Intestinal cell diversity and treatment responses in a parasitic nematode at single cell resolution.

BACKGROUND: Parasitic nematodes, significant pathogens for humans, animals, and plants, depend on diverse organ systems for intra-host survival. Understanding the cellular diversity and molecular variations underlying these functions holds promise for developing novel therapeutics, with specific emphasis on the neuromuscular system's functional diversity. The nematode intestine, crucial for anthelmintic therapies, exhibits diverse cellular phenotypes, and unraveling this diversity at the single-cell level is essential for advancing knowledge in anthelmintic research across various organ systems. RESULTS: Here, using novel single-cell transcriptomics datasets, we delineate cellular diversity within the intestine of adult female Ascaris suum, a parasitic nematode species that infects animals and people. Gene transcripts expressed in individual nuclei of untreated intestinal cells resolved three phenotypic clusters, while lower stringency resolved additional subclusters and more potential diversity. Clusters 1 and 3 phenotypes displayed variable congruence with scRNA phenotypes of C. elegans intestinal cells, whereas the A. suum cluster 2 phenotype was markedly unique. Distinct functional pathway enrichment characterized each A. suum intestinal cell cluster. Cluster 2 was distinctly enriched for Clade III-associated genes, suggesting it evolved within clade III nematodes. Clusters also demonstrated differential transcriptional responsiveness to nematode intestinal toxic treatments, with Cluster 2 displaying the least responses to short-term intra-pseudocoelomic nematode intestinal toxin treatments. CONCLUSIONS: This investigation presents advances in knowledge related to biological differences among major cell populations of adult A. suum intestinal cells. For the first time, diverse nematode intestinal cell populations were characterized, and associated biological markers of these cells were identified to support tracking of constituent cells under experimental conditions. These advances will promote better understanding of this and other parasitic nematodes of global importance, and will help to guide future anthelmintic treatments.

Identification of genes that promote PI3K pathway activation and prostate tumour formation.

We have performed a functional in vivo mutagenesis screen to identify genes that, when altered, cooperate with a heterozygous Pten mutation to promote prostate tumour formation. Two genes, Bzw2 and Eif5a2, which have been implicated in the process of protein translation, were selected for further validation. Using prostate organoid models, we show that either Bzw2 downregulation or EIF5A2 overexpression leads to increased organoid size and in vivo prostate growth. We show that both genes impact the PI3K pathway and drive a sustained increase in phospho-AKT expression, with PTEN protein levels reduced in both models. Mechanistic studies reveal that EIF5A2 is directly implicated in PTEN protein translation. Analysis of patient datasets identified EIF5A2 amplifications in many types of human cancer, including the prostate. Human prostate cancer samples in two independent cohorts showed a correlation between increased levels of EIF5A2 and upregulation of a PI3K pathway gene signature. Consistent with this, organoids with high levels of EIF5A2 were sensitive to AKT inhibitors. Our study identified novel genes that promote prostate cancer formation through upregulation of the PI3K pathway, predicting a strategy to treat patients with genetic aberrations in these genes particularly relevant for EIF5A2 amplified tumours.

Autophagy promotes efficient T cell responses to restrict high-dose Mycobacterium tuberculosis infection in mice.

Although autophagy sequesters Mycobacterium tuberculosis (Mtb) in in vitro cultured macrophages, loss of autophagy in macrophages in vivo does not result in susceptibility to a standard low-dose Mtb infection until late during infection, leaving open questions regarding the protective role of autophagy during Mtb infection. Here we report that loss of autophagy in lung macrophages and dendritic cells results in acute susceptibility of mice to high-dose Mtb infection, a model mimicking active tuberculosis. Rather than observing a role for autophagy in controlling Mtb replication in macrophages, we find that autophagy suppresses macrophage responses to Mtb that otherwise result in accumulation of myeloid-derived suppressor cells and subsequent defects in T cell responses. Our finding that the pathogen-plus-susceptibility gene interaction is dependent on dose has important implications both for understanding how Mtb infections in humans lead to a spectrum of outcomes and for the potential use of autophagy modulators in clinical medicine.

Thio-2 inhibits key signaling pathways required for the development and progression of castration resistant prostate cancer.

