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Suppression of apoptosis by expression of antiapoptotic BCL2 family members is a hallmark of acute myeloblastic leukemia (AML). Induced myeloid leukemia cell differentiation protein (MCL1), an antiapoptotic BCL2 family member, is commonly upregulated in AML cells and is often a primary mode of resistance to treatment with the BCL2 inhibitor venetoclax. Here, we describe VU661013, a novel, potent, selective MCL1 inhibitor that destabilizes BIM/MCL1 association, leads to apoptosis in AML, and is active in venetoclax-resistant cells and patient-derived xenografts. In addition, VU661013 was safely combined with venetoclax for synergy in murine models of AML. Importantly, BH3 profiling of patient samples and drug-sensitivity testing ex vivo accurately predicted cellular responses to selective inhibitors of MCL1 or BCL2 and showed benefit of the combination. Taken together, these data suggest a strategy of rationally using BCL2 and MCL1 inhibitors in sequence or in combination in AML clinical trials.Significance:Targeting antiapoptotic proteins in AML is a key therapeutic strategy, and MCL1 is a critical antiapoptotic oncoprotein. Armed with novel MCL1 inhibitors and the potent BCL2 inhibitor venetoclax, it may be possible to selectively induce apoptosis by combining or thoughtfully sequencing these inhibitors based on a rational evaluation of AML.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494

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Purpose: The purpose of this preclinical study was to determine the effectiveness of RAF265, a multikinase inhibitor, for treatment of human metastatic melanoma and to characterize traits associated with drug response.Experimental Design: Advanced metastatic melanoma tumors from 34 patients were orthotopically implanted to nude mice. Tumors that grew in mice (17 of 34) were evaluated for response to RAF265 (40 mg/kg, every day) over 30 days. The relation between patient characteristics, gene mutation profile, global gene expression profile, and RAF265 effects on tumor growth, mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation, proliferation, and apoptosis markers was evaluated.Results: Nine of the 17 tumors that successfully implanted (53%) were mutant BRAF (BRAFV600E/K), whereas eight of 17 (47%) tumors were BRAF wild type (BRAFWT). Tumor implants from 7 of 17 patients (41%) responded to RAF265 treatment with more than 50% reduction in tumor growth. Five of the 7 (71%) responders were BRAFWT, of which 1 carried c-KITL576P and another N-RASQ61R mutation, while only 2 (29%) of the responding tumors were BRAFV600E/K. Gene expression microarray data from nonimplanted tumors revealed that responders exhibited enriched expression of genes involved in cell growth, proliferation, development, cell signaling, gene expression, and cancer pathways. Although response to RAF265 did not correlate with pERK1/2 reduction, RAF265 responders did exhibit reduced pMEK1, reduced proliferation based upon reduced Ki-67, cyclin D1 and polo-like kinase1 levels, and induction of the apoptosis mediator BCL2-like 11.Conclusions: Orthotopic implants of patient tumors in mice may predict prognosis and treatment response for melanoma patients. A subpopulation of human melanoma tumors responds to RAF265 and can be characterized by gene mutation and gene expression profiles. Clin Cancer Res; 18(8); 2184–98. ©2012 AACR.

Background Obesity is a risk factor for the development of asthma. However, pharmacologic therapeutic strategies that specifically target obese asthmatics have not been identified. We hypothesize that glucagon‐like peptide‐1 receptor agonist (GLP‐1RA) treatment inhibits aeroallergen‐induced early innate airway inflammation in a mouse model of asthma in the setting of obesity. Methods SWR (lean) and TALLYHO (obese) mice were challenged intranasally with Alternaria alternata extract (Alt‐Ext) or PBS for 4 consecutive days concurrent with GLP‐1RA or vehicle treatment. Results TALLYHO mice had greater Alt‐Ext‐induced airway neutrophilia and lung protein expression of IL‐5, IL‐13, CCL11, CXCL1, and CXCL5, in addition to ICAM‐1 expression on lung epithelial cells compared with SWR mice, and all endpoints were reduced by GLP‐1RA treatment. Alt‐Ext significantly increased BALF IL‐33 in both TALLYHO and SWR mice compared to PBS challenge, but there was no difference in the BALF IL‐33 levels between these two strains. However, TALLYHO, but not SWR, mice had significantly higher airway TSLP in BALF following Alt‐Ext challenge compared to PBS, and BALF TSLP was significantly greater in TALLYHO mice compared to SWR mice following airway Alt‐Ext challenge. GLP‐1RA treatment significantly decreased the Alt‐Ext‐induced TSLP and IL‐33 release in TALLYHO mice. While TSLP or ST2 inhibition with a neutralizing antibody decreased airway eosinophils, they did not reduce airway neutrophils in TALLYHO mice. Conclusions These results suggest that GLP‐1RA treatment may be a novel pharmacologic therapeutic strategy for obese persons with asthma by inhibiting aeroallergen‐induced neutrophilia, a feature not seen with either TSLP or ST2 inhibition., Polygenic obese mice have larger level of Altrernaria extract‐induced TSLP, the number of lung ILC2, neutrophils in the airway, and ICAM‐1 on lung epithelial cells compared with lean mice. GLP‐1RA decreases the Altrernaria extract‐induced IL‐33 and TSLP release, type‐2 inflammation mediated by ILC2, eosinophilia and neutrophilia in the airway, and airway responsiveness in polygenic obese mice. Our findings indicate the potential for a new pharmacological therapeutic strategy to patients with asthma in the setting of obesity. Abbreviations: ICAM‐1, intercellular adhesion molecule 1; ILC2, group 2 innate lymphoid cells; GLP‐1RA, glucagon‐like peptide‐1 agonist

BackgroundCurrent melphalan-based intravitreal regimens for retinoblastoma (RB) vitreous seeds cause retinal toxicity. We assessed the efficacy and toxicity of topotecan monotherapy compared with melphalan in our rabbit model and patient cohort.MethodsRabbit experiments: empiric pharmacokinetics were determined following topotecan injection. For topotecan (15 μg or 30 µg), melphalan (12.5 µg) or saline, toxicity was evaluated by serial electroretinography (ERG) and histopathology, and efficacy against vitreous seed xenografts was measured by tumour cell reduction and apoptosis induction. Patients: retrospective cohort study of 235 patients receiving 990 intravitreal injections of topotecan or melphalan.ResultsIntravitreal topotecan 30 µg (equals 60 µg in humans) achieved the IC90 across the rabbit vitreous. Three weekly topotecan injections (either 15 µg or 30 µg) caused no retinal toxicity in rabbits, whereas melphalan 12.5 µg (equals 25 µg in humans) reduced ERG amplitudes 42%–79%. Intravitreal topotecan 15 µg was equally effective to melphalan to treat WERI-Rb1 cell xenografts in rabbits (96% reduction for topotecan vs saline (p=0.004), 88% reduction for melphalan vs saline (p=0.004), topotecan vs melphalan, p=0.15). In our clinical study, patients received 881 monotherapy injections (48 topotecan, 833 melphalan). Patients receiving 20 µg or 30 µg topotecan demonstrated no significant ERG reductions; melphalan caused ERG reductions of 7.6 μV for every injection of 25 µg (p=0.03) or 30 µg (pConclusionsTaken together, these experiments suggest that intravitreal topotecan monotherapy for the treatment of RB vitreous seeds is non-toxic and effective.

Purpose: The BCL2 inhibitor, venetoclax, has transformed clinical care in acute myeloid leukemia (AML). However, subsets of patients do not respond or eventually acquire resistance. Venetoclax-based regimens can lead to considerable marrow suppression in some patients. Bromodomain and extraterminal inhibitors (BETi) are potential treatments for AML, as regulators of critical AML oncogenes. We tested the efficacy of novel BET inhibitor INCB054329, and its synergy with venetoclax to reduce AML without induction of hematopoietic toxicity. Experimental Design: INCB054329 efficacy was assessed by changes in cell cycle and apoptosis in treated AML cell lines. In vivo efficacy was assessed by tumor reduction in MV-4-11 cell line–derived xenografts. Precision run-on and sequencing (PRO-seq) evaluated effects of INCB054329. Synergy between low-dose BETi and venetoclax was assessed in cell lines and patient samples in vitro and in vivo while efficacy and toxicity was assessed in patient-derived xenograft (PDX) models. Results: INCB054329 induced dose-dependent apoptosis and quiescence in AML cell lines. PRO-seq analysis evaluated the effects of INCB054329 on transcription and confirmed reduced transcriptional elongation of key oncogenes, MYC and BCL2, and genes involved in the cell cycle and metabolism. Combinations of BETi and venetoclax led to reduced cell viability in cell lines and patient samples. Low-dose combinations of INCB054329 and venetoclax in cell line and PDX models reduced AML burden, regardless of the sensitivity to monotherapy without development of toxicity. Conclusions: Our findings suggest low dose combinations of venetoclax and BETi may be more efficacious for patients with AML than either monotherapy, potentially providing a longer, more tolerable dosing regimen.

The cyclooxygenase (COX) metabolic pathway regulates immune responses and inflammation. The effect of the COX pathway on innate pulmonary inflammation induced by protease-containing fungal allergens, such as Alternaria alternata, is not fully defined. In this study, we tested the hypothesis that COX inhibition augments Alternaria-induced pulmonary group 2 innate lymphoid cell (ILC2) responses and IL-33 release. Mice were treated with the COX inhibitors indomethacin, flurbiprofen, or vehicle and challenged intranasally with Alternaria extract for four consecutive days to induce innate lung inflammation. We found that indomethacin and flurbiprofen significantly increased the numbers of ILC2 and IL-5 and IL-13 expression by ILC2 in the lung. Indomethacin also increased ILC2 proliferation, the percentages of eosinophils, and mucus production in the lung. Both indomethacin and flurbiprofen augmented the release of IL-33 in bronchoalveolar lavage fluid after Alternaria challenge, suggesting that more IL-33 was available for ILC2 activation and that a COX product(s) inhibited IL-33 release. This is supported by the in vitro finding that the COX product PGE2 and the PGI2 analogs cicaprost decreased Alternaria extract–induced IL-33 release by human bronchial epithelial cells. Although contrasting effects of PGD2, PGE2, and PGI2 on ILC2 responses have been previously reported, the overall effect of the COX pathway on ILC2 function is inhibitory in Alternaria-induced innate airway inflammation.

Fatty liver involves ectopic lipid accumulation and dysregulated hepatic oxidative metabolism, which can progress to a state of elevated inflammation and fibrosis referred to as nonalcoholic steatohepatitis (NASH). The factors that control progression from simple steatosis to NASH are not fully known. Here, we tested the hypothesis that dietary vitamin E (VitE) supplementation would prevent NASH progression and associated metabolic alterations induced by a Western diet (WD). Hyperphagic melanocortin-4 receptor-deficient (MC4R(−/−)) mice were fed chow, chow+VitE, WD, or WD+VitE starting at 8 or 20 weeks of age. All groups exhibited extensive hepatic steatosis by the end of the study (28 weeks of age). WD feeding exacerbated liver disease severity without inducing proportional changes in liver triglycerides. Eight weeks of WD accelerated liver pyruvate cycling, and 20 weeks of WD extensively upregulated liver glucose and oxidative metabolism assessed by (2)H/(13)C flux analysis. VitE supplementation failed to reduce the histological features of NASH. Rather, WD+VitE increased the abundance and saturation of liver ceramides and accelerated metabolic flux dysregulation compared with 8 weeks of WD alone. In summary, VitE did not limit NASH pathogenesis in genetically obese mice, but instead increased some indicators of metabolic dysfunction.

Background DNA methyltransferase inhibitors (DNMTis) improve survival for patients with myelodysplastic syndromes (MDS) and those with acute myeloid leukemia (AML) unable to receive standard cytotoxic chemotherapy and are, accordingly, the backbone of standard-of-care treatment for these conditions. Standard regimens with DNMTIs, decitabine (DEC) or azacitidine (AZA) include daily subcutaneous (s.c.) or intravenous (i.v.) administration for 5–7 consecutive days. Attempts to provide the therapy orally have been limited given rapid clearance of the agents by the enzyme cytidine deaminase (CDA), which is ubiquitous in the gut and liver as part of first-pass metabolism. Recently, cedazuridine (CDZ), an oral inhibitor of CDA, was successfully combined with DEC to approximate the pharmacokinetics of i.v. DEC in patients. Objective To determine if an oral dosing strategy might be feasible in the clinic with AZA, we attempted to increase the bioavailability of oral AZA through the use of CDZ, in a murine model. Methods Following pharmacokinetic and pharmacodynamic assessment of oral AZA dosed with CDZ in murine and monkey models, we tested this regimen in vivo with a human cell line-derived xenograft transplantation experiment (CDX). Following this we combined the regimen with venetoclax (VEN) to test the efficacy of an all-oral regimen in a patient-derived xenograft (PDX) model. Results Parenteral AZA and oral AZA + CDZ exhibited similar pharmacokinetic profiles, and efficacy against human AML cells. Tumor regression was seen with AZA + CDZ in MOLM-13 CDX and PDX models. Conclusions We conclude that oral AZA when combined with CDZ achieves successful tumor regression in both CDX and PDX models. Furthermore, the combination of AZA + CDZ with VEN in a PDX model emulated responses seen with VEN + AZA in the clinic, implying a potential all-oral VEN-based therapy opportunity in myeloid diseases.

OBJECTIVE: To determine the impact of autoimmunity in the absence of glycemic alterations on pregnancy in type 1 diabetes (T1D). DESIGN: Because nonobese diabetic (NOD) mice experience autoimmunity before the onset of hyperglycemia, we studied pregnancy outcomes in prediabetic NOD mice using flow cytometry and enzyme-linked immunosorbent assays. Once we determined that adverse events in pregnancy occurred in euglycemic mice, we performed an exploratory study using electronic health records to better understand pregnancy complications in humans with T1D and normal hemoglobin A1c levels. SETTING: University Medical Center. PATIENT(S)/ANIMAL(S): Nonobese diabetic mice and electronic health records from Vanderbilt University Medical Center. INTERVENTION(S): Nonobese diabetic mice were administered 200 μg of an anti-interleukin 6 (IL-6) antibody every other day starting on day 5 of gestation. MAIN OUTCOME MEASURE(S): Changes in the number of abnormal and reabsorbed pups in NOD mice and odds of vascular complications in pregnancy in T1D in relation to A1c. RESULT(S): Prediabetic NOD mice had increased adverse pregnancy outcomes compared with nonautoimmune mice; blockade of IL-6, which was secreted by endothelial cells, decreased the number of reabsorbed and abnormal fetuses. Similarly, vascular complications were increased in pregnant patients with T1D across all A1c values. CONCLUSION(S): The vascular secretion of IL-6 drives adverse pregnancy outcomes in prediabetic NOD mice. Pregnant patients with T1D have increased vascular complications even with normal hemoglobin A1cs, indicating a potential effect of autoimmunity on the placental vasculature.

Acinetobacter baumannii is a nosocomial pathogen that exhibits substantial genomic plasticity. Here, the identification of two variants of A. baumannii ATCC 17978 that differ based on the presence of a 44-kb accessory locus, named AbaAL44 (A. baumannii accessory locus 44 kb), is described. Analyses of existing deposited data suggest that both variants are found in published studies of A. baumannii ATCC 17978 and that American Type Culture Collection (ATCC)-derived laboratory stocks comprise a mix of these two variants. Yet, each variant exhibits distinct interactions with the host in vitro and in vivo. Infection with the variant that harbors AbaAL44 (A. baumannii 17978 UN) results in decreased bacterial burdens and increased neutrophilic lung inflammation in a mouse model of pneumonia, and affects the production of interleukin 1 beta (IL-1β) and IL-10 by infected macrophages. AbaAL44 harbors putative pathogenesis genes, including those predicted to encode a type I pilus cluster, a catalase, and a cardiolipin synthase. The accessory catalase increases A. baumannii resistance to oxidative stress and neutrophil-mediated killing in vitro. The accessory cardiolipin synthase plays a dichotomous role by promoting bacterial uptake and increasing IL-1β production by macrophages, but also by enhancing bacterial resistance to cell envelope stress. Collectively, these findings highlight the phenotypic consequences of the genomic dynamism of A. baumannii through the evolution of two variants of a common type strain with distinct infection-related attributes.

To study the complex processes involved in liver injuries, researchers rely on animal investigations, using chemically or surgically induced liver injuries, to extrapolate findings and infer human health risks. However, this presents obvious challenges in performing a detailed comparison and validation between the highly controlled animal models and development of liver injuries in humans. Furthermore, it is not clear whether there are species-dependent and -independent molecular initiating events or processes that cause liver injury before they eventually lead to end-stage liver disease. Here, we present a side-by-side study of rats and guinea pigs using thioacetamide to examine the similarities between early molecular initiating events during an acute-phase liver injury. We exposed Sprague Dawley rats and Hartley guinea pigs to a single dose of 25 or 100 mg/kg thioacetamide and collected blood plasma for metabolomic analysis and liver tissue for RNA-sequencing. The subsequent toxicogenomic analysis identified consistent liver injury trends in both genomic and metabolomic data within 24 and 33 h after thioacetamide exposure in rats and guinea pigs, respectively. In particular, we found species similarities in the key injury phenotypes of inflammation and fibrogenesis in our gene module analysis for liver injury phenotypes. We identified expression of several common genes (e.g., SPP1, TNSF18, SERPINE1, CLDN4, TIMP1, CD44, and LGALS3), activation of injury-specific KEGG pathways, and alteration of plasma metabolites involved in amino acid and bile acid metabolism as some of the key molecular processes that changed early upon thioacetamide exposure and could play a major role in the initiation of acute liver injury.

Perinatal infection with Streptococcus agalactiae, or Group B Streptococcus (GBS), is associated with preterm birth, neonatal sepsis, and stillbirth. Here, we study the interactions of GBS with macrophages, essential sentinel immune cells that defend the gravid reproductive tract. Transcriptional analyses of GBS-macrophage co-cultures reveal enhanced expression of a gene encoding a putative metal resistance determinant, cadD. Deletion of cadD reduces GBS survival in macrophages, metal efflux, and resistance to metal toxicity. In a mouse model of ascending infection during pregnancy, the ΔcadD strain displays attenuated bacterial burden, inflammation, and cytokine production in gestational tissues. Furthermore, depletion of host macrophages alters cytokine expression and decreases GBS invasion in a cadD-dependent fashion. Our results indicate that GBS cadD plays an important role in metal detoxification, which promotes immune evasion and bacterial proliferation in the pregnant host.

Background Approximately 10% of patients with SCLC develop a paraneoplastic syndrome (PNS). Neurologic PNS are thought to improve prognosis, which we hypothesized is related to increased tumor-infiltrating lymphocytes and immune recognition. Methods We queried 2,512,042 medical records from a single institution to identify patients who have SCLC with and without PNS and performed manual, retrospective chart review. We then performed multiplexed fluorescence immunohistochemistry and automated quantitative analysis (AQUA Technology) on tumors to assess CD3, CD4, and CD8 T cell infiltrates and programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) interactions. T cell infiltrates and PD-1/PD-L1 interaction scores were compared among patients with neurologic PNS, endocrinologic PNS, and a control group without PNS. Clinical outcomes were analyzed using the Kaplan-Meier method and Cox proportional hazards models. Results We evaluated 145 SCLC patients: 55 with PNS (25 neurologic and 30 endocrinologic) and 90 controls. Patients with neurologic PNS experienced improved overall survival compared to patients with endocrinologic PNS and controls (median overall survival of 24 months versus 12 months versus 13 months, respectively). Of the 145 patients, we identified tumor tissue from 34 patients that was adequate for AQUA analysis. Among 37 specimens from these 34 patients, patients with neurologic PNS had increased T cell infiltrates (p = 0.033) and PD-1/PD-L1 interaction (p = 0.014) compared to tumors from patients with endocrinologic PNS or controls. Conclusions Tumor tissue from patients with SCLC with neurologic PNS showed increased tumor-infiltrating lymphocytes and PD-1/PD-L1 interaction consistent with an inflamed tumor microenvironment.

Calprotectin is a heterodimer of the proteins S100A8 and S100A9, and it is an abundant innate immune protein associated with inflammation. In humans, calprotectin transcription and protein abundance are associated with asthma and disease severity. However, mechanistic studies in experimental asthma models have been inconclusive, identifying both protective and pathogenic effects of calprotectin. To clarify the role of calprotectin in asthma, calprotectin-deficient S100A9-/- and wild-type (WT) C57BL/6 mice were compared in a murine model of allergic airway inflammation. Mice were intranasally challenged with extracts of the clinically relevant allergen, Alternaria alternata (Alt Ext), or PBS every third day over 9 days. On Day 10, BAL fluid and lung tissue homogenates were harvested and allergic airway inflammation was assessed. Alt Ext challenge induced release of S100A8/S100A9 to the alveolar space and increased protein expression in the alveolar epithelium of WT mice. Compared with WT mice, S100A9-/- mice displayed significantly enhanced allergic airway inflammation, including production of IL-13, CCL11, CCL24, serum IgE, eosinophil recruitment, and airway resistance and elastance. In response to Alt Ext, S100A9-/- mice accumulated significantly more IL-13+IL-5+CD4+ T-helper type 2 cells. S100A9-/- mice also accumulated a significantly lower proportion of CD4+ T regulatory (Treg) cells in the lung that had significantly lower expression of CD25. Calprotectin enhanced WT Treg cell suppressive activity in vitro. Therefore, this study identifies a role for the innate immune protein, S100A9, in protection from CD4+ T-helper type 2 cell hyperinflammation in response to Alt Ext. This protection is mediated, at least in part, by CD4+ Treg cell function.

The ventricular–subventricular zone (V‐SVZ) of the mammalian brain is a site of adult neurogenesis. Within the V‐SