Analysis of bronchoalveolar lavage transcriptome profiles of asthmatic horses by single-cell mRNA sequencing

Severe equine asthma (SEA) is a common respiratory condition of horses, whose underlying immune mechanisms remain to be elucidated. In this thesis project, we took advantage of the recently developed single-cell mRNA (scRNA-seq) technology to investigate the immunological landscape of equine bronchoalveolar lavage fluid (BALF) cells in both health and disease. Initially, we conducted a pilot experiment involving three horses to demonstrate the feasibility of scRNA-seq on cryopreserved equine BALF samples. Although the experiment was successful, the proportion of reads aligning to the annotated equine reference transcriptome was suboptimal. To address this, we generated a custom equine BALF transcriptome using long-read sequencing, aiming to improve the quality of 3'-UTR annotation and document BALF-specific isoforms. While we identified several novel isoforms, the read mapping percentage did not improve when aligning our scRNA-seq transcripts to the custom transcriptome. By extending the 3'-UTRs of the existing reference annotation, we achieved a satisfactory read mapping percentage, enabling subsequent qualitative downstream analysis. Our scRNA-seq dataset encompassed six major cell populations: monocytes-macrophages, neutrophils, T cells, B cells and dendritic cells. Within the monocyte-macrophage and T cell groups, we identified previously uncharacterized cell subtypes. Encouraged by these findings, we applied our optimized experimental protocol and analysis pipeline to study SEA. ScRNA-seq analysis of cryopreserved BALF cells from 6 asthmatic and 5 healthy controls revealed the same major cell populations as observed in the pilot study. In addition to T cells and monocytes-macrophages, we characterized several cell subtypes within the B cell, dendritic cell and neutrophil populations. Differential gene expression analysis revealed a strong T helper (Th)17 signature in SEA, primarily driven by monocytes-macrophages and T cells. Notably, BALF from SEA horses was enriched in B cells, with a lower proportion of activated plasma cells. Neutrophils in the SEA group displayed increased migratory capacity and a heightened propensity to form neutrophil extracellular traps (NETs). An intriguing finding in both scRNA-seq experiments was the detection of a dual monocyte-lymphocyte population, potentially representing genuine cellular complexes engaged in an immunological synapse. In summary, this thesis project represents pioneering work employing scRNA-seq in the field of equine pulmonology. Our findings support a predominant Th17 immune pathway in SEA, necessitating further investigation to improve diagnostic tools and therapeutic management of severely asthmatic horses

Long-Read Transcriptome of Equine Bronchoalveolar Cells

We used Pacific Biosciences long-read isoform sequencing to generate full-length transcript sequences in equine bronchoalveolar lavage fluid (BALF) cells. Our dataset consisted of 313,563 HiFi reads comprising 805 Mb of polished sequence information. The resulting equine BALF transcriptome consisted of 14,234 full-length transcript isoforms originating from 7017 unique genes. These genes consisted of 6880 previously annotated genes and 137 novel genes. We identified 3428 novel transcripts in addition to 10,806 previously known transcripts. These included transcripts absent from existing genome annotations, transcripts mapping to putative novel (unannotated) genes and fusion transcripts incorporating exons from multiple genes. We provide transcript-level data for equine BALF cells as a resource to the scientific community

Single-cell gene expression analysis of cryopreserved equine bronchoalveolar cells

The transcriptomic profile of a cell population can now be studied at the cellular level using single-cell mRNA sequencing (scRNA-seq). This novel technique provides the unprecedented opportunity to explore the cellular composition of the bronchoalveolar lavage fluid (BALF) of the horse, a species for which cell type markers are poorly described. Here, scRNA-seq technology was applied to cryopreserved equine BALF cells. Analysis of 4,631 cells isolated from three asthmatic horses in remission identified 16 cell clusters belonging to six major cell types: monocytes/macrophages, T cells, B/plasma cells, dendritic cells, neutrophils and mast cells. Higher resolution analysis of the constituents of the major immune cell populations allowed deep annotation of monocytes/macrophages, T cells and B/plasma cells. A significantly higher lymphocyte/macrophage ratio was detected with scRNA-seq compared to conventional cytological differential cell count. For the first time in horses, we detected a transcriptomic signature consistent with monocyte-lymphocyte complexes. Our findings indicate that scRNA-seq technology is applicable to cryopreserved equine BALF cells, allowing the identification of its major (cytologically differentiated) populations as well as previously unexplored T cell and macrophage subpopulations. Single-cell gene expression analysis has the potential to facilitate understanding of the immunological mechanisms at play in respiratory disorders of the horse, such as equine asthma

Severe platelet dysfunction in NHL patients receiving ibrutinib is absent in patients receiving acalabrutinib

The Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib induces platelet dysfunction and causes increased risk of bleeding. Off-target inhibition of Tec is believed to contribute to platelet dysfunction and other side-effects of ibrutinib. The second generation Btk inhibitor acalabrutinib was developed with improved specificity for Btk over Tec. We investigated platelet function in patients with Non-Hodgkin Lymphoma (NHL) receiving ibrutinib or acalabrutinib by aggregometry and by measuring thrombus formation on collagen under arterial shear. Both patient groups had similarly dysfunctional aggregation responses to collagen and collagen-related peptide (CRP-XL) and comparison with mechanistic experiments in which platelets from healthy donors were treated with the Btk inhibitors suggested that both drugs inhibit platelet Btk and Tec at physiological concentrations. Only ibrutinib caused dysfunctional thrombus formation, while size and morphology of thrombi following acalabrutinib treatment were of normal size and morphology. We found that ibrutinib but not acalabrutinib inhibited SFKs and that SFKs have a critical role in platelet adhesion to collagen that is likely to underpin unstable thrombus formation observed in ibrutinib patients. We found that platelet function was enhanced by increasing levels of vWF and FVIII ex vivo by addition of intermediate purity FVIII (haemate P) to blood from patients, resulting in consistently larger thrombi. We conclude that acalabrutinib avoids major platelet dysfunction associated with ibrutinib therapy, and platelet function may be enhanced in patients with B-cell NHL by increasing plasma vWF and FVIII

Delivery of costimulatory blockade to lymph nodes promotes transplant acceptance in mice

The lymph node (LN) is the primary site of alloimmunity activation and regulation during transplantation. Here, we investigated how fibroblastic reticular cells (FRCs) facilitate the tolerance induced by anti-CD40L in a murine model of heart transplantation. We found that both the absence of LNs and FRC depletion abrogated the effect of anti-CD40L in prolonging murine heart allograft survival. Depletion of FRCs impaired homing of T cells across the high endothelial venules (HEVs) and promoted formation of alloreactive T cells in the LNs in heart-transplanted mice treated with anti-CD40L. Single-cell RNA sequencing of the LNs showed that anti-CD40L promotes a Madcam1+ FRC subset. FRCs also promoted the formation of regulatory T cells (Tregs) in vitro. Nanoparticles (NPs) containing anti-CD40L were selectively delivered to the LNs by coating them with MECA-79, which binds to peripheral node addressin (PNAd) glycoproteins expressed exclusively by HEVs. Treatment with these MECA-79-anti-CD40L-NPs markedly delayed the onset of heart allograft rejection and increased the presence of Tregs. Finally, combined MECA-79-anti-CD40L-NPs and rapamycin treatment resulted in markedly longer allograft survival than soluble anti-CD40L and rapamycin. These data demonstrate that FRCs are critical to facilitating costimulatory blockade. LN-targeted nanodelivery of anti-CD40L could effectively promote heart allograft acceptance

Cucurbitacins elicit anti-platelet activity via perturbation of the actin cytoskeleton and integrin function

Cucurbitacins are dietary compounds that have been shown to elicit a range of anti-tumour, anti-inflammatory and anti-atherosclerotic activities. Originally identified as STAT inhibitors, a variety of mechanisms of action have since been described, including dysregulation of the actin cytoskeleton and disruption of integrin function. Integrin outside-in signalling and cytoskeletal rearrangements are critical for the propagation of stable thrombus formation and clot retraction following platelet adhesion at the site of vessel damage. The effects of cucurbitacins on platelet function and thrombus formation are unknown. We report for the first time anti-platelet and anti-thrombotic effects of cucurbitacins B, E and I in human platelets. Treatment of platelets with cucurbitacins resulted in attenuation of platelet aggregation, secretion and fibrinogen binding following stimulation by ADP, TRAP6, collagen and CRP-XL. Cucurbitacins were also found to potently inhibit other integrin- and cytoskeleton-mediated events, including adhesion, spreading and clot retraction. Further investigation of cytoskeletal dynamics found treatment with cucurbitacins altered cofilin phosphorylation, enhanced activation, and increased F actin polymerisation and microtubule assembly. Disruption to cytoskeletal dynamics has been previously shown to impair integrin activation, platelet spreading and clot retraction. Anti-platelet properties of cucurbitacins were found to extend to a disruption of stable thrombus formation, with an increase in thrombi instability and de-aggregation under flow. Our research identifies novel, anti-platelet and anti-thrombotic actions of cucurbitacins that appear to be linked to dysregulation of cytoskeletal dynamics and integrin function

Bringing People Back into Public Health Data: Community Feedback on a Set of Visualization Tools - Summary Report

This course-based study is a product of the University of Denver’s Spring 2022 The Social Determination of Health (ANTH 2424) class. The study aimed to understand how well a set of public health visualization tools tells the data stories about people in Colorado, and about important public health problems. For this, a team of almost sixty undergraduate students taking the class, coordinated by three graduate teaching assistants, and directed by the course instructor interviewed a total of fifty-six people from Colorado, qualitatively analyzed those interviews, and wrote reports that draw conclusions and recommendations

Hepatic Stem-like Phenotype and Interplay of Wnt/β-Catenin and Myc Signaling in Aggressive Childhood Liver Cancer

SummaryHepatoblastoma, the most common pediatric liver cancer, is tightly linked to excessive Wnt/β-catenin signaling. Here, we used microarray analysis to identify two tumor subclasses resembling distinct phases of liver development and a discriminating 16-gene signature. β-catenin activated different transcriptional programs in the two tumor types, with distinctive expression of hepatic stem/progenitor markers in immature tumors. This highly proliferating subclass was typified by gains of chromosomes 8q and 2p and upregulated Myc signaling. Myc-induced hepatoblastoma-like tumors in mice strikingly resembled the human immature subtype, and Myc downregulation in hepatoblastoma cells impaired tumorigenesis in vivo. Remarkably, the 16-gene signature discriminated invasive and metastatic hepatoblastomas and predicted prognosis with high accuracy