Therapies that abrogate persistent androgen receptor (AR) signaling in castration resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain (NTD) of the AR that drives transcriptional activity in CRPC remains a challenging therapeutic target. Herein we demonstrate that BAG-1 mRNA is highly expressed and associates with signaling pathways, including AR signaling, that are implicated in the development and progression of CRPC. In addition, interrogation of geometric and physiochemical properties of the BAG domain of BAG-1 isoforms identifies it to be a tractable but challenging drug target. Furthermore, through BAG-1 isoform mouse knockout studies we confirm that BAG-1 isoforms regulate hormone physiology and that therapies targeting the BAG domain will be associated with limited 'on-target' toxicity. Importantly, the postulated inhibitor of BAG-1 isoforms, Thio-2, suppressed AR signaling and other important pathways implicated in the development and progression of CRPC to reduce the growth of treatment resistant prostate cancer cell lines and patient derived models. However, the mechanism by which Thio-2 elicits the observed phenotype needs further elucidation since the genomic abrogation of BAG-1 isoforms was unable to recapitulate the Thio-2 mediated phenotype. Overall, these data support the interrogation of related compounds with improved drug-like properties as a novel therapeutic approach in CRPC, and further highlight the clinical potential of treatments that block persistent AR signaling which are currently undergoing clinical evaluation in CRPC.

Cell-type-specific tumour sensitivity identified with a bromodomain targeting PROTAC in adenoid cystic carcinoma.

Salivary gland adenoid cystic carcinoma (ACC) is a rare malignancy with limited treatment options. The development of novel therapies is hindered by a lack of preclinical models. We have generated ACC patient-derived xenograft (PDX) lines that retain the physical and genetic properties of the original tumours, including the presence of the common MYB::NFIB or MYBL1::NFIB translocations. We have developed the conditions for the generation of both 2D and 3D tumour organoid patient-derived ACC models that retain MYB expression and can be used for drug studies. Using these models, we show in vitro and in vivo sensitivity of ACC cells to the bromodomain degrader, dBET6. Molecular studies show a decrease in BRD4 and MYB protein levels and target gene expression with treatment. The most prominent effect of dBET6 on tumours in vivo was a change in the relative composition of ACC cell types expressing either myoepithelial or ductal markers. We show that dBET6 inhibits the progenitor function of ACC cells, particularly in the myoepithelial marker-expressing population, revealing a cell-type-specific sensitivity. These studies uncover a novel mechanistic effect of bromodomain inhibitors on tumours and highlight the need to impact both cell-type populations for more effective treatments in ACC patients. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Single-cell atlas of healthy human blood unveils age-related loss of NKG2C+GZMB-CD8+ memory T cells and accumulation of type 2 memory T cells.

Extensive, large-scale single-cell profiling of healthy human blood at different ages is one of the critical pending tasks required to establish a framework for the systematic understanding of human aging. Here, using single-cell RNA/T cell receptor (TCR)/BCR-seq with protein feature barcoding, we profiled 317 samples from 166 healthy individuals aged 25-85 years old. From this, we generated a dataset from ∼2 million cells that described 55 subpopulations of blood immune cells. Twelve subpopulations changed with age, including the accumulation of GZMK+CD8+ T cells and HLA-DR+CD4+ T cells. In contrast to other T cell memory subsets, transcriptionally distinct NKG2C+GZMB-CD8+ T cells counterintuitively decreased with age. Furthermore, we found a concerted age-associated increase in type 2/interleukin (IL)4-expressing memory subpopulations across CD4+ and CD8+ T cell compartments (CCR4+CD8+ Tcm and Th2 CD4+ Tmem), suggesting a systematic functional shift in immune homeostasis with age. Our work provides novel insights into healthy human aging and a comprehensive annotated resource.

IL-15 Priming Alters IFN-γ Regulation in Murine NK Cells.

