Syngeneic murine models with distinct immune microenvironments represent subsets of human intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a poorly immunogenic malignancy associated with limited survival. Syngeneic immunocompetent mouse models of CCA are an essential tool to elucidate the tumor immune microenvironment (TIME), understand mechanisms of tumor immune evasion, and test novel immunotherapeutic strategies. The scope of this study was to develop and characterize immunocompetent CCA models with distinct genetic drivers, and correlate tumor genomics, immunobiology, and therapeutic response. METHODS: A multifaceted approach including scRNA-seq, CITE-seq, whole exome and bulk RNA sequencing was employed. FDA-approved PD-1/PD-L1 antibodies were tested in humanized PD-1/PD-L1 mice (HuPD-H1). RESULTS: A genetic mouse model of intrahepatic CCA (iCCA) driven by intrabiliary transduction of Fbxw7ΔF/Akt that mimics human iCCA was generated. From the Fbxw7ΔF/Akt tumors, a murine cell line (FAC) and syngeneic model with genetic and phenotypic characteristics of human iCCA were developed. Established SB1 (YAPS127A/Akt) and KPPC (KrasG12Dp53L/L) models were compared to the FAC model. Although the models had transcriptomic similarities, they had substantial differences as well. Mutation patterns of FAC, SB1, and KPPC cells matched different mutational signatures in Western and Japanese CCA patient cohorts. KPPC tumors had a high tumor mutation burden. FAC tumors had a T cell-infiltrated TIME, while SB1 tumors had a preponderance of suppressive myeloid cells. FAC, SB1, and KPPC tumors matched different immune signatures in human iCCA cohorts. Moreover, FAC, SB1, and KPPC tumor-bearing HuPD-H1 mice displayed differential responses to nivolumab or durvalumab. CONCLUSIONS: Syngeneic iCCA models display a correlation between tumor genotype and TIME phenotype, with differential responses to FDA-approved immunotherapies. This study underscores the importance of leveraging multiple preclinical models to understand responses to immunotherapy in different genetic subsets of human CCA. IMPACT AND IMPLICATIONS: Understanding the relationship between tumor genotype and the phenotype of the immune microenvironment is an unmet need in cholangiocarcinoma (CCA). Herein, we use syngeneic murine models of intrahepatic CCA with different genetic drivers to demonstrate a correlation between tumor genotype and immune microenvironment phenotype in murine models, which is associated with differential responses to FDA-approved immunotherapies. This information will help guide other preclinical studies. Additionally, it emphasizes that immune checkpoint inhibition in patients with CCA is not a "one-size-fits-all" approach. Our observations suggest that, as for targeted therapies, patients should be stratified and selected for treatment according to their tumor genetics.

WITHDRAWN: Neoadjuvant therapy leads to objective response in intrahepatic cholangiocarcinoma.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, https://doi.org/10.1016/j.hpb.2024.04.011. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/policies/article-withdrawal.

Neoadjuvant therapy leads to objective response in intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is the second most common hepatic malignancy and has a poor prognosis. Surgical resection is the standard of care for patients with resectable disease, representing 30-40% of cases. Increasingly, neoadjuvant systemic therapy is being utilized in patients due to high-risk anatomic or biologic considerations. However, data on the clinical effect of this approach are limited. We performed a cohort study to evaluate the effect of neoadjuvant therapy in patients with oncologically high-risk iCCA. METHODS: iCCA patients (n = 181) between the years 2014-2020 were reviewed for clinical, histopathologic, treatment, and outcome-related data. Tumor regression grade was scored per CAP criteria for gastrointestinal carcinomas. RESULTS: 47 iCCA patients received neoadjuvant therapy and 72 did not. Neoadjuvant treatment led to objective response and tumor regression by CAP score. After adjustment for age, clinical stage, and tumor size, the outcomes of patients who had neoadjuvant therapy followed by surgery were not significantly different from those patients who had surgery first. DISCUSSION: In conclusion, neoadjuvant therapy in iCCA facilitated surgical care. The progression-free and overall survival for surgical patients with and without neoadjuvant therapy were not significantly different suggesting this approach needs further exploration as an effective treatment paradigm.

