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INTRODUCTION: Digital education tools are a cornerstone in the evolution to CBME through EPAs. Successful implementation requires understanding the variable impacts of EHR-driven delivery of EPAs, flexible digital device access to EPAs, and user-behavior trends. METHODS: Through a HIPAA compliant, flexible-device accessible, surgical education platform, general surgery training programs at 21 institutions collected EPA from July 2023 to April 2024. At 5 EHR-integrated institutions (EHR+), EPA were created for clinical activities based on the OR schedule, automatically pushed to attendings and residents with built in completion reminders. At 16 institutions without EHR integration (EHR-), EPA were initiated manually. To improve user experience, care phases were bundled (cEPA). We compared the EHR+ and EHR- groups, computing descriptive statistics on the cEPAs completed and user behavior metrics. RESULTS: We collected 4187 cEPAs in total, with 82% at EHR+ institutions and 18% at EHR- institutions. Platform triggering dramatically drove cEPA completion for both faculty and residents, 88% and 81%, respectively. Only 3% were initiated by the faculty or resident. Faculty at EHR+ institutions strongly preferred the automated OR-triggered workflow to start their EPAs (Chi-squared test, p ≈ 0). Faculty completed all 3 care phases nearly 80% of the time. Time reminders specifically drive EPA completion for residents and faculty on weekdays and build habits on weekends. 71% of cEPAs completed were by computer, and 29% by phone. More comments were provided when computers were used. Residents reviewed feedback with a median lag of 1 hour and 29 min after results were available. CONCLUSIONS: EHR-driven delivery of EPA leads to a 4.6-fold increase in EPAs completed. EPA initiation is the most critical phase in the workflow and EHR-data driven alerts drive this action. These alerts are also effective drivers of habit formation. Flexible device access is important to increase EPAs completed and improve the usefulness through comments for residents.
ABSTRACT
Heterotopic ossifications (HOs) are the pathologic process by which bone inappropriately forms outside of the skeletal system. Despite HOs being a persistent clinical problem in the general population, there are no definitive strategies for their prevention and treatment due to a limited understanding of the cellular and molecular mechanisms contributing to lesion development. One disease in which the development of heterotopic subcutaneous ossifications (SCOs) leads to morbidity is Albright hereditary osteodystrophy (AHO). AHO is caused by heterozygous inactivation of GNAS, the gene that encodes the α-stimulatory subunit (Gαs) of G proteins. Previously, we had shown using our laboratory's AHO mouse model that SCOs develop around hair follicles (HFs). Here we show that SCO formation occurs due to inappropriate expansion and differentiation of HF-resident stem cells into osteoblasts. We also show in AHO patients and mice that Secreted Frizzled Related Protein 2 (SFRP2) expression is upregulated in regions of SCO formation and that elimination of Sfrp2 in male AHO mice exacerbates SCO development. These studies provide key insights into the cellular and molecular mechanisms contributing to SCO development and have implications for potential therapeutic modalities not only for AHO patients but also for patients suffering from HOs with other etiologies.
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Contact dermatitis (CD) is a common cutaneous inflammatory condition that affects millions of people worldwide. Xenobiotic agents are frequently encountered in substances used in everyday life, making it difficult to avoid personal and occupational exposure. Toll-like receptors (TLRs) are transmembrane receptors that modulate the innate immune system in response to tissue injury or infection. TLRs play a key role in the pathophysiology of contact dermatitis. TLR signaling is involved in three major forms of CD: protein CD, allergic contact dermatitis (ACD), and irritant CD. Of the 10 TLRs found in humans, three play an important role in ACD. This makes TLRs a useful potential therapeutic target to consider against CD. In this review, we discuss the role of TLRs in CD and summarize current and emerging treatments for CD that target TLRs.