NK effector functions can be triggered by inflammatory cytokines and engagement of activating receptors. NK cell production of IFN-γ, an important immunoregulatory cytokine, exhibits activation-specific IFN-γ regulation. Resting murine NK cells exhibit activation-specific metabolic requirements for IFN-γ production, which are reversed for activating receptor-mediated stimulation following IL-15 priming. Although both cytokine and activating receptor stimulation leads to similar IFN-γ protein production, only cytokine stimulation upregulates Ifng transcript, suggesting that protein production is translationally regulated after receptor stimulation. Based on these differences in IFN-γ regulation, we hypothesized that ex vivo IL-15 priming of murine NK cells allows a switch to IFN-γ transcription upon activating receptor engagement. Transcriptional analysis of primed NK cells compared with naive cells or cells cultured with low-dose IL-15 demonstrated that primed cells strongly upregulated Ifng transcript following activating receptor stimulation. This was not due to chromatin accessibility changes in the Ifng locus or changes in ITAM signaling, but was associated with a distinct transcriptional signature induced by ITAM stimulation of primed compared with naive NK cells. Transcriptional analyses identified a common signature of c-Myc (Myc) targets associated with Ifng transcription. Although Myc marked NK cells capable of Ifng transcription, Myc itself was not required for Ifng transcription using a genetic model of Myc deletion. This work highlights altered regulatory networks in IL-15-primed cells, resulting in distinct gene expression patterns and IFN-γ regulation in response to activating receptor stimulation.

The medical humanities at United States medical schools: a mixed method analysis of publicly assessable information on 31 schools.

INTRODUCTION: There have been increasing efforts to integrate the arts and humanities into medical education, particularly during undergraduate medical education (UME). Previous studies, however, have focused on courses and curricular programming without rigorous characterization of the associated paracurricular environment or infrastructure enabling or facilitating these offerings. METHODS: To assess opportunities for students to engage the arts and humanities during their medical education as well as the institutional resources to support those opportunities, we developed the Humanities and Arts Programming Scale (HARPS): an 18-point scale involving eight sub-domains (Infrastructure, Curricular Opportunities, Extracurricular Engagement, Opportunities for Immersion, Faculty Engagement, Staff Support, Student Groups, and Scholarship). This scale was used to evaluate the top-31 ranked United States medical schools as determined by US News and World Report's (USWNR) Medical School Research Rankings using information derived from public-facing, online information. RESULTS: Mean cumulative HARPS score was 11.26, with a median score of 12, a standard deviation of 4.32 and a score range of 3-17. Neither USWNR ranking nor private/public institution status were associated with the cumulative score (p = 0.121, p = 0.739). 52% of institutions surveyed had a humanities-focused center/division with more than 70% of the schools having significant (> 5) faculty engaged in the medical humanities. 65% of schools offered 10 or more paracurricular medical humanities events annually, while 68% of the institutions had more than 5 medical humanities student organizations. While elective, non-credit courses are available, only 3 schools required instruction in the arts and humanities, and comprehensive immersive experiences in the medical humanities were present in only 29% of the schools. CONCLUSIONS: Although there is a significant presence of the medical humanities in UME, there is a need for integration of the arts and humanities into required UME curricula and into immersive pathways for engaging the medical humanities.

IL-15 priming alters IFN-γ regulation in murine NK cells.

Natural killer (NK) effector functions can be triggered by inflammatory cytokines and engagement of activating receptors. NK cell production of IFN-γ, an important immunoregulatory cytokine, exhibits activation-specific IFN-γ regulation. Resting murine NK cells exhibit activation-specific metabolic requirements for IFN-γ production, which are reversed for activating receptor-mediated stimulation following IL-15 priming. While both cytokine and activating receptor stimulation leads to similar IFN-γ protein production, only cytokine stimulation upregulates Ifng transcript, suggesting that protein production is translationally regulated after receptor stimulation. Based on these differences in IFN-γ regulation, we hypothesized that ex vivo IL-15 priming of murine NK cells allows a switch to IFN-γ transcription upon activating receptor engagement. Transcriptional analysis of primed NK cells compared to naïve cells or cells cultured with low-dose IL-15 demonstrated that primed cells strongly upregulated Ifng transcript following activating receptor stimulation. This was not due to chromatin accessibility changes in the Ifng locus or changes in ITAM signaling, but was associated with a distinct transcriptional signature induced by ITAM stimulation of primed compared to naïve NK cells. Transcriptional analyses identified a common signature of c-Myc (Myc) targets associated with Ifng transcription. While Myc marked NK cells capable of Ifng transcription, Myc itself was not required for Ifng transcription using a genetic model of Myc deletion. This work highlights altered regulatory networks in IL-15 primed cells, resulting in distinct gene expression patterns and IFN-γ regulation in response to activating receptor stimulation.