Tyrosine phosphorylation of YAP-1 in biliary epithelial cells mediates posthepatectomy liver regeneration and is affected by serotonin.

Experimental data suggested activation of yes-associated protein (YAP-1) as a critical regulator of liver regeneration (LR). Serotonin (5-HT) promotes LR in rodent models and has been proposed to act via YAP-1. How 5-HT affects LR is incompletely understood. A possible mechanism how 5-HT affects human LR was explored. Sixty-one patients were included. Tissue samples prior and 2 h after induction of LR were collected. Circulating levels of 5-HT and osteopontin (OPN) were assessed. YAP-1, its phosphorylation states, cytokeratin 19 (CK-19) and OPN were assessed using immunofluorescence. A mouse model of biliary epithelial cells (BECs) specific deletion of YAP/TAZ was developed. YAP-1 increased as early as 2 h after induction of LR (p = 0.025) predominantly in BECs. BEC specific deletion of YAP/TAZ reduced LR after 70% partial hepatectomy in mice (Ki67%, p < 0.001). SSRI treatment, depleting intra-platelet 5-HT, abolished YAP-1 and OPN induction upon LR. Portal vein 5-HT levels correlated with intrahepatic YAP-1 expression upon LR (R = 0.703, p = 0.035). OPN colocalized with YAP-1 in BECs and its circulating levels increased in the liver vein 2 h after induction of LR (p = 0.017). In the context of LR tyrosine-phosphorylated YAP-1 significantly increased (p = 0.042). Stimulating BECs with 5-HT resulted in increased YAP-1 activation via tyrosine-phosphorylation and subsequently increased OPN expression. BECs YAP-1 appears to be critical for LR in mice and humans. Our evidence suggests that 5-HT, at least in part, exerts its pro-regenerative effects via YAP-1 tyrosine-phosphorylation in BECs and subsequent OPN-dependent paracrine immunomodulation.

LCK inhibition downregulates YAP activity and is therapeutic in patient-derived models of cholangiocarcinoma.

BACKGROUND & AIMS: There is an unmet need to develop novel, effective medical therapies for cholangiocarcinoma (CCA). The Hippo pathway effector, Yes-associated protein (YAP), is oncogenic in CCA, but has historically been difficult to target therapeutically. Recently, we described a novel role for the LCK proto-oncogene, Src family tyrosine kinase (LCK) in activating YAP through tyrosine phosphorylation. This led to the hypothesis that LCK is a viable therapeutic target in CCA via regulation of YAP activity. METHODS: A novel tyrosine kinase inhibitor with relative selectivity for LCK, NTRC 0652-0, was pharmacodynamically profiled in vitro and in CCA cells. A panel of eight CCA patient-derived organoids were characterized and tested for sensitivity to NTRC 0652-0. Two patient-derived xenograft models bearing fibroblast growth factor receptor 2 (FGFR2)-rearrangements were utilized for in vivo assessment of pharmacokinetics, toxicity, and efficacy. RESULTS: NTRC 0652-0 demonstrated selectivity for LCK inhibition in vitro and in CCA cells. LCK inhibition with NTRC 0652-0 led to decreased tyrosine phosphorylation, nuclear localization, and co-transcriptional activity of YAP, and resulted in apoptotic cell death in CCA cell lines. A subset of tested patient-derived organoids demonstrated sensitivity to NTRC 0652-0. CCAs with FGFR2 fusions were identified as a potentially susceptible and clinically relevant genetic subset. In patient-derived xenograft models of FGFR2 fusion-positive CCA, daily oral treatment with NTRC 0652-0 resulted in stable plasma and tumor drug levels, acceptable toxicity, decreased YAP tyrosine phosphorylation, and significantly decreased tumor growth. CONCLUSIONS: A novel LCK inhibitor, NTRC 0652-0, inhibited YAP signaling and demonstrated preclinical efficacy in CCA cell lines, and patient-derived organoid and xenograft models. IMPACT AND IMPLICATIONS: Although aberrant YAP activation is frequently seen in CCA, YAP targeted therapies are not yet clinically available. Herein we show that a novel LCK-selective tyrosine kinase inhibitor (NTRC 0652-0) effectively inhibits YAP tyrosine phosphorylation and cotranscriptional activity and is well tolerated and cytotoxic in multiple preclinical models. The data suggest this approach may be effective in CCA with YAP dependence or FGFR2 fusions, and these findings warrant further investigation in phase I clinical trials.