ABSTRACT
Despite over a century of observations, the obligate insect parasites within the order Entomophthorales remain poorly characterized at the genetic level. In this manuscript, we present a genome for a laboratory-tractable Entomophthora muscae isolate that infects fruit flies. Our E. muscae assembly is 1.03 Gb, consists of 7810 contigs and contains 81.3% complete fungal BUSCOs. Using a comparative approach with recent datasets from entomophthoralean fungi, we show that giant genomes are the norm within Entomophthoraceae owing to extensive, but not recent, Ty3 retrotransposon activity. In addition, we find that E. muscae and its closest allies possess genes that are likely homologs to the blue-light sensor white-collar 1, a Neurospora crassa gene that has a well-established role in maintaining circadian rhythms. We uncover evidence that E. muscae diverged from other entomophthoralean fungi by expansion of existing families, rather than loss of particular domains, and possesses a potentially unique suite of secreted catabolic enzymes, consistent with E. muscae's species-specific, biotrophic lifestyle. Finally, we offer a head-to-head comparison of morphological and molecular data for species within the E. muscae species complex that support the need for taxonomic revision within this group. Altogether, we provide a genetic and molecular foundation that we hope will provide a platform for the continued study of the unique biology of entomophthoralean fungi.
Subject(s)
Entomophthora , Genome, Fungal , Animals , Entomophthora/genetics , DNA Transposable Elements/genetics , Phylogeny , Circadian Rhythm/genetics , Entomophthorales/genetics , Entomophthorales/physiologyABSTRACT
Bacterial pericarditis is a rare phenomenon that progresses rapidly and carries high mortality. Patients presenting with new pericardial effusions are often evaluated for concomitant rheumatologic, oncologic, and infectious diseases. We present a complex case of purulent pericarditis with pneumopericardium.
ABSTRACT
Resveratrol prevents various neurodegenerative diseases in animal models despite reaching only low nanomolar concentrations in the brain after oral administration. In this study, based on the quenching of intrinsic tryptophan fluorescence and molecular docking, we found that trans-resveratrol, its conjugates (glucuronide and sulfate), and dihydro-resveratrol (intestinal microbial metabolite) bind with high affinities (Kd, 0.2-2 nm) to the peptide G palindromic sequence (near glycosaminoglycan-binding motif) of the 67-kDa laminin receptor (67LR). Preconditioning with low concentrations (0.01-10 nm) of these polyphenols, especially resveratrol-glucuronide, protected neuronal cells from death induced by serum withdrawal via activation of cAMP-mediated signaling pathways. This protection was prevented by a 67LR-blocking antibody, suggesting a role for this cell-surface receptor in neuroprotection by resveratrol metabolites.
Subject(s)
Neuroprotective Agents , Receptors, Laminin , Resveratrol , Resveratrol/pharmacology , Resveratrol/metabolism , Resveratrol/chemistry , Receptors, Laminin/metabolism , Receptors, Laminin/genetics , Humans , Neuroprotective Agents/pharmacology , Neuroprotective Agents/metabolism , Molecular Docking Simulation , Animals , Protein Binding , Neurons/metabolism , Neurons/drug effects , Stilbenes/pharmacology , Stilbenes/metabolism , Stilbenes/chemistry , Neuroprotection/drug effects , Signal Transduction/drug effects , Binding Sites , Glucuronides/metabolism , Glucuronides/chemistry , Ribosomal ProteinsABSTRACT
Despite over a century of observations, the obligate insect parasites within the order Entomophthorales remain poorly characterized at the genetic level. This is in part due to their large genome sizes and difficulty in obtaining sequenceable material. In this manuscript, we leveraged a recently-isolated, laboratory-tractable Entomophthora muscae isolate and improved long-read sequencing to obtain a largely-complete entomophthoralean genome. Our E. muscae assembly is 1.03 Gb, consists of 7,810 contigs and contains 81.3% complete fungal BUSCOs. Using a comparative approach with other available (transcriptomic and genomic) datasets from entomophthoralean fungi, we provide new insight into the biology of these understudied pathogens. We offer a head-to-head comparison of morphological and molecular data for species within the E. muscae species complex. Our findings suggest that substantial taxonomic revision is needed to define species within this group and we provide recommendations for differentiating strains and species in the context of the existing body of E. muscae scientific literature. We show that giant genomes are the norm within Entomophthoraceae owing to extensive, but not recent, Ty3 retrotransposon activity, despite the presence of machinery to defend against transposable elements(RNAi). In addition, we find that E. muscae and its closest allies are enriched for M16A peptidases and possess genes that are likely homologs to the blue-light sensor white-collar 1, a Neurospora crassa gene that has a well-established role in maintaining circadian rhythms. We find that E. muscae has an expanded group of acid-trehalases, consistent with trehalose being the primary sugar component of fly (and insect) hemolymph. We uncover evidence that E. muscae diverged from other entomophthoralean fungi by expansion of existing families, rather than loss of particular domains, and possesses a potentially unique suite of secreted catabolic enzymes, consistent with E. muscae's species-specific, biotrophic lifestyle. Altogether, we provide a genetic and molecular foundation that we hope will provide a platform for the continued study of the unique biology of entomophthoralean fungi.
