Disruption of Circadian Clock Induces Abnormal Mammary Morphology and Aggressive Basal Tumorigenesis by Enhancing LILRB4 Signaling.

Epidemiological studies have shown that circadian rhythm disruption (CRD) is associated with the risk of breast cancer. However, the role of CRD in mammary gland morphology and aggressive basal mammary tumorigenesis and the molecular mechanisms underlying CRD and cancer risk remain unknown. To investigate the effect of CRD on aggressive tumorigenesis, a genetically engineered mouse model that recapitulates the human basal type of breast cancer was used for this study. The effect of CRD on mammary gland morphology was investigated using wild-type mice model. The impact of CRD on the tumor microenvironment was investigated using the tumors from LD12:12 and CRD mice via scRNA seq. ScRNA seq was substantiated by multiplexing immunostaining, flow cytometry, and realtime PCR. The effect of LILRB4 immunotherapy on CRD-induced tumorigenesis was also investigated. Here we identified the impact of CRD on basal tumorigenesis and mammary gland morphology and identified the role of LILRB4 on CRD-induced lung metastasis. We found that chronic CRD disrupted mouse mammary gland morphology and increased tumor burden, and lung metastasis and induced an immunosuppressive tumor microenvironment by enhancing LILRB4a expression. Moreover, CRD increased the M2-macrophage and regulatory T-cell populations but decreased the M1-macrophage populations. Furthermore, targeted immunotherapy against LILRB4 reduced CRD-induced immunosuppressive microenvironment and lung metastasis. These findings identify and implicate LILRB4a as a link between CRD and aggressive mammary tumorigenesis. This study also establishes the potential role of the targeted LILRB4a immunotherapy as an inhibitor of CRD-induced lung metastasis.

Beyond Trauma: High-Volume Critical Care Medicine in a Military Medical Center-Based Military-Civilian Partnership.

INTRODUCTION: Critical Care Internal Medicine (CCIM) is vital to the U.S. Military as evidenced by the role CCIM played in the COVID-19 pandemic response and wartime operations. Although the proficiency needs of military surgeons have been well studied, this has not been the case for CCIM. The objective of this study was to compare the patient volume and acuity of military CCIM physicians working solely at Military Treatment Facilities (MTFs) with those at MTFs also working part-time in a military-civilian partnership (MCP) at the University Medical Center of Southern Nevada (UMC). MATERIALS AND METHODS: We analyzed FY2019 critical care coding data from the Military Health System and UMC comparing the number of critical care encounters, the number of high-acuity critical care encounters, and the Abilities/Activity component of the Knowledge, Skills, and Abilities/Clinical Activity (KSA) score. This analysis was restricted to critical care encounters defined by Current Procedural Terminology codes for critical care (99291 and 99292). A critical care encounter was considered high acuity if the patient had ICD-10 codes for shock, respiratory failure, or cardiac arrest or had at least three codes for critical care in the same episode. RESULTS: The five AF CCIM physicians in the MCP group performed 2,019 critical care encounters in 206 days, with 63.1% (1,273) being defined as high acuity. The total number of MTF critical care encounters was 16,855 across all providers and services, with 28.9% (4,864) of encounters defined as high acuity. When limited to CCIM encounters, MTFs had 6,785 critical care encounters, with 32.0% being high acuity (2,171). Thus, the five AF CCIM physicians, while working 206 days at the UMC, equated to 12.0% (2,019/16,855) of the total critical care MTF encounters, 27.2% (1,273/4,684) of the total high-acuity MTF critical care encounters, and 29.8% (2,019/6,785) of the MTF CCIM encounters, with 58.6% (1,273/2,171) of the MTF CCIM high-acuity encounters.The USAF CCIM physicians in the MCP group performed 454,395 KSAs in 206 days, with a KSA density per day of 2,206. In the MTF group, CCIM providers generated 2,344,791 total KSAs over 10,287 days, with a KSA density per day of 227.9. Thus, the five CCIM physicians at the UMC accounted for 19.38% of the MTF CCIM KSAs, with a KSA density over 10 times higher (2,206 vs. 227.9). CONCLUSIONS: The volume and acuity of critical care at MTFs may be insufficient to maintain CCIM proficiency under the current system. Military-civilian partnerships are invaluable in maintaining clinical proficiency for military CCIM physicians and can be done on a part-time basis while maintaining beneficiary care at an MTF. Future CCIM expeditionary success is contingent on CCIM physicians and team members having the required CCIM exposure to grow and maintain clinical proficiency.Limitations of this study include the absence of off-duty employment (moonlighting) data and difficulty filtering military data down to just CCIM physicians, which likely caused the MTF CCIM data to be overestimated.