Orthorexic eating behaviors related to exercise addiction and internal motivations in a sample of university students.

PURPOSE: This research explored the exercise tendencies and motivations of individuals varying in orthorexia symptomatology. METHOD: Participants were 411 university students, who completed the Eating Habits Questionnaire alongside measures of exercise activity and addiction in Study 1 (a modified version of the Leisure-Time Exercise Questionnaire, the Exercise Addiction Inventory, and the Compulsive Exercise Test) and various exercise motivations in Study 2 (the Behavioural Regulations in Exercise Questionnaire and the Exercise Motivations Inventory-2). RESULTS: Orthorexia symptomatology was positively correlated with aerobic and strength-training exercise levels; all measures of exercise addiction; all measures of internal exercise motivation; and nearly all measures of exercise motivation for the purposes of psychological, social, health, and body improvement. Symptomatology was not significantly related to either measure that specifically assessed external motivation to exercise. CONCLUSION: Individuals high in orthorexia symptomatology are internally driven to exercise for the purposes of improving their physical and mental health, but these strong motivations also lead to exercise addiction characterized by a compulsive need to follow a rigid schedule of intensive exercise even in the face of injury, illness, or other problems. LEVEL OF EVIDENCE: Level V, descriptive cross-sectional study.

Soluble vascular endothelial growth factor receptor 3 is essential for corneal alymphaticity.

Corneal transparency is a prerequisite for optimal vision and in turn relies on an absence of blood and lymphatic vessels, which is remarkable given the cornea's proximity to vascularized tissues. Membrane-bound vascular endothelial growth factor receptor 3 (VEGFR-3), with its cognate ligand vascular endothelial growth factor C (VEGF-C), is a major mediator of lymphangiogenesis. Here, we demonstrate that the cornea expresses a novel truncated isoform of this molecule, soluble VEGFR-3 (sVEGFR-3), which is critical for corneal alymphaticity, by sequestering VEGF-C. sVEGFR-3 binds and sequesters VEGF-C, thereby blocking signaling through VEGFR-3 and suppressing lymphangiogenesis induced by VEGF-C. sVEGFR-3 knockdown leads to lymphangiogenesis and hemangiogenesis in the mouse cornea, while overexpression of sVEGFR-3 inhibits lymphangiogenesis and hemangiogenesis in a murine suture injury model. Pax6(+/-) mice spontaneously develop corneal and lymphatic vessels and are deficient in sVEGFR-3. sVEGFR-3 suppresses hemangiogenesis by blocking VEGF-C-induced phosphorylation of VEGFR-2. Overexpression of sVEGFR-3 leads to a 5-fold increase in corneal transplant survival in mouse models. sVEGFR-3 holds promise as a molecule to control and regress lymphatic-vessel-based dysfunction. Therefore, sVEGFR-3 has the potential to protect the injured cornea from opacification secondary to infection, inflammation, or transplant rejection.

Heated humidified high-flow nasal cannula versus low-flow nasal cannula as weaning mode from nasal CPAP in infants ≤28 weeks of gestation.

UNLABELLED: Despite the paucity of evidence, the practice of weaning nasal continuous positive airway pressure (NCPAP) is widespread. However, the most clinically effective non-invasive ventilatory support strategy remains to be determined. We compared the outcome of very premature infants with respiratory distress syndrome treated with a combination of NCPAP and heated humidified high-flow nasal cannula (HHFNC) versus NCPAP and low-flow nasal cannula (LFNC). Between 2004 and 2008, patients ≤28 weeks of gestation and <1,250 g of birth weight were treated with NCPAP + HHFNC or NCPAP + LFNC. Their respiratory and non-respiratory outcome including cost-effectiveness was compared after matching for antenatal steroid doses, mode of delivery, birth plurality, gestational age, birth weight, gender, surfactant doses, length of mechanical ventilation and clinical risk index for babies-II (CRIB-II) score. Thirty-nine infants received HHFNC + NCPAP, and 40 received NCPAP + LFNC. Median gestational age and birth weight were 27 weeks and 930 g and 27 weeks and 980 g, respectively. The total number of NCPAP days was significantly reduced by 50 % in the HHFNC group. Thirteen percent of the patients on NCPAP suffered from nasal bridge lesions compared to none on HHFNC. Respiratory and non-respiratory outcome was not significantly different otherwise. Combination of NCPAP and HHFNC reduced costs by 33 %. CONCLUSIONS: HHFNC shortens NCPAP time without increasing overall length of non-invasive respiratory support in very preterm infants. Unlike NCPAP, HHFNC does not seem to increase the risk of nasal trauma and appears to improve cost-effectiveness whilst producing otherwise equal respiratory and non-respiratory outcomes.

A small molecule MST1/2 inhibitor accelerates murine liver regeneration with improved survival in models of steatohepatitis.

Dysfunctional liver regeneration following surgical resection remains a major cause of postoperative mortality and has no therapeutic options. Without targeted therapies, the current treatment paradigm relies on supportive therapy until homeostasis can be achieved. Pharmacologic acceleration of regeneration represents an alternative therapeutic avenue. Therefore, we aimed to generate a small molecule inhibitor that could accelerate liver regeneration with an emphasis on diseased models, which represent a significant portion of patients who require surgical resection and are often not studied. Utilizing a clinically approved small molecule inhibitor as a parent compound, standard medicinal chemistry approaches were utilized to generate a small molecule inhibitor targeting serine/threonine kinase 4/3 (MST1/2) with reduced off-target effects. This compound, mCLC846, was then applied to preclinical models of murine partial hepatectomy, which included models of diet-induced metabolic dysfunction-associated steatohepatitis (MASH). mCLC846 demonstrated on target inhibition of MST1/2 and reduced epidermal growth factor receptor inhibition. The inhibitory effects resulted in restored pancreatic beta-cell function and survival under diabetogenic conditions. Liver-specific cell-line exposure resulted in Yes-associated protein activation. Oral delivery of mCLC846 perioperatively resulted in accelerated murine liver regeneration and improved survival in diet-induced MASH models. Bulk transcriptional analysis of regenerating liver remnants suggested that mCLC846 enhanced the normal regenerative pathways and induced them following liver resection. Overall, pharmacological acceleration of liver regeneration with mCLC846 was feasible, had an acceptable therapeutic index, and provided a survival benefit in models of diet-induced MASH.

Checking Our Blind Spots: Examining Characteristics of Interviewees Versus Matriculants to a Hepatopancreatobiliary Surgical Fellowship Program.

OBJECTIVE: Racial and gender biases exist within academic surgery; bias negatively impacts patient care, reimbursement, student training, and staff retention. Few studies have investigated the potential for bias in surgical fellowship recruitment. We aimed to compare the racial and gender diversity at our hepatopancreatobiliary (HPB) surgery fellowship program to nationwide standards. We further aimed to investigate differences in the demographics of resident interviewees versus matriculants to our HPB fellowship. DESIGN: Retrospective review. SETTING: North American HPB fellowship training programs. PARTICIPANTS: Mayo Clinic's HPB surgery fellowship interviewees and North American HPB surgery fellowship graduates from 2013 to 2020. RESULTS: When compared to general surgery residency graduates during the study period (in 2019), a lower proportion of North American HPB surgery fellowship graduates were female (26% HPB fellowship graduates vs. 43.1% residents, p = 0.005), with no difference in proportion of racially under-represented in medicine (rURM) HPB fellowship graduates (10.7%) compared to rURM proportion of general surgery residents nationally (14.5%). There was an upward trend in female representation among North American HPB fellowship graduates from 11% in 2013 to 32% in 2020, but proportions of rURM HPB fellows remained steadily low. When comparing HPB interviewees at our institution to national general surgery residents, no differences were observed in proportions of female (34.4% interviewees vs. 43.1% residents, p = 0.17) or rURM (interviewees = 6.8%, residents = 14.5%, p = 0.09) applicants. Additionally, there was no significant difference between the proportion of female or rURM interviewees and matriculants to our HPB program. CONCLUSIONS: While fewer female graduating surgeons are pursuing HPB fellowship training than male graduates, this gender gap has narrowed over time. In contrast, the national percentage of rURM HPB fellowship graduates has remained low, mirroring stagnant proportions of rURM surgical residency graduates. When comparing HPB fellowship interviewees at our own institution to North American fellowship graduates, we observed similar proportions of female interviewees but lower proportions of rURM interviewees. Locally, these data will drive process change toward more intentional examination of our interview selection process. Nationally, more work is needed to increase the racial diversity of surgical residency and fellowship trainees to best reflect and serve our diverse patient populations.

