3D histology reveals that immune response to pancreatic precancers is heterogeneous and depends on global pancreas structure.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer for which few effective therapies exist. Immunotherapies specifically are ineffective in pancreatic cancer, in part due to its unique stromal and immune microenvironment. Pancreatic intraepithelial neoplasia, or PanIN, is the main precursor lesion to PDAC. Recently it was discovered that PanINs are remarkably abundant in the grossly normal pancreas, suggesting that the vast majority will never progress to cancer. Here, through construction of 48 samples of cm3-sized human pancreas tissue, we profiled the immune microenvironment of 1,476 PanINs in 3D and at single-cell resolution to better understand the early evolution of the pancreatic tumor microenvironment and to determine how inflammation may play a role in cancer progression. We found that bulk pancreatic inflammation strongly correlates to PanIN cell fraction. We found that the immune response around PanINs is highly heterogeneous, with distinct immune hotspots and cold spots that appear and disappear in a span of tens of microns. Immune hotspots generally mark locations of higher grade of dysplasia or locations near acinar atrophy. The immune composition at these hotspots is dominated by naïve, cytotoxic, and regulatory T cells, cancer associated fibroblasts, and tumor associated macrophages, with little similarity to the immune composition around less-inflamed PanINs. By mapping FOXP3+ cells in 3D, we found that regulatory T cells are present at higher density in larger PanIN lesions compared to smaller PanINs, suggesting that the early initiation of PanINs may not exhibit an immunosuppressive response. This analysis demonstrates that while PanINs are common in the pancreases of most individuals, inflammation may play a pivotal role, both at the bulk and the microscopic scale, in demarcating regions of significance in cancer progression.

Micro-Computed Tomography Analysis of the Knee in Aged Dunkin-Hartley Guinea Pigs after Intra Articular Injection.

The purpose of this protocol is to guide researchers in performing a palpation-guided technique of intra-articular knee injection in guinea pigs and assessment using micro-computed tomography. Dunkin-Hartley guinea pigs are robust models for osteoarthritis research as they spontaneously develop osteoarthritis in their knees. Intra-articular drug delivery is a common method to study the effects of an investigational drug in vivo. In humans, therapeutic agents administered via intra-articular injection can offer pain relief and delay further progression of osteoarthritis. As with any species, the introduction of a needle into a joint space has the potential to cause injury, which can result in pain, lameness, or infection. Such adverse events can compromise animal welfare, confound study results, and necessitate additional animals to achieve study objectives. As such, it is imperative to develop proper injection techniques to prevent complications, especially in longitudinal studies that require multiple, repeated intra-articular injections. Using the presented methodology, five guinea pigs received bilateral knee injections under general anesthesia. Seven days after injection, animals were humanely euthanized for analysis of osteoarthritis severity. No adverse events occurred following anesthesia or knee injections, including limping, pain, or infection. X-ray micro-computed tomography analysis of the knee can detect pathologic changes associated with osteoarthritis. Micro-computed tomography data indicates osteoarthritis is more severe in older animals, as indicated by increased bone mineral density and trabecular thickness with age. These results are consistent with histologic changes and Modified Mankin scores, an established and widely used scoring system to assess arthritis severity in these same animals. This protocol can be utilized to refine intra-articular injections in guinea pigs.

Power-law growth models explain incidences and sizes of pancreatic cancer precursor lesions and confirm spatial genomic findings.

Pancreatic ductal adenocarcinoma is a rare but lethal cancer. Recent evidence suggests that pancreatic intraepithelial neoplasia (PanIN), a microscopic precursor lesion that gives rise to pancreatic cancer, is larger and more prevalent than previously believed. Better understanding of the growth-law dynamics of PanINs may improve our ability to understand how a miniscule fraction makes the transition to invasive cancer. Here, using three-dimensional tissue mapping, we analyzed >1000 PanINs and found that lesion size is distributed according to a power law. Our data suggest that in bulk, PanIN size can be predicted by general growth behavior without consideration for the heterogeneity of the pancreatic microenvironment or an individual's age, history, or lifestyle. Our models suggest that intraductal spread and fusing of lesions drive our observed size distribution. This analysis lays the groundwork for future mathematical modeling efforts integrating PanIN incidence, morphology, and molecular features to understand tumorigenesis and demonstrates the utility of combining experimental measurement with dynamic modeling in understanding tumorigenesis.