Career practitioners' response to career development concerns in the time of COVID-19.

The COVID-19 pandemic has had a world-wide impact on all areas of individuals' health, including physical, psychological, financial, familial, social, and vocational. In the United States, the unemployment rate rose from 3.5% (5.8 million) to 13.3% (21 million) in May 2020 before dropping to 7.9% in October 2020. Cognitive information processing (CIP)is one career theory that addresses career needs of clients and society. In this article, we examine the impact of COVID-19 on mental health and wellness, highlight differences for marginalized groups, and demonstrate how CIP theoretical elements may have been impacted by COVID-19, and provide strategies enhancing client growth in these domains during a time when largescale social and physical distancing is recommended. The CIP-based differentiated service delivery model is also described as a means for extending and providing access to career services.

Telangiectasia Macularis Eruptiva Perstans of the Scalp Mimicking Angiosarcoma.

ABSTRACT: We present a case of a 74-year-old man with marked photodamage who was ultimately diagnosed with telangiectasia macularis eruptiva perstans (TMEP) of the scalp. The diagnosis was made more difficult because of the clinical and histological similarity of this case with an early angiosarcoma. TMEP is a benign and indolent rare subtype of cutaneous mastocytosis presenting clinically with red-brown telangiectatic macules, usually symmetrically distributed over the trunk and extremities. Although most cases are limited to the skin, systemic involvement can occur, and this can be a potentially life-threatening disease. Although also rare, in contrast to TMEP, cutaneous angiosarcoma is a highly malignant vascular tumor with a poor prognosis. This case highlights the importance of including TMEP on the differential diagnosis where vascular lesions of the scalp are observed.

Age-associated mitochondrial DNA mutations cause metabolic remodelling that contributes to accelerated intestinal tumorigenesis.

Oxidative phosphorylation (OXPHOS) defects caused by somatic mitochondrial DNA (mtDNA) mutations increase with age in human colorectal epithelium and are prevalent in colorectal tumours, but whether they actively contribute to tumorigenesis remains unknown. Here we demonstrate that mtDNA mutations causing OXPHOS defects are enriched during the human adenoma/carcinoma sequence, suggesting they may confer a metabolic advantage. To test this we deleted the tumour suppressor Apc in OXPHOS deficient intestinal stem cells in mice. The resulting tumours were larger than in control mice due to accelerated cell proliferation and reduced apoptosis. We show that both normal crypts and tumours undergo metabolic remodelling in response to OXPHOS deficiency by upregulating the de novo serine synthesis pathway (SSP). Moreover, normal human colonic crypts upregulate the SSP in response to OXPHOS deficiency prior to tumorigenesis. Our data show that age-associated OXPHOS deficiency causes metabolic remodelling that can functionally contribute to accelerated intestinal cancer development.

Synergy between EphA2-ILs-DTXp, a Novel EphA2-Targeted Nanoliposomal Taxane, and PD-1 Inhibitors in Preclinical Tumor Models.