Systematic Review of Procedural Skill Simulation in Health Care in Low- and Middle-Income Countries.

ABSTRACT: Low- and middle-income countries (LMICs) have adopted procedural skill simulation, with researchers increasingly investigating simulation efforts in resource-strained settings. We aim to summarize the current state of procedural skill simulation research in LMICs focusing on methodology, clinical area, types of outcomes and cost, cost-effectiveness, and overall sustainability. We performed a comprehensive literature review of original articles that assessed procedural skill simulation from database inception until April 2022.From 5371 screened articles, 262 were included in this review. All included studies were in English. Most studies were observational cohort studies (72.9%) and focused on obstetrics and neonatal medicine (32.4%). Most measured outcome was the process of task performance (56.5%). Several studies mentioned cost (38.9%) or sustainability (29.8%). However, few articles included actual monetary cost information (11.1%); only 1 article assessed cost-effectiveness. Based on our review, future research of procedural skill simulation in LMICS should focus on more rigorous research, cost assessments, and on less studied areas.

Post-injury outcomes of children with behavioral health disorders.

BACKGROUND: The impact of Behavioral Health Disorders (BHDs) on pediatric injury is poorly understood. We investigated the relationship between BHDs and outcomes following pediatric trauma. METHODS: We analyzed injured children (age 5-15) from 2014 to 2016 using the Pediatric Trauma Quality Improvement Program. The primary outcome was in-hospital mortality. Univariable and multivariable analyses compared children with and without a comorbid BHD. RESULTS: Of 69,305 injured children, 3,448 (5%) had a BHD. These 3,448 children had a median of 1 [IQR: 1, 1] BHD diagnosis: ADHD (n = 2491), major psychiatric disorder (n = 1037), drug use disorder (n = 250), and alcohol use disorder (n = 29). A higher proportion of injured children with BHDs suffered intentional and penetrating injury. Firearm injuries were more common for BHD patients (3% vs 1%, p<0.001). Children with BHDs were more likely to have an ISS>25 compared to children without (5% vs 3%, p<0.001). While median LOS was longer for BHD patients (2 [1, 3] vs 2 [1, 4], p<0.001), mortality was similar (1% vs 1%, p = 0.76) and complications were less frequent (7% vs 8%, p = 0.002). BHD was associated with lower risk of mortality (OR 0.45, 95%CI [0.30, 0.69]) after controlling for age, sex, race, trauma type, and injury intent and severity. CONCLUSION: Children with BHDs experienced lower in-hospital mortality risk after traumatic injury despite more severe injury upon presentation. Intentional and penetrating injuries are particularly concerning, and future work should assess prevention efforts in this vulnerable group.

SHP2 inhibition enhances Yes-associated protein-mediated liver regeneration in murine partial hepatectomy models.