Surgical Protocol for Partial Heart Transplantation in Growing Piglets.

Partial heart transplantation is a new approach to deliver growing heart valve implants. Partial heart transplants differ from heart transplants because only the part of the heart containing the necessary heart valve is transplanted. This allows partial heart transplants to grow, similar to the valves in heart transplants. However, the transplant biology of partial heart transplantation remains unexplored. This is a critical barrier to progress of the field. Without knowledge about the specific transplant biology of partial heart transplantation, children with partial heart transplants are empirically treated like children with heart transplants because the valves in heart transplants are known to grow. In order to progress the field, an animal model for partial heart transplantation is necessary. Here, we contribute our surgical protocol for partial heart transplantation in growing piglets. All aspects of partial heart transplantation, including the donor procedure, the recipient procedure, and recipient perioperative care are described in detail. There are important nuances in the conduct of virtually all aspects of open heart surgery that differs in piglets from humans. Our surgical protocol, which is based on our experience with 34 piglets, will allow other investigators to leverage our experience to seek fundamental knowledge about the nature of partial heart transplants. This is significant because the partial heart transplant model in piglets is complex and very resource intensive.

PanIN or IPMN? Redefining Lesion Size in 3 Dimensions.

Pancreatic ductal adenocarcinoma (PDAC) develops from 2 known precursor lesions: a majority (∼85%) develops from pancreatic intraepithelial neoplasia (PanIN), and a minority develops from intraductal papillary mucinous neoplasms (IPMNs). Clinical classification of PanIN and IPMN relies on a combination of low-resolution, 3-dimensional (D) imaging (computed tomography, CT), and high-resolution, 2D imaging (histology). The definitions of PanIN and IPMN currently rely heavily on size. IPMNs are defined as macroscopic: generally >1.0 cm and visible in CT, and PanINs are defined as microscopic: generally <0.5 cm and not identifiable in CT. As 2D evaluation fails to take into account 3D structures, we hypothesized that this classification would fail in evaluation of high-resolution, 3D images. To characterize the size and prevalence of PanINs in 3D, 47 thick slabs of pancreas were harvested from grossly normal areas of pancreatic resections, excluding samples from individuals with a diagnosis of an IPMN. All patients but one underwent preoperative CT scans. Through construction of cellular resolution 3D maps, we identified >1400 ductal precursor lesions that met the 2D histologic size criteria of PanINs. We show that, when 3D space is considered, 25 of these lesions can be digitally sectioned to meet the 2D histologic size criterion of IPMN. Re-evaluation of the preoperative CT images of individuals found to possess these large precursor lesions showed that nearly half are visible on imaging. These findings demonstrate that the clinical classification of PanIN and IPMN fails in evaluation of high-resolution, 3D images, emphasizing the need for re-evaluation of classification guidelines that place significant weight on 2D assessment of 3D structures.

Bacteria Endocarditis Caused by Mycoplasma hyorhinis in a Juvenile, Immunosuppressed Pig (Sus scrofa domesticus) following Partial Heart Transplantation.

Domestic swine (Sus scrofa domesticus) are important translational models for cardiovascular transplant studies. This can be attributed to the anatomic and physiologic similarities of their cardiovascular system to humans. Transplant studies frequently employ clinically relevant immunosuppression regimens to prevent organ rejection postoperatively. Immunosuppression can lead to opportunistic infection, including presentations that are novel or poorly described in immunocompetent hosts. In this study, we describe the first case of Mycoplasma hyorhinis -induced endocarditis affecting the pulmonary valve in a juvenile, immunosuppressed pig following a partial heart transplantation procedure. Clinical signs of infection began at 15 d postoperation, were consistent with a variety of infectious agents, including Mycoplasma hyorhinis, and included lethargy, respiratory signs, and elevated white blood cell counts. By 28 d post procedure, lameness and soft tissue swelling around the left tarsus developed. Joint fluid obtained by arthrocentesis was PCR positive for Mycoplasma hyorhinis and negative for other tested pathogens. Despite antimicrobial treatment, the transplanted pulmonary valve developed leaflet thickening, stenosis, and insufficiency starting at 30 d after the procedure. At 86 d posttransplantation, the pig reached experimental endpoints and was humanely euthanized for necropsy and histopathology. The pulmonary valve had numerous dark red vegetative expansions of all 3 leaflets. Postmortem testing of a vegetative lesion was positive for Mycoplasma hyorhinis, confirming the etiologic agent responsible for endocarditis. Mycoplasma hyorhinis -induced endocarditis of an orthotopic transplanted pulmonary valve has yet to be described in swine. This case report demonstrates that infections following immunosuppression may present with novel or undercharacterized clinical signs.

