dutch Province Limburg “LINK”, European Union ERA-NET TRANSCAN2-643638.
Authors
Klára Ščupáková, Oluwatobi T. Adelaja, Benjamin Balluff, Vinay Ayyappan, Caitlin M. Tressler, Nicole M. Jenkinson, Britt S.R. Claes, Andrew P. Bowman, Ashley M. Cimino-Mathews, Marissa J. White, Pedram Argani, Ron M.A. Heeren, Kristine Glunde
Abstract
Controlled human malaria infection (CHMI) provides a highly informative means to investigate host-pathogen interactions and enable in vivo proof-of-concept efficacy testing of new drugs and vaccines. However, unlike Plasmodium falciparum, well-characterized P. vivax parasites that are safe and suitable for use in modern CHMI models are limited. Here, two healthy malaria-naïve UK adults with universal donor blood group were safely infected with a clone of P. vivax from Thailand by mosquito-bite CHMI. Parasitemia developed in both volunteers and, prior to treatment, each volunteer donated blood to produce a cryopreserved stabilate of infected red blood cells. Following stringent safety screening, the parasite stabilate from one of these donors (“PvW1”) was thawed and used to inoculate six healthy malaria-naïve UK adults by blood-stage CHMI, at three different dilutions. Parasitemia developed in all volunteers, who were then successfully drug treated. PvW1 parasite DNA was isolated and sequenced to produce a high quality genome assembly by using a hybrid assembly method. We analysed leading vaccine candidate antigens and multigene families, including the Vivax interspersed repeat (VIR) genes of which we identified 1145 in the PvW1 genome. Our genomic analysis will guide future assessment of candidate vaccines and drugs, as well as experimental medicine studies.
Authors
Angela M. Minassian, Yrene Themistocleous, Sarah E. Silk, Jordan R. Barrett, Alison Kemp, Doris Quinkert, Carolyn M. Nielsen, Nick J. Edwards, Thomas A. Rawlinson, Fernando Ramos Lopez, Wanlapa Roobsoong, Katherine J.D. Ellis, Jee-Sun Cho, Eerik Aunin, Thomas D. Otto, Adam J. Reid, Florian A. Bach, Geneviève M.C. Labbé, Ian D. Poulton, Arianna Marini, Marija Zaric, Margaux Mulatier, Raquel Lopez Ramon, Megan Baker, Celia H. Mitton, Jason C. Sousa, Nattawan Rachaphaew, Chalermpon Kumpitak, Nongnuj Maneechai, Chayanut Suansomjit, Tianrat Piteekan, Mimi M. Hou, Baktash Khozoee, Kirsty McHugh, David J. Roberts, Alison M. Lawrie, Andrew M. Blagborough, Fay L. Nugent, Iona J. Taylor, Kimberly J. Johnson, Philip J. Spence, Jetsumon Sattabongkot, Sumi Biswas, Julian C. Rayner, Simon J. Draper
Abstract
COVID-19, caused by SARS-CoV-2, has spread worldwide with a dire disaster situation. To urgently investigate the pathogenicity of COVID-19 and develop vaccines and therapeutics, animal models that are highly susceptible to SARS-CoV-2 infection are needed. In the present study, we established an animal model highly susceptible to SARS-CoV-2 via the intratracheal tract infection in CAG-promoter-driven human angiotensin-converting enzyme 2 transgenic (CAG-hACE2) mice. The CAG-hACE2 mice showed several severe symptoms of SARS-CoV-2 infection, with definitive weight loss and subsequent death. Acute lung injury with elevated cytokine and chemokine levels was observed at an early stage of infection in CAG-hACE2 mice infected with SARS-CoV-2. The analysis of the hACE2 gene in CAG-hACE2 mice revealed that more than 15 copies of hACE2 genes were tandemly integrated into the mouse genome, supporting the high susceptibility to SARS-CoV-2. In the developed model, immunization with viral antigen or injection of plasma from immunized mice prevented body weight loss and lethality due to infection with SARS-CoV-2. These results indicate that a highly susceptible model of SARS-CoV-2 infection in CAG-hACE2 mice via the intratracheal tract is suitable for evaluating vaccines and therapeutic medicines.
Authors
Masamitsu N. Asaka, Daichi Utsumi, Haruhiko Kamada, Satoshi Nagata, Yutaka Nakachi, Tomokazu Yamaguchi, Yoshihiro Kawaoka, Keiji Kuba, Yasuhiro Yasutomi
Abstract
Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and β cells and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled >40,000 cells from normal human islets by scRNA-seq and stratified α and β cells based on combinatorial TF expression. Subpopulations of islet cells co-expressing ARX/MAFB (α cells) and MAFA/MAFB (β cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-seq, MAFA/MAFB co-expressing β cells showed enhanced electrophysiological activity. Thus, these results indicate combinatorial TF expression in islet α and β cells predicts highly functional, mature subpopulations.
Authors
Shristi Shrestha, Diane C. Saunders, John T. Walker, Joan Camunas-Soler, Xiao-Qing Dai, Rachana Haliyur, Radhika Aramandla, Greg Poffenberger, Nripesh Prasad, Rita Bottino, Roland Stein, Jean-Philippe Cartailler, Stephen C.J. Parker, Patrick E. MacDonald, Shawn E. Levy, Alvin C. Powers, Marcela Brissova
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
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