Disrupted liver regeneration following hepatectomy represents an "undruggable" clinical challenge associated with poor patient outcomes. Yes-associated protein (YAP), a transcriptional coactivator that is repressed by the Hippo pathway, is instrumental in liver regeneration. We have previously described an alternative, Hippo-independent mechanism of YAP activation mediated by downregulation of protein tyrosine phosphatase nonreceptor type 11 (PTPN11, also known as SHP2) inhibition. Herein, we examined the effects of YAP activation with a selective SHP1/SHP2 inhibitor, NSC-87877, on liver regeneration in murine partial hepatectomy models. In our studies, NSC-87877 led to accelerated hepatocyte proliferation, improved liver regeneration, and decreased markers of injury following partial hepatectomy. The effects of NSC-87877 were lost in mice with hepatocyte-specific Yap/Taz deletion, and this demonstrated dependence on these molecules for the enhanced regenerative response. Furthermore, administration of NSC-87877 to murine models of nonalcoholic steatohepatitis was associated with improved survival and decreased markers of injury after hepatectomy. Evaluation of transcriptomic changes in the context of NSC-87877 administration revealed reduction in fibrotic signaling and augmentation of cell cycle signaling. Cytoprotective changes included downregulation of Nr4a1, an apoptosis inducer. Collectively, the data suggest that SHP2 inhibition induces a pro-proliferative and cytoprotective enhancement of liver regeneration dependent on YAP.

Lunar Crater Identification in Digital Images.

It is often necessary to identify a pattern of observed craters in a single image of the lunar surface and without any prior knowledge of the camera's location. This so-called "lost-in-space" crater identification problem is common in both crater-based terrain relative navigation (TRN) and in automatic registration of scientific imagery. Past work on crater identification has largely been based on heuristic schemes, with poor performance outside of a narrowly defined operating regime (e.g., nadir pointing images, small search areas). This work provides the first mathematically rigorous treatment of the general crater identification problem. It is shown when it is (and when it is not) possible to recognize a pattern of elliptical crater rims in an image formed by perspective projection. For the cases when it is possible to recognize a pattern, descriptors are developed using invariant theory that provably capture all of the viewpoint invariant information. These descriptors may be pre-computed for known crater patterns and placed in a searchable index for fast recognition. New techniques are also developed for computing pose from crater rim observations and for evaluating crater rim correspondences. These techniques are demonstrated on both synthetic and real images.

The search for lunar mantle rocks exposed on the surface of the Moon.

The lunar surface is ancient and well-preserved, recording Solar System history and planetary evolution processes. Ancient basin-scale impacts excavated lunar mantle rocks, which are expected to remain present on the surface. Sampling these rocks would provide insight into fundamental planetary processes, including differentiation and magmatic evolution. There is contention among lunar scientists as to what lithologies make up the upper lunar mantle, and where they may have been exposed on the surface. We review dynamical models of lunar differentiation in the context of recent experiments and spacecraft data, assessing candidate lithologies, their distribution, and implications for lunar evolution.

Optimized production and analysis of the staphylococcal multidrug efflux protein QacA.

The plasmid-encoded QacA multidrug transport protein confers high-level resistance to a range of commonly used antimicrobials and is carried by widespread clinical strains of the human pathogen Staphylococcus aureus making it a potential target for future drug therapies. In order to obtain a sufficient yield of QacA protein for structural and biophysical studies, an optimized strategy for QacA overexpression was developed. QacA expression, directed from several vector systems in Escherichia coli, was tested under various growth and induction conditions and a synthetic qacA gene, codon-optimized for expression in E. coli was developed. Despite the extreme hydrophobicity and potential toxicity of the QacA secondary transport protein, a strategy based on the pBAD expression system, yielding up to four milligrams of approximately 95% pure QacA protein per litre of liquid culture, was devised. Purified QacA protein was examined using circular dichroism spectroscopy and displayed a secondary structure akin to that predicted from in silico analyses. Additionally, detergent solubilized QacA protein was shown to bind its fluorescent substrate rhodamine 6G with micro-molar affinity using a fluorescence polarization-based binding assay, similar to other multidrug transport proteins. To check the applicability of the expression/purification system described for QacA to other staphylococcal secondary transporters, the gene encoding the TetA(K) tetracycline efflux protein, which was previously recalcitrant to overexpression, was incorporated into the pBAD-based system and shown to be readily produced at easily detectable levels. Therefore, this expression system could be of general use for the production of secondary transport proteins in E. coli.