3D genomic mapping reveals multifocality of human pancreatic precancers.

Pancreatic intraepithelial neoplasias (PanINs) are the most common precursors of pancreatic cancer, but their small size and inaccessibility in humans make them challenging to study1. Critically, the number, dimensions and connectivity of human PanINs remain largely unknown, precluding important insights into early cancer development. Here, we provide a microanatomical survey of human PanINs by analysing 46 large samples of grossly normal human pancreas with a machine-learning pipeline for quantitative 3D histological reconstruction at single-cell resolution. To elucidate genetic relationships between and within PanINs, we developed a workflow in which 3D modelling guides multi-region microdissection and targeted and whole-exome sequencing. From these samples, we calculated a mean burden of 13 PanINs per cm3 and extrapolated that the normal intact adult pancreas harbours hundreds of PanINs, almost all with oncogenic KRAS hotspot mutations. We found that most PanINs originate as independent clones with distinct somatic mutation profiles. Some spatially continuous PanINs were found to contain multiple KRAS mutations; computational and in situ analyses demonstrated that different KRAS mutations localize to distinct cell subpopulations within these neoplasms, indicating their polyclonal origins. The extensive multifocality and genetic heterogeneity of PanINs raises important questions about mechanisms that drive precancer initiation and confer differential progression risk in the human pancreas. This detailed 3D genomic mapping of molecular alterations in human PanINs provides an empirical foundation for early detection and rational interception of pancreatic cancer.

Inhibition of complement C3 prevents osteoarthritis progression in guinea pigs by blocking STAT1 activation.

Osteoarthritis (OA) is one of the leading causes of disability, affecting over 500 million adults worldwide. Previous studies have found that various inflammatory factors can contribute to the pathogenesis of OA, including complement factors in the synovial fluid of OA patients. However, the pathogenesis of this disease is still not known, and the only therapy of severe OA is total joint replacements. Total joint replacements are invasive, expensive, and affect quality of life. Here we show that when human articular chondrocytes are stimulated with pro-inflammatory mediator interleukin-1ß (IL-1ß) there is an increase in inflammatory factors including complement component 3 (C3). We also found the transcription factor, signal transducer and activator of transcription 1 (STAT1), is responsible for increased C3 expression after IL-1ß stimulation in human articular chondrocytes. A specific STAT1 inhibitor, fludarabine, attenuates the hyper-expression of C3 and delays/prevents spontaneous OA in Dunkin-Hartley guinea pigs. Since fludarabine is already clinically used for chemotherapy, this study has great translational potential as a unique disease-modifying osteoarthritis drug (DMOAD) in treating primary OA.

Establishing New Isosexual Pairs in Adult Male Guinea Pigs (Cavia porcellus) to Facilitate Social Housing.

Guinea pigs (Cavia porcellus) are a commonly used species in biomedical research. As social creatures, compatible guinea pigs should be housed together unless scientific objectives or veterinary care require otherwise. Extensive literature suggests that adult male guinea pigs are highly aggressive in the presence of females, but data are lacking regarding the compatibility of cohoused adult males in the absence of females. Most studies that use adult males do not report housing densities. We used serial wound scoring and observations of behavior to determine whether unfamiliar adult male guinea pigs will develop stable, prosocial isosexual pairs. Wound scoring was performed before and 24 h after pairing. Serial behavioral observations assessed affiliative and agonistic behaviors at 0.5, 2, 24, and 48 h after pairing. Wound scoring and behavioral observations continued weekly for 1 mo and monthly thereafter. Wound scores were significantly higher at 24 h after pairing as compared with baseline and all other time points. Wounding was rare after week 2, indicating reduced aggression. Furthermore, affiliative behaviors significantly increased over time while agonistic behaviors were rare. Together, these data suggest that unfamiliar adult male guinea pigs establish stable prosocial pairs after an acclimation period. As was done in the present study, providing ample space, separate shelters for each animal, and the absence of female guinea pigs will likely facilitate successful pairing. We recommend consideration of a social housing program for adult male guinea pigs to provide companionship and enrich their housing environment.

Three-dimensional assessments are necessary to determine the true, spatially-resolved composition of tissues.

Methods for spatially resolved cellular profiling using thinly cut sections have enabled in-depth quantitative tissue mapping to study inter-sample and intra-sample differences in normal human anatomy and disease onset and progression. These methods often profile extremely limited regions, which may impact the evaluation of heterogeneity due to tissue sub-sampling. Here, we applied CODA, a deep learning-based tissue mapping platform, to reconstruct the three-dimensional (3D) microanatomy of grossly normal and cancer-containing human pancreas biospecimens obtained from individuals who underwent pancreatic resection. To compare inter- and intra-sample heterogeneity, we assessed bulk and spatially resolved tissue composition in a cohort of two-dimensional (2D) whole slide images (WSIs) and a cohort of thick slabs of pancreas tissue that were digitally reconstructed in 3D from serial sections. To demonstrate the marked under sampling of 2D assessments, we simulated the number of WSIs and tissue microarrays (TMAs) necessary to represent the compositional heterogeneity of 3D data within 10% error to reveal that tens of WSIs and hundreds of TMA cores are sometimes needed. We show that spatial correlation of different pancreatic structures decay significantly within a span of microns, demonstrating that 2D histological sections may not be representative of their neighboring tissues. In sum, we demonstrate that 3D assessments are necessary to accurately assess tissue composition in normal and abnormal specimens and in order to accurately determine neoplastic content. These results emphasize the importance of intra-sample heterogeneity in tissue mapping efforts.

Power-law growth models explain incidences and sizes of pancreatic cancer precursor lesions and confirm spatial genomic findings.

Pancreatic ductal adenocarcinoma is a rare but lethal cancer. Recent evidence reveals that pancreatic intraepithelial neoplasms (PanINs), the microscopic precursor lesions in the pancreatic ducts that can give rise to invasive pancreatic cancer, are significantly larger and more prevalent than previously believed. Better understanding of the growth law dynamics of PanINs may improve our ability to understand how a miniscule fraction of these lesions makes the transition to invasive cancer. Here, using artificial intelligence (AI)-based three-dimensional (3D) tissue mapping method, we measured the volumes of >1,000 PanIN and found that lesion size is distributed according to a power law with a fitted exponent of -1.7 over > 3 orders of magnitude. Our data also suggest that PanIN growth is not very sensitive to the pancreatic microenvironment or an individual's age, family history, and lifestyle, and is rather shaped by general growth behavior. We analyze several models of PanIN growth and fit the predicted size distributions to the observed data. The best fitting models suggest that both intraductal spread of PanIN lesions and fusing of multiple lesions into large, highly branched structures drive PanIN growth patterns. This work lays the groundwork for future mathematical modeling efforts integrating PanIN incidence, morphology, genomic, and transcriptomic features to understand pancreas tumorigenesis, and demonstrates the utility of combining experimental measurement of human tissues with dynamic modeling for understanding cancer tumorigenesis.

Three-dimensional genomic mapping of human pancreatic tissue reveals striking multifocality and genetic heterogeneity in precancerous lesions.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS, but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia.

Tissue clearing and 3D reconstruction of digitized, serially sectioned slides provide novel insights into pancreatic cancer.