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Bisphosphonates are widely used to treat osteoporosis and malignant tumors due to their effectiveness in increasing bone density and inhibiting bone resorption. However, their association with bisphosphonate-related osteonecrosis of the jaws (BRONJ) following invasive dental procedures poses a significant challenge. This review explores the functions, mechanisms, and side effects of bisphosphonates, emphasizing their impact on dental procedures. Dental patients receiving bisphosphonate treatment are at higher risk of BRONJ, necessitating dentists' awareness of these risks. Topical bisphosphonate applications enhance dental implant success, by promoting osseointegration and preventing osteoclast apoptosis, and is effective in periodontal treatment. Yet, systemic administration (intravenous or intraoral) significantly increases the risk of BRONJ following dental procedures, particularly in inflamed conditions. Prevention and management of BRONJ involve maintaining oral health, considering alternative treatments, and careful pre-operative and post-operative follow-ups. Future research could focus on finding bisphosphonate alternatives with fewer side effects or developing combinations that reduce BRONJ risk. This review underscores the need for further exploration of bisphosphonates and their implications in dental procedures.
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3D spheroids have emerged as powerful drug discovery tools given their high-throughput screening (HTS) compatibility. Here, we describe a method for generating functional neural spheroids by cell-aggregation of differentiated human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes at cell type compositions mimicking specific regions of the human brain. Recordings of intracellular calcium oscillations were used as functional assays, and the utility of this spheroids system was shown through disease modeling, drug testing, and formation of assembloids to model neurocircuitry. As a proof of concept, we generated spheroids incorporating neurons with Alzheimer's disease-associated alleles, as well as opioid use disorder modeling spheroids induced by chronic treatment of a mu-opioid receptor agonist. We reversed baseline functional deficits in each pilot disease model with clinically approved treatments and showed that assembloid activity can be chemogenetically manipulated. Here, we lay the groundwork for brain region-specific neural spheroids as a robust functional assay platform for HTS studies.
Subject(s)
Induced Pluripotent Stem Cells , Humans , Brain , Cell Differentiation/physiology , Neurons , High-Throughput Screening Assays/methodsABSTRACT
The National Center for Advancing Translational Sciences (NCATS) Assay Guidance Manual (AGM) Workshop on 3D Tissue Models for Antiviral Drug Development, held virtually on 7-8 June 2022, provided comprehensive coverage of critical concepts intended to help scientists establish robust, reproducible, and scalable 3D tissue models to study viruses with pandemic potential. This workshop was organized by NCATS, the National Institute of Allergy and Infectious Diseases, and the Bill and Melinda Gates Foundation. During the workshop, scientific experts from academia, industry, and government provided an overview of 3D tissue models' utility and limitations, use of existing 3D tissue models for antiviral drug development, practical advice, best practices, and case studies about the application of available 3D tissue models to infectious disease modeling. This report includes a summary of each workshop session as well as a discussion of perspectives and challenges related to the use of 3D tissues in antiviral drug discovery.
Subject(s)
Antiviral Agents , Drug Discovery , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Biological AssayABSTRACT
Quercetin, a dietary flavonoid, has been shown to protect against various neurodegenerative diseases with mechanisms largely unknown. After oral administration, quercetin is rapidly conjugated, and the aglycone is not detectable in the plasma and brain. However, its glucuronide and sulfate conjugates are present only at low nanomolar concentrations in the brain. Since quercetin and its conjugates have limited antioxidant capability at low nanomolar concentrations, it is crucial to determine whether they induce neuroprotection by binding to high-affinity receptors. Previously we found that (-)-epigallocatechin-3-gallate (EGCG), a polyphenol from green tea, induces neuroprotection by binding to the 67-kDa laminin receptor (67LR). Therefore, in this study, we determined whether quercetin and its conjugates bind 67LR to induce neuroprotection and compared their ability with EGCG. Based on the quenching of intrinsic tryptophan fluorescence of peptide G (residues 161-180 in 67LR), we found quercetin, quercetin-3-O-glucuronide, and quercetin-3-O-sulfate bind to this peptide with a high affinity comparable to EGCG. Molecular docking using the crystal structure of 37-kDa laminin receptor precursor supported the high-affinity binding of all these ligands to the site corresponding to peptide G. A pretreatment with quercetin (1-1000 nM) did not effectively protect Neuroscreen-1 cells from death induced by serum starvation. Contrarily, a pretreatment with low concentrations (1-10 nM) of quercetin conjugates better protected these cells than quercetin and EGCG. The 67LR-blocking antibody substantially prevented neuroprotection by all these agents, suggesting the role of 67LR in this process. Collectively, these studies reveal that quercetin induces neuroprotection primarily through its conjugates via high affinity binding to 67LR.
Subject(s)
Catechin , Flavonoids , Flavonoids/pharmacology , Quercetin/pharmacology , Glucuronides/pharmacology , Sulfates , Molecular Docking Simulation , Polyphenols/pharmacology , Receptors, Laminin/metabolism , Catechin/pharmacology , Cell Adhesion Molecules , Cell DeathABSTRACT
Background: Established mouse models of HER2+ cancer are based on the over-expression of rodent Neu/Erbb2 homologues, which are incompatible with human HER2 (huHER2) targeted therapeutics. Additionally, the use of immune-deficient xenograft or transgenic models precludes assessment of native anti-tumour immune responses. These hurdles have been a challenge for our understanding of the immune mechanisms behind huHER2-targeting immunotherapies. Methods: To assess the immune impacts of our huHER2-targeted combination strategy, we generated a syngeneic mouse model of huHER2+ breast cancer, using a truncated form of huHER2, HER2T. Following validation of this model, we next treated tumour-bearing with our immunotherapy strategy: oncolytic vesicular stomatitis virus (VSVΔ51) with clinically approved antibody-drug conjugate targeting huHER2, trastuzumab emtansine (T-DM1). We assessed efficacy through tumour control, survival, and immune analyses. Results: The generated truncated HER2T construct was non-immunogenic in wildtype BALB/c mice upon expression in murine mammary carcinoma 4T1.2 cells. Treatment of 4T1.2-HER2T tumours with VSVΔ51+T-DM1 yielded robust curative efficacy compared to controls, and broad immunologic memory. Interrogation of anti-tumour immunity revealed tumour infiltration by CD4+ T cells, and activation of B, NK, and dendritic cell responses, as well as tumour-reactive serum IgG. Conclusions: The 4T1.2-HER2T model was used to evaluate the anti-tumour immune responses following our complex pharmacoviral treatment strategy. These data demonstrate utility of the syngeneic HER2T model for assessment of huHER2-targeted therapies in an immune-competent in vivo setting. We further demonstrated that HER2T can be implemented in multiple other syngeneic tumour models, including but not limited to colorectal and ovarian models. These data also suggest that the HER2T platform may be used to assess a range of surface-HER2T targeting approaches, such as CAR-T, T-cell engagers, antibodies, or even retargeted oncolytic viruses.