Transcriptomic atlas and interaction networks of brain cells in mouse CNS demyelination and remyelination.

Demyelination is a hallmark of multiple sclerosis, leukoencephalopathies, cerebral vasculopathies, and several neurodegenerative diseases. The cuprizone mouse model is widely used to simulate demyelination and remyelination occurring in these diseases. Here, we present a high-resolution single-nucleus RNA sequencing (snRNA-seq) analysis of gene expression changes across all brain cells in this model. We define demyelination-associated oligodendrocytes (DOLs) and remyelination-associated MAFBhi microglia, as well as astrocytes and vascular cells with signatures of altered metabolism, oxidative stress, and interferon response. Furthermore, snRNA-seq provides insights into how brain cell types connect and interact, defining complex circuitries that impact demyelination and remyelination. As an explicative example, perturbation of microglia caused by TREM2 deficiency indirectly impairs the induction of DOLs. Altogether, this study provides a rich resource for future studies investigating mechanisms underlying demyelinating diseases.

Human early-onset dementia caused by DAP12 deficiency reveals a unique signature of dysregulated microglia.

The TREM2-DAP12 receptor complex sustains microglia functions. Heterozygous hypofunctional TREM2 variants impair microglia, accelerating late-onset Alzheimer's disease. Homozygous inactivating variants of TREM2 or TYROBP-encoding DAP12 cause Nasu-Hakola disease (NHD), an early-onset dementia characterized by cerebral atrophy, myelin loss and gliosis. Mechanisms underpinning NHD are unknown. Here, single-nucleus RNA-sequencing analysis of brain specimens from DAP12-deficient NHD individuals revealed a unique microglia signature indicating heightened RUNX1, STAT3 and transforming growth factor-ß signaling pathways that mediate repair responses to injuries. This profile correlated with a wound healing signature in astrocytes and impaired myelination in oligodendrocytes, while pericyte profiles indicated vascular abnormalities. Conversely, single-nuclei signatures in mice lacking DAP12 signaling reflected very mild microglial defects that did not recapitulate NHD. We envision that DAP12 signaling in microglia attenuates wound healing pathways that, if left unchecked, interfere with microglial physiological functions, causing pathology in human. The identification of a dysregulated NHD microglia signature sparks potential therapeutic strategies aimed at resetting microglia signaling pathways.

Characterisation of a Novel Cell Line (ICR-SS-1) Established from a Patient-Derived Xenograft of Synovial Sarcoma.

Synovial sarcoma is a rare translocation-driven cancer with poor survival outcomes, particularly in the advanced setting. Previous synovial sarcoma preclinical studies have relied on a small panel of cell lines which suffer from the limitation of genomic and phenotypic drift as a result of being grown in culture for decades. Patient-derived xenografts (PDX) are a valuable tool for preclinical research as they retain many histopathological features of their originating human tumour; however, this approach is expensive, slow, and resource intensive, which hinders their utility in large-scale functional genomic and drug screens. To address some of these limitations, in this study, we have established and characterised a novel synovial sarcoma cell line, ICR-SS-1, which is derived from a PDX model and is amenable to high-throughput drug screens. We show that ICR-SS-1 grows readily in culture, retains the pathognomonic SS18::SSX1 fusion gene, and recapitulates the molecular features of human synovial sarcoma tumours as shown by proteomic profiling. Comparative analysis of drug response profiles with two other established synovial sarcoma cell lines (SYO-1 and HS-SY-II) finds that ICR-SS-1 harbours intrinsic resistance to doxorubicin and is sensitive to targeted inhibition of several oncogenic pathways including the PI3K-mTOR pathway. Collectively, our studies show that the ICR-SS-1 cell line model may be a valuable preclinical tool for studying the biology of anthracycline-resistant synovial sarcoma and identifying new salvage therapies following failure of doxorubicin.

Assessment of Androgen Receptor Splice Variant-7 as a Biomarker of Clinical Response in Castration-Sensitive Prostate Cancer.

PURPOSE: Therapies targeting the androgen receptor (AR) have improved the outcome for patients with castration-sensitive prostate cancer (CSPC). Expression of the constitutively active AR splice variant-7 (AR-V7) has shown clinical utility as a predictive biomarker of AR-targeted therapy resistance in castration-resistant prostate cancer (CRPC), but its importance in CSPC remains understudied. EXPERIMENTAL DESIGN: We assessed different approaches to quantify AR-V7 mRNA and protein in prostate cancer cell lines, patient-derived xenograft (PDX) models, publicly available cohorts, and independent institutional clinical cohorts, to identify reliable approaches for detecting AR-V7 mRNA and protein and its association with clinical outcome. RESULTS: In CSPC and CRPC cohorts, AR-V7 mRNA was much less abundant when detected using reads across splice boundaries than when considering isoform-specific exonic reads. The RM7 AR-V7 antibody had increased sensitivity and specificity for AR-V7 protein detection by immunohistochemistry (IHC) in CRPC cohorts but rarely identified AR-V7 protein reactivity in CSPC cohorts, when compared with the EPR15656 AR-V7 antibody. Using multiple CRPC PDX models, we demonstrated that AR-V7 expression was exquisitely sensitive to hormonal manipulation. In CSPC institutional cohorts, AR-V7 protein quantification by either assay was associated neither with time to development of castration resistance nor with overall survival, and intense neoadjuvant androgen-deprivation therapy did not lead to significant AR-V7 mRNA or staining following treatment. Neither pre- nor posttreatment AR-V7 levels were associated with volumes of residual disease after therapy. CONCLUSIONS: This study demonstrates that further analytical validation and clinical qualification are required before AR-V7 can be considered for clinical use in CSPC as a predictive biomarker.

Myeloid cell interferon responses correlate with clearance of SARS-CoV-2.

Emergence of mutant SARS-CoV-2 strains associated with an increased risk of COVID-19-related death necessitates better understanding of the early viral dynamics, host responses and immunopathology. Single cell RNAseq (scRNAseq) allows for the study of individual cells, uncovering heterogeneous and variable responses to environment, infection and inflammation. While studies have reported immune profiling using scRNAseq in terminal human COVID-19 patients, performing longitudinal immune cell dynamics in humans is challenging. Macaques are a suitable model of SARS-CoV-2 infection. Our longitudinal scRNAseq of bronchoalveolar lavage (BAL) cell suspensions from young rhesus macaques infected with SARS-CoV-2 (n = 6) demonstrates dynamic changes in transcriptional landscape 3 days post- SARS-CoV-2-infection (3dpi; peak viremia), relative to 14-17dpi (recovery phase) and pre-infection (baseline) showing accumulation of distinct populations of both macrophages and T-lymphocytes expressing strong interferon-driven inflammatory gene signature at 3dpi. Type I interferon response is induced in the plasmacytoid dendritic cells with appearance of a distinct HLADR+CD68+CD163+SIGLEC1+ macrophage population exhibiting higher angiotensin-converting enzyme 2 (ACE2) expression. These macrophages are significantly enriched in the lungs of macaques at 3dpi and harbor SARS-CoV-2 while expressing a strong interferon-driven innate anti-viral gene signature. The accumulation of these responses correlated with decline in viremia and recovery.

Drawing for Visual Communication in Medicine: A Novel Pilot Course for Senior Medical Students.

OBJECTIVE: Visual aids such as drawings have been reported to improve patient comprehension, retention, and adherence. We sought to determine the feasibility of teaching live drawing for clinical communication to medical students. DESIGN: We designed a course to teach basic drawing skills and visual communication of health information to senior medical students. Data was gathered from both an intervention and control group via written pre- and post-course surveys. The intervention group also completed a survey six months after the course. SETTING: The course was offered as an elective at the University of Pennsylvania Perelman School of Medicine during February 2020. PARTICIPANTS: The intervention group consisted of 17 enrolled students, while 17 students not taking the course served as a control group. Third year, fourth year, and research year medical students were invited to enroll in the course. RESULTS: The intervention group had significantly greater comfort with visual communication for patient care and increased objective drawing and visual communication scores compared to the control group. Visual abilities not targeted by the curriculum did not change between the intervention and control groups. At 6-months follow-up, course participants reported persistently elevated comfort in visual communication, as well as utilization of visual communication skills in their clinical practice. CONCLUSIONS: These data provide initial evidence of the efficacy of an elective course aimed at developing the skill and confidence to draw for visual communication in medicine as well as support for continued efforts to further develop and disseminate this type of curriculum.

Protein kinase A drives paracrine crisis and WNT4-dependent testis tumor in Carney complex.

Large-cell calcifying Sertoli cell tumors (LCCSCTs) are among the most frequent lesions occurring in male Carney complex (CNC) patients. Although they constitute a key diagnostic criterion for this rare multiple neoplasia syndrome resulting from inactivating mutations of the tumor suppressor PRKAR1A, leading to unrepressed PKA activity, LCCSCT pathogenesis and origin remain elusive. Mouse models targeting Prkar1a inactivation in all somatic populations or separately in each cell type were generated to decipher the molecular and paracrine networks involved in the induction of CNC testis lesions. We demonstrate that the Prkar1a mutation was required in both stromal and Sertoli cells for the occurrence of LCCSCTs. Integrative analyses comparing transcriptomic, immunohistological data and phenotype of mutant mouse combinations led to the understanding of human LCCSCT pathogenesis and demonstrated PKA-induced paracrine molecular circuits in which the aberrant WNT4 signal production is a limiting step in shaping intratubular lesions and tumor expansion both in a mouse model and in human CNC testes.

Enhanced epigenetic profiling of classical human monocytes reveals a specific signature of healthy aging in the DNA methylome.

The impact of healthy aging on molecular programming of immune cells is poorly understood. Here, we report comprehensive characterization of healthy aging in human classical monocytes, with a focus on epigenomic, transcriptomic, and proteomic alterations, as well as the corresponding proteomic and metabolomic data for plasma, using healthy cohorts of 20 young and 20 older males (~27 and ~64 years old on average). For each individual, we performed eRRBS-based DNA methylation profiling, which allowed us to identify a set of age-associated differentially methylated regions (DMRs) - a novel, cell-type specific signature of aging in DNA methylome. Hypermethylation events were associated with H3K27me3 in the CpG islands near promoters of lowly-expressed genes, while hypomethylated DMRs were enriched in H3K4me1 marked regions and associated with age-related increase of expression of the corresponding genes, providing a link between DNA methylation and age-associated transcriptional changes in primary human cells.

HER3 Is an Actionable Target in Advanced Prostate Cancer.

It has been recognized for decades that ERBB signaling is important in prostate cancer, but targeting ERBB receptors as a therapeutic strategy for prostate cancer has been ineffective clinically. However, we show here that membranous HER3 protein is commonly highly expressed in lethal prostate cancer, associating with reduced time to castration resistance (CR) and survival. Multiplex immunofluorescence indicated that the HER3 ligand NRG1 is detectable primarily in tumor-infiltrating myelomonocytic cells in human prostate cancer; this observation was confirmed using single-cell RNA sequencing of human prostate cancer biopsies and murine transgenic prostate cancer models. In castration-resistant prostate cancer (CRPC) patient-derived xenograft organoids with high HER3 expression as well as mouse prostate cancer organoids, recombinant NRG1 enhanced proliferation and survival. Supernatant from murine bone marrow-derived macrophages and myeloid-derived suppressor cells promoted murine prostate cancer organoid growth in vitro, which could be reversed by a neutralizing anti-NRG1 antibody and ERBB inhibition. Targeting HER3, especially with the HER3-directed antibody-drug conjugate U3-1402, exhibited antitumor activity against HER3-expressing prostate cancer. Overall, these data indicate that HER3 is commonly overexpressed in lethal prostate cancer and can be activated by NRG1 secreted by myelomonocytic cells in the tumor microenvironment, supporting HER3-targeted therapeutic strategies for treating HER3-expressing advanced CRPC. SIGNIFICANCE: HER3 is an actionable target in prostate cancer, especially with anti-HER3 immunoconjugates, and targeting HER3 warrants clinical evaluation in prospective trials.