Pancreatic cancer is currently the third leading cause of cancer death in the United States. The clinical hallmarks of this disease include abdominal pain that radiates to the back, the presence of a hypoenhancing intrapancreatic lesion on imaging, and widespread liver metastases. Technologies such as tissue clearing and three-dimensional (3D) reconstruction of digitized serially sectioned hematoxylin and eosin-stained slides can be used to visualize large (up to 2- to 3-centimeter cube) tissues at cellular resolution. When applied to human pancreatic cancers, these 3D visualization techniques have provided novel insights into the basis of a number of the clinical characteristics of this disease. Here, we describe the clinical features of pancreatic cancer, review techniques for clearing and the 3D reconstruction of digitized microscope slides, and provide examples that illustrate how 3D visualization of human pancreatic cancer at the microscopic level has revealed features not apparent in 2D microscopy and, in so doing, has closed the gap between bench and bedside. Compared with animal models and 2D microscopy, studies of human tissues in 3D can reveal the difference between what can happen and what does happen in human cancers.

Intraparenchymal metastases as a cause for local recurrence of pancreatic cancer.

Using CODA, a technique for three-dimensional reconstruction of large tissues, Kiemen et al. report observation of a microscopic focus of pancreatic cancer found in the vasculature of grossly normal human pancreas tissue resected adjacent to a large tumour. They use TP53 and SMAD4 staining to relate the small focus to the primary tumour. This report describes a represents a probable case of intraparenchymal metastasis of pancreatic cancer, revealing a probable cause of local recurrence.

CODA: quantitative 3D reconstruction of large tissues at cellular resolution.

A central challenge in biology is obtaining high-content, high-resolution information while analyzing tissue samples at volumes relevant to disease progression. We address this here with CODA, a method to reconstruct exceptionally large (up to multicentimeter cubed) tissues at subcellular resolution using serially sectioned hematoxylin and eosin-stained tissue sections. Here we demonstrate CODA's ability to reconstruct three-dimensional (3D) distinct microanatomical structures in pancreas, skin, lung and liver tissues. CODA allows creation of readily quantifiable tissue volumes amenable to biological research. As a testbed, we assess the microanatomy of the human pancreas during tumorigenesis within the branching pancreatic ductal system, labeling ten distinct structures to examine heterogeneity and structural transformation during neoplastic progression. We show that pancreatic precancerous lesions develop into distinct 3D morphological phenotypes and that pancreatic cancer tends to spread far from the bulk tumor along collagen fibers that are highly aligned to the 3D curves of ductal, lobular, vascular and neural structures. Thus, CODA establishes a means to transform broadly the structural study of human diseases through exploration of exhaustively labeled 3D microarchitecture.

Progression and Resolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection in Golden Syrian Hamsters.

To catalyze severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research, including development of novel interventive and preventive strategies, the progression of disease was characterized in a robust coronavirus disease 2019 (COVID-19) animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at 2, 4, 7, 14, and 28 days after inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal time points, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 after inoculation, corresponding with widespread necrosis and inflammation. At day 7, when the presence of infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 after inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

Hamsters as a Model of Severe Acute Respiratory Syndrome Coronavirus-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), rapidly spread across the world in late 2019, leading to a pandemic. While SARS-CoV-2 infections predominately affect the respiratory system, severe infections can lead to renal and cardiac injury and even death. Due to its highly transmissible nature and severe health implications, animal models of SARS-CoV-2 are critical to developing novel therapeutics and preventatives. Syrian hamsters (Mesocricetus auratus) are an ideal animal model of SARS-CoV-2 infections because they recapitulate many aspects of human infections. After inoculation with SARS-CoV-2, hamsters become moribund, lose weight, and show varying degrees of respiratory disease, lethargy, and ruffled fur. Histopathologically, their pulmonary lesions are consistent with human infections including interstitial to broncho-interstitial pneumonia, alveolar hemorrhage and edema, and granulocyte infiltration. Similar to humans, the duration of clinical signs and pulmonary pathology are short lived with rapid recovery by 14 d after infection. Immunocompromised hamsters develop more severe infections and mortality. Preclinical studies in hamsters have shown efficacy of therapeutics, including convalescent serum treatment, and preventatives, including vaccination, in limiting or preventing clinical disease. Although hamster studies have contributed greatly to our understanding of the pathogenesis and progression of disease after SARS-CoV-2 infection, additional studies are required to better characterize the effects of age, sex, and virus variants on clinical outcomes in hamsters. This review aims to describe key findings from studies of hamsters infected with SARS-CoV-2 and to highlight areas that need further investigation.

Sex Differences in Lung Imaging and SARS-CoV-2 Antibody Responses in a COVID-19 Golden Syrian Hamster Model.

In the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males than in females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8 to 10 weeks of age) were inoculated intranasally with 105 50% tissue culture infective dose (TCID50) of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developed more extensive pneumonia as noted on chest computed tomography, and recovered more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including interferon-ß (IFN-ß) and tumor necrosis factor-α (TNF-α), were comparable between the sexes. However, during the recovery phase of infection, females mounted 2-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole-inactivated SARS-CoV-2 and mutant S-RBDs as well as virus-neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2-associated sex differences seen in the human population. IMPORTANCE Men experience more severe outcomes from coronavirus disease 2019 (COVID-19) than women. Golden Syrian hamsters were used to explore sex differences in the pathogenesis of a human isolate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). After inoculation, male hamsters experienced greater sickness, developed more severe lung pathology, and recovered more slowly than females. Sex differences in disease could not be reversed by estradiol treatment in males and were not explained by either virus replication kinetics or the concentrations of inflammatory cytokines in the lungs. During the recovery period, antiviral antibody responses in the respiratory tract and plasma, including to newly emerging SARS-CoV-2 variants, were greater in female than in male hamsters. Greater lung pathology during the acute phase combined with lower antiviral antibody responses during the recovery phase of infection in males than in females illustrate the utility of golden Syrian hamsters as a model to explore sex differences in the pathogenesis of SARS-CoV-2 and vaccine-induced immunity and protection.

Sex differences in lung imaging and SARS-CoV-2 antibody responses in a COVID-19 golden Syrian hamster model.

In the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males compared with females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8-10 weeks of age) were inoculated intranasally with 10 5 TCID 50 of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developing more extensive pneumonia as noted on chest computed tomography, and recovering more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including IFNb and TNFa, were comparable between the sexes. However, during the recovery phase of infection, females mounted two-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole inactivated SARS-CoV-2 and mutant S-RBDs, as well as virus neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2 associated sex differences seen in the human population.

Multiregion whole-exome sequencing of intraductal papillary mucinous neoplasms reveals frequent somatic KLF4 mutations predominantly in low-grade regions.

OBJECTIVE: Intraductal papillary mucinous neoplasms (IPMNs) are non-invasive precursor lesions that can progress to invasive pancreatic cancer and are classified as low-grade or high-grade based on the morphology of the neoplastic epithelium. We aimed to compare genetic alterations in low-grade and high-grade regions of the same IPMN in order to identify molecular alterations underlying neoplastic progression. DESIGN: We performed multiregion whole exome sequencing on tissue samples from 17 IPMNs with both low-grade and high-grade dysplasia (76 IPMN regions, including 49 from low-grade dysplasia and 27 from high-grade dysplasia). We reconstructed the phylogeny for each case, and we assessed mutations in a novel driver gene in an independent cohort of 63 IPMN cyst fluid samples. RESULTS: Our multiregion whole exome sequencing identified KLF4, a previously unreported genetic driver of IPMN tumorigenesis, with hotspot mutations in one of two codons identified in >50% of the analyzed IPMNs. Mutations in KLF4 were significantly more prevalent in low-grade regions in our sequenced cases. Phylogenetic analyses of whole exome sequencing data demonstrated diverse patterns of IPMN initiation and progression. Hotspot mutations in KLF4 were also identified in an independent cohort of IPMN cyst fluid samples, again with a significantly higher prevalence in low-grade IPMNs. CONCLUSION: Hotspot mutations in KLF4 occur at high prevalence in IPMNs. Unique among pancreatic driver genes, KLF4 mutations are enriched in low-grade IPMNs. These data highlight distinct molecular features of low-grade and high-grade dysplasia and suggest diverse pathways to high-grade dysplasia via the IPMN pathway.