Subject(s)
Breast Neoplasms , Rhabdoviridae , Humans , Mice , Animals , Female , Ado-Trastuzumab Emtansine/therapeutic use , Breast Neoplasms/metabolism , Trastuzumab/pharmacology , Trastuzumab/therapeutic use , Antibodies, Monoclonal, Humanized/therapeutic use , Disease Models, AnimalABSTRACT
For at least two centuries, scientists have been enthralled by the "zombie" behaviors induced by mind-controlling parasites. Despite this interest, the mechanistic bases of these uncanny processes have remained mostly a mystery. Here, we leverage the Entomophthora muscae-Drosophila melanogaster "zombie fly" system to reveal the mechanistic underpinnings of summit disease, a manipulated behavior evoked by many fungal parasites. Using a high-throughput approach to measure summiting, we discovered that summiting behavior is characterized by a burst of locomotion and requires the host circadian and neurosecretory systems, specifically DN1p circadian neurons, pars intercerebralis to corpora allata projecting (PI-CA) neurons and corpora allata (CA), the latter being solely responsible for juvenile hormone (JH) synthesis and release. Using a machine learning classifier to identify summiting animals in real time, we observed that PI-CA neurons and CA appeared intact in summiting animals, despite invasion of adjacent regions of the "zombie fly" brain by E. muscae cells and extensive host tissue damage in the body cavity. The blood-brain barrier of flies late in their infection was significantly permeabilized, suggesting that factors in the hemolymph may have greater access to the central nervous system during summiting. Metabolomic analysis of hemolymph from summiting flies revealed differential abundance of several compounds compared to non-summiting flies. Transfusing the hemolymph of summiting flies into non-summiting recipients induced a burst of locomotion, demonstrating that factor(s) in the hemolymph likely cause summiting behavior. Altogether, our work reveals a neuro-mechanistic model for summiting wherein fungal cells perturb the fly's hemolymph, activating a neurohormonal pathway linking clock neurons to juvenile hormone production in the CA, ultimately inducing locomotor activity in their host.
Subject(s)
Drosophila , Parasites , Animals , Drosophila melanogaster/physiology , Corpora Allata/metabolism , Juvenile Hormones/metabolismABSTRACT
BACKGROUND: The 2018 adult heart allocation policy sought to improve waitlist risk stratification, reduce waitlist mortality, and increase organ access. This system prioritized patients at greatest risk for waitlist mortality, especially individuals requiring temporary mechanical circulatory support (tMCS). Posttransplant complications are significantly higher in patients on tMCS before transplantation, and early posttransplant complications impact long-term mortality. We sought to determine if policy change affected early posttransplant complication rates of rejection, infection, and hospitalization. METHODS: We included all adult, heart-only, single-organ heart transplant recipients from the UNOS registry with pre-policy (PRE) individuals transplanted between November 1, 2016, and October 31, 2017, and post-policy (POST) between November 1, 2018, and October 31, 2019. We used a multivariable logistic regression analysis to assess the effect of policy change on posttransplant rejection, infection, and hospitalization. Two COVID-19 eras (2019-2020, 2020-2021) were included in our analysis. RESULTS: The majority of baseline characteristics were comparable between PRE and POST era recipients. The odds of treated rejection (p = 0.8), hospitalization (p = 0.69), and hospitalization due to rejection (p = 0.76) and infection (p = 0.66) were similar between PRE and POST eras; there was a trend towards reduced odds of rejection (p = 0.08). In both COVID eras, there was a clear reduction in rejection and treated rejection with no effect on hospitalization for rejection or infection. Odds of all-cause hospitalization was increased in both COVID eras. CONCLUSIONS: The UNOS policy change improves access to heart transplantation for higher acuity patients without increasing early posttransplant rates of treated rejection or hospitalization for rejection or infection, factors which portend risk for long-term posttransplant mortality.
Subject(s)
COVID-19 , Heart Transplantation , Adult , Humans , Patient Readmission , COVID-19/epidemiology , Heart Transplantation/adverse effects , Hospitalization , Policy , Waiting Lists , Retrospective StudiesABSTRACT
Introduction: The increased incidence of cutaneous melanoma (CM) and Merkel cell carcinoma (MCC) in patients with hematologic malignancies (HM) is well established. While the risk of CM has been assessed in some subtypes of HM including cutaneous T-cell lymphoma, the incidence in patients with primary cutaneous B-cell lymphoma (PCBCL) has not been interrogated. Methods: Here we evaluated the standardized incidence ratio (SIR) of CM and MCC in 5,179 PCBCL patients compared to approximately 1.5 billion individuals in the general population using the Surveillance, Epidemiology and End Results (SEER) database. Among patients with PCBCL, we identified subgroups that were at increased risk for CM or MCC as a second primary cancer. Results: We found 36 cases of CM in the PCBCL cohort (SIR, 1.35; 95% CI, 0.94-1.86), among which SIR was significantly elevated for non-Hispanic White patients compared to the general population (SIR, 1.48; 95% CI, 1.03-2.06). Males had a significantly increased risk of developing CM after a diagnosis of PCBCL (SIR, 1.60; 95% CI, 1.10-2.26). We found that males in the age group of 50-59 were at increased risk for CM development (SIR, 3.02; 95% CI, 1.11-6.58). Males were at increased risk of CM 1-5 years after PCBCL diagnosis (SIR, 2.06; 95% CI, 1.18-3.34). Patients were at greater risk of developing MCC within 1 year of diagnosis of PCBCL (SIR, 23.60; 95% CI, 2.86-85.27), particularly in patients who were over the age of 80 (SIR, 46.50; 95% CI, 5.63-167.96). Males aged 60-69 with PCBCL, subtype marginal zone, were also at increased risk for MCC (SIR, 42.71; 95% CI, 1.08-237.99). Conclusion: There is an increased incidence of CM in White, middle-aged males within 5 years of diagnosis of PCBCL and an increased risk of MCC in elderly patients within 1 year of PCBCL diagnosis. These data suggest that certain subgroups of patients with PCBCL may require more rigid surveillance for CM and MCC.
ABSTRACT
BACKGROUND: Albright hereditary osteodystrophy (AHO) is caused by heterozygous inactivating mutations in GNAS. Patients with maternally-inherited mutations develop pseudohypoparathyroidism type 1A (PHP1A) with multi-hormone resistance and aberrant craniofacial and skeletal development among other abnormalities. Chiari malformation type 1 (CM1), a condition in which brain tissue extends into the spinal canal when the skull is too small, has been reported in isolated cases of PHP1A. It has been hypothesized to be associated with growth hormone (GH) deficiency. Given the adverse clinical sequelae that can occur if CM1 goes unrecognized, we investigated the previously undetermined prevalence of CM1, as well as any potential correlations with GH status, given the known increased prevalence of GH deficiency in PHP1A. We also investigated these metrics for low lying cerebellar tonsils (LLCT), defined as tonsillar descent less than 5 mm below the foramen magnum. In addition, we investigated possible correlations of CM1/LLCT with advanced hand/wrist bone ages and craniofacial abnormalities known to occur in PHP1A to determine whether premature chondrocyte differentiation and/or aberrant craniofacial development could be potential etiologies of CM1/LLCT through both human studies and investigations of our AHO mouse model. METHODS: We examined patients with PHP1A in our clinic and noticed CM1 more frequently than expected. Therefore, we set out to determine the true prevalence of CM1 and LLCT in a cohort of 54 mutation-confirmed PHP1A participants who had clinically-indicated brain imaging. We examined potential correlations with GH status, clinical features, biological sex, genotype, and hand/wrist bone age determinations. In addition, we investigated the craniofacial development in our mouse model of AHO (Gnas E1+/-m) by histologic analyses, dynamic histomorphometry, and micro-computerized tomographic imaging (MCT) in order to determine potential etiologies of CM1/LLCT in PHP1A. RESULTS: In our cohort of PHP1A, the prevalence of CM1 is 10.8%, which is at least 10-fold higher than in the general population. If LLCT is included, the prevalence increases to 21.7%. We found no correlation with GH status, biological sex, genotype, or hand/wrist bone age. Through investigations of our Gnas E1+/-m mice, the correlate to PHP1A, we identified a smaller cranial vault and increased cranial dome angle with evidence of hyperostosis due to increased osteogenesis. We also demonstrated that there was premature closure of the spheno-occipital synchondrosis (SOS), a cartilaginous structure essential to the development of the cranial base. These findings lead to craniofacial abnormalities and could contribute to CM1 and LLCT development in PHP1A. CONCLUSION: The prevalence of CM1 is at least 10-fold higher in PHP1A compared to the general population and 20-fold higher when including LLCT. This is independent of the GH deficiency that is found in approximately two-thirds of patients with PHP1A. In light of potential serious consequences of CM1, clinicians should have a low threshold for brain imaging. Investigations of our AHO mouse model revealed aberrant cranial formation including a smaller cranium, increased cranial dome angle, hyperostosis, and premature SOS closure rates, providing a potential etiology for the increased prevalence of CM1 and LLCT in PHP1A.
Subject(s)
Arnold-Chiari Malformation , Pseudohypoparathyroidism , Humans , Animals , Mice , Prevalence , Pseudohypoparathyroidism/epidemiology , Pseudohypoparathyroidism/genetics , Pseudohypoparathyroidism/complications , GTP-Binding Protein alpha Subunits, Gs/genetics , Bone Development , Genotype , Arnold-Chiari Malformation/diagnostic imaging , Arnold-Chiari Malformation/epidemiology , Arnold-Chiari Malformation/genetics , Chromogranins/geneticsABSTRACT
Colorectal cancer is the third most diagnosed cancer and the second leading cause of cancer mortality worldwide, highlighting an urgent need for new therapeutic options and combination strategies for patients. The orchestration of potent T cell responses against human cancers is necessary for effective antitumour immunity. However, regression of a limited number of cancers has been induced by immune checkpoint inhibitors, T cell engagers (TCEs) and/or oncolytic viruses. Although one TCE has been FDA-approved for the treatment of hematological malignancies, many challenges exist for the treatment of solid cancers. Here, we show that TCEs targeting CEACAM5 and CD3 stimulate robust activation of CD4 and CD8-positive T cells in in vitro co-culture models with colorectal cancer cells, but in vivo efficacy is hindered by a lack of TCE retention in the tumour microenvironment and short TCE half-life, as demonstrated by HiBiT bioluminescent TCE-tagging technology. To overcome these limitations, we engineered Bispecific Engager Viruses, or BEVirs, a novel tumour-targeted vaccinia virus platform for intra-tumour delivery of these immunomodulatory molecules. We characterized virus-mediated TCE-secretion, TCE specificity and functionality from infected colorectal cancer cells and patient tumour samples, as well as TCE cytotoxicity in spheroid models, in the presence and absence of T cells. Importantly, we show regression of colorectal tumours in both syngeneic and xenograft mouse models. Our data suggest that a different profile of cytokines may contribute to the pro-inflammatory and immune effects driven by T cells in the tumour microenvironment to provide long-lasting immunity and abscopal effects. We establish combination regimens with immune checkpoint inhibitors for aggressive colorectal peritoneal metastases. We also observe a significant reduction in lung metastases of colorectal tumours through intravenous delivery of our oncolytic virus driven T-cell based combination immunotherapy to target colorectal tumours and FAP-positive stromal cells or CTLA4-positive Treg cells in the tumour microenvironment. In summary, we devised a novel combination strategy for the treatment of colorectal cancers using oncolytic vaccinia virus to enhance immune-payload delivery and boost T cell responses within tumours.
Subject(s)
Colorectal Neoplasms , Oncolytic Virotherapy , Oncolytic Viruses , Humans , Mice , Animals , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Immunotherapy , Vaccinia virus , Disease Models, Animal , Colorectal Neoplasms/therapy , Tumor MicroenvironmentABSTRACT
Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.
Subject(s)
Bacteriophages , COVID-19 , Single-Domain Antibodies , Antibodies, Neutralizing , Antibodies, Viral , Bacteriophages/metabolism , Humans , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, CoronavirusABSTRACT
Cutaneous leishmaniasis is a parasitic infection that causes significant maternal morbidity, and even fetal mortality, during pregnancy, yet there are limited therapeutic options. Here, we report a case of leishmaniasis in a pregnant immigrant with exuberant mucocutaneous lesions with favorable response to liposomal amphotericin B.