Combinations of chemotherapy with immunotherapy have seen recent clinical success, including two approvals of anti-PD-1/L1 agents in combination with taxane-based chemotherapy in non-small cell lung cancer and triple-negative breast cancer. Here, we present a study on the combination activity and mechanistic rationale of a novel EphA2-targeted liposomal taxane (EphA2-ILs-DTXp) and anti-PD-1. This combination was highly active in mouse syngeneic tumor models, with complete responses observed in 3 of 5 models. In the EMT-6 tumor model, combination of EphA2-ILs-DTXp with anti-PD-1 resulted in a 60% complete response rate, with durable responses that were resistant to rechallenge. These responses were not observed in the absence of CD8+ T cells. Characterization of the immune infiltrates in EMT-6 tumors reveals increased CD8+ T cells, increased CD8+ IFNγ+ CTLs, and an increased CD8/regulatory T-cell (Treg) ratio. These immunomodulatory effects were not observed in mice treated with a combination of docetaxel and anti-PD-1. Pharmacokinetic analysis revealed that the AUC of docetaxel was increased 15 times, from 52.1 to 785 ng/mL/hour, when delivered by EphA2-ILs-DTXp. A dose reduction study of EphA2-ILs-DTXp showed a dose-response relationship for both tumor growth inhibition and the CD8/Treg ratio. Our data indicate that synergism between docetaxel and anti-PD-1 is achievable with nanoliposomal delivery.

Antibody-mediated targeting of TNFR2 activates CD8+ T cells in mice and promotes antitumor immunity.

Tumor necrosis factor receptor 2 (TNFR2) is the alternate receptor for TNF and can mediate both pro- and anti-inflammatory activities of T cells. Although TNFR2 has been linked to enhanced suppressive activity of regulatory T cells (Tregs) in autoimmune diseases, the viability of TNFR2 as a target for cancer immunotherapy has been underappreciated. Here, we show that new murine monoclonal anti-TNFR2 antibodies yield robust antitumor activity and durable protective memory in multiple mouse cancer cell line models. The antibodies mediate potent Fc-dependent T cell costimulation and do not result in significant depletion of Tregs Corresponding human agonistic monoclonal anti-TNFR2 antibodies were identified and also had antitumor effects in humanized mouse models. Anti-TNFR2 antibodies could be developed as a novel treatment option for patients with cancer.

Critical Appraisal of Programmed Death Ligand 1 Reflex Diagnostic Testing: Current Standards and Future Opportunities.

INTRODUCTION: Patient suitability to anti-programmed death ligand 1 (PD-L1) immune checkpoint inhibition is key to the treatment of NSCLC. We present, applied to PD-L1 testing: a comprehensive cross-validation of two immunohistochemistry (IHC) clones; our descriptive experience in diagnostic reflex testing; the concordance of IHC to in situ RNA (RNA-ISH); and application of digital pathology. METHODS: Eight hundred thirteen NSCLC tumor samples collected from 564 diagnostic samples were analyzed prospectively, and 249 diagnostic samples analyzed retrospectively in tissue microarray format. Validated methods for IHC and RNA-ISH were tested in tissue microarrays and full sections and the QuPath system were used for digital pathology analysis. RESULTS: Antibody concordance of clones SP263 and 22C3 validation was 97% to 98% in squamous cell carcinoma and adenocarcinomas, respectively. Clinical NSCLC cases were reported as PD-L1-negative (48%), 1% to 49% (23%), and more than 50% (29%), with differences associated to tissue-type and EGFR status. Comparison of IHC and RNA-ISH was highly concordant in both subgroups. Comparison of digital assessment versus manual assessment was highly concordant. Discrepancies were mostly around the 1% clinical threshold. Challenging IHC interpretation included 1) calculating the total tumor cell denominator and the nature of PD-L1 expressing cell aggregates in cytology samples; 2) peritumoral expression of positive immune cells; 3) calculation of positive tumor percentages around clinical thresholds; and 4) relevance of the 100 malignant cell rule. CONCLUSIONS: Sample type and EGFR status dictate differences in the expected percentage of PD-L1 expression. Analysis of PD-L1 is challenging, and interpretative guidelines are discussed. PD-L1 evaluations by RNA-ISH and digital pathology appear reliable, particularly in adenocarcinomas.

Collagen VII deficient mice show morphologic and histologic corneal changes that phenotypically mimic human dystrophic epidermolysis bullosa of the eye.

BACKGROUND: Absence of collagen VII causes blistering of the skin, eyes and many other tissues. This disease is termed dystrophic epidermolysis bullosa (DEB). Corneal fibrosis occurs in up to 41% and vision loss in up to 64% of patients. Standard treatments are supportive and there is no cure. The hypomorphic mouse model for DEB shows production of collagen VII at 10% of wild type levels in skin and spleen, but the eyes have not been described. Our purpose is to characterize the corneas to determine if this is an appropriate model for study of ocular therapeutics. METHODS: Western blot analysis (WB) and immunohistochemistry (IHC) were performed to assess presence and location of collagen VII protein within the hypomorphic mouse cornea. Additional IHC for inflammatory and fibrotic biomarkers transforming growth factor-beta-1 (TGF-ß1), alpha-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), proteinase 3, tenascin C and collagen III were performed. Clinical photographs documenting corneal opacification were assessed and scored independently by 2 examiners. Histology was then used to investigate morphologic changes. RESULTS: IHC and WB confirmed that hypomorphic mice produce less collagen VII production at the level of the basement membrane when compared with wild-types. IHC showed anomalous deposition of collagen III throughout the stroma. Of the 5 biomarkers tested, TGF-ß1 showed the strongest and most consistently staining. Photographs documented corneal opacities only in mice older than 10 weeks, opacities were not seen in younger animals. Histology showed multiple abnormalities, including epithelial hyperplasia, ulceration, fibrosis, edema, dysplasia, neovascularization and bullae formation. CONCLUSIONS: The collagen VII hypomorphic mouse shows reduced collagen VII production at the level of the corneal basement membrane. Corneal changes are similar to pathology seen in humans with this disease. The presence of anomalous stromal collagen III and TGF-ß1 appear to be the most consistent and strongest staining biomarkers in diseased mice. This mouse appears to mimic human corneal disease. It is an appropriate model for testing of therapeutics to treat EB ocular disease.

Use of open and endovascular surgical techniques to manage vascular injuries in the trauma setting: A review of the American Association for the Surgery of Trauma PROspective Observational Vascular Injury Trial registry.

BACKGROUND: Vascular trauma data have been submitted to the American Association for the Surgery of Trauma PROspective Observational Vascular Injury Trial (PROOVIT) database since 2013. We present data to describe current use of endovascular surgery in vascular trauma. METHODS: Registry data from March 2013 to December 2016 were reviewed. All trauma patients who had an injury to a named artery, except the forearm and lower leg, were included. Arteries were grouped into anatomic regions and by compressible and noncompressible region for analysis. This review focused on patients with noncompressible transection, partial transection, or flow-limiting defect injuries. Bivariate and multivariate analyses were used to assess the relationships between study variables. RESULTS: One thousand one hundred forty-three patients from 22 institutions were included. Median age was 32 years (interquartile range, 23-48) and 76% (n = 871) were male. Mechanisms of injury were 49% (n = 561) blunt, 41% (n = 464) penetrating, and 1.8% (n = 21) of mixed aetiology. Gunshot wounds accounted for 73% (n = 341) of all penetrating injuries. Endovascular techniques were used least often in limb trauma and most commonly in patients with blunt injuries to more than one region. Penetrating wounds to any region were preferentially treated with open surgery (74%, n = 341/459). The most common indication for endovascular treatment was blunt noncompressible torso injuries. These patients had higher Injury Severity Scores and longer associated hospital stays, but required less packed red blood cells, and had lower in hospital mortality than those treated with open surgery. On multivariate analysis, admission low hemoglobin concentration and abdominal injury were independent predictors of mortality. CONCLUSION: Our review of PROOVIT registry data demonstrates a high utilization of endovascular therapy among severely injured blunt trauma patients primarily with noncompressible torso hemorrhage. This is associated with a decreased need for blood transfusion and improved survival despite longer length of stay. LEVEL OF EVIDENCE: Therapeutic/care management, level III.

Comparison of direct site endovascular repair utilizing expandable polytetrafluoroethylene stent grafts versus standard vascular shunts in a porcine (Sus scrofa) model.

INTRODUCTION: The small diameter of temporary vascular shunts for vascular trauma management may restrict flow and result in ischemia or early thrombosis. We have previously reported a clinical experience with direct, open surgical reconstruction using expandable polytetrafluoroethylene stent grafts to create a "sutureless" anastomosis as an alternative to standard temporary vascular shunts. We sought to characterize patency and flow characteristics of these grafts compared with standard shunts in a survival model of porcine vascular injury. METHODS: Twelve Yorkshire-cross swine received a 2-cm-long near-circumferential defect in the bilateral iliac arteries. A 14 Fr Argyle shunt was inserted into one randomly assigned artery, with a self-expanding expandable polytetrafluoroethylene stent deployed in the other. At 72 hours, conduit patency was evaluated by angiography. Arterial flow measurements were obtained at baseline, immediately after intervention, and after 72 hours via direct measurement with perivascular flow meters. Blood pressure proximal and distal to the conduits and arterial samples for histopathology were obtained during the terminal procedure. RESULTS: Angiography revealed no difference in patency at 72 hours (p = 1.0). While there was no difference in baseline arterial flow between arteries (p = 0.63), the stent grafts demonstrated significantly improved blood flow compared with shunts both immediately after intervention (390 ± 36 mL/min vs. 265 ± 25 mL/min, p = 0.002) and at 72 hours (261 ± 29 mL/min vs. 170 ± 36 mL/min, p = 0.005). The pressure gradient across the shunts was greater than that of the stent grafts (11.5 mm Hg [interquartile range, 3-19 mm Hg] vs. 3 mm Hg [interquartile range, 3-5 mm Hg], p = 0.013). The speed of deployment was similar between the two devices. CONCLUSIONS: Open "sutureless" direct site repair using commercially available stent grafts to treat vascular injury is a technically feasible strategy for damage control management of peripheral vascular injury and offers increased blood flow when compared with temporary shunts. Furthermore, stent grafts may offer improved durability to extend the window until definitive vascular repair. The combination of these traits may improve outcomes after vascular injury. LEVEL OF EVIDENCE: Epidemiologic/Prognostic, level III.

Direct-site endovascular repair (DSER): A novel approach to vascular trauma.

Peripheral vascular injuries carry significant risk for permanent functional impairment, limb loss, and death. Definitive correction of these injuries requires significant operative time and has traditionally been resource and skill set intensive. In the initial surgical treatment of the physiologically depleted trauma patient, faster techniques may prove more appropriate. Damage control techniques, including vascular shunting, rapidly restore distal flow but require additional vascular intervention and risk shunt thrombosis with prolonged use. To address these challenges, we present a technique, using an off-the-shelf endovascular device, for treatment of peripheral arterial injuries. Direct-site endovascular repair (DSER) is an open vascular surgical reconstruction technique using conventional endovascular stent grafts to create a "sutureless" anastomosis. We believe this technique to be a valuable adjunct to current repair options.The values of this technique are that it is (1) rapid, (2) of low technical complexity, (3) requires very little equipment, and (4) may offer extended durability in damage control scenarios.We describe three patients where this technique was used. In the first case, the technique was used to provide a temporary arterial shunt in a patient with a local infection and arterial disruption. In the second case, DSER was used for definitive repair of an injured artery after penetrating trauma. The third case involves DSER for definitive of both an artery and vein after penetrating trauma.

Galectin-8 promotes regulatory T-cell differentiation by modulating IL-2 and TGFß signaling.

Galectins (Gals) have emerged as potent immunoregulatory molecules that control chronic inflammation through distinct mechanisms. Gal-8, a tandem-repeat type Gal with unique preference for α2,3-sialylated glycans, is ubiquitously expressed, but little is known about its role in T-cell differentiation. Here we report that Gal-8 promotes the polyclonal differentiation of primary mouse regulatory T (Treg) cells in vitro. We further show that Gal-8 also facilitates antigen-specific differentiation of Treg cells, and that Treg cells polarized in the presence of Gal-8 express cytotoxic T-lymphocyte antigen-4 and interleukin (IL)-10 at a higher frequency than control Treg cells, and efficiently inhibit proliferation of activated T-cells in vitro. Investigation of the mechanism by which Gal-8 promotes Treg conversion revealed that Gal-8 activates transforming growth factor-ß signaling and promotes sustained IL-2R signaling. Taken together, these data suggest that Gal-8 promotes the differentiation of highly suppressive Treg cells, which has implications for the treatment of inflammatory and autoimmune diseases.

Galectin-8 Ameliorates Murine Autoimmune Ocular Pathology and Promotes a Regulatory T Cell Response.

Galectins have emerged as potent immunoregulatory agents that control chronic inflammation through distinct mechanisms. Here, we report that treatment with Galectin-8 (Gal-8), a tandem-repeat member of the galectin family, reduces retinal pathology and prevents photoreceptor cell damage in a murine model of experimental autoimmune uveitis. Gal-8 treatment increased the number of regulatory T cells (Treg) in both the draining lymph node (dLN) and the inflamed retina. Moreover, a greater percentage of Treg cells in the dLN and retina of Gal-8 treated animals expressed the inhibitory coreceptor cytotoxic T lymphocyte antigen (CTLA)-4, the immunosuppressive cytokine IL-10, and the tissue-homing integrin CD103. Treg cells in the retina of Gal-8-treated mice were primarily inducible Treg cells that lack the expression of neuropilin-1. In addition, Gal-8 treatment blunted production of inflammatory cytokines by retinal T helper type (TH) 1 and TH17 cells. The effect of Gal-8 on T cell differentiation and/or function was specific for tissues undergoing an active immune response, as Gal-8 treatment had no effect on T cell populations in the spleen. Given the need for rational therapies for managing human uveitis, Gal-8 emerges as an attractive therapeutic candidate not only for treating retinal autoimmune diseases, but also for other TH1- and TH17-mediated inflammatory disorders.

IL-17A-mediated protection against Acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a very painful and vision-impairing infection of the cornea that is difficult to treat. Although past studies have indicated a critical role of neutrophils and macrophages in AK, the relative contribution of the proinflammatory cytokine, IL-17A, that is essential for migration, activation, and function of these cells into the cornea is poorly defined. Moreover, the role of the adaptive immune response, particularly the contribution of CD4(+) T cell subsets, Th17 and regulatory T cells , in AK is yet to be understood. In this report, using a mouse corneal intrastromal injection-induced AK model, we show that Acanthamoeba infection induces a strong CD4(+) T effector and regulatory T cell response in the cornea and local draining lymph nodes. We also demonstrate that corneal Acanthamoeba infection induces IL-17A expression and that IL-17A is critical for host protection against severe AK pathology. Accordingly, IL-17A neutralization in Acanthamoeba-infected wild-type mice or Acanthamoeba infection of mice lacking IL-17A resulted in a significantly increased corneal AK pathology, increased migration of inflammatory cells at the site of inflammation, and a significant increase in the effector CD4(+) T cell response in draining lymph nodes. Thus, in sharp contrast with other corneal infections such as herpes and Pseudomonas aeruginosa keratitis where IL-17A exacerbates corneal pathology and inflammation, the findings presented in this article suggest that IL-17A production after Acanthamoeba infection plays an important role in host protection against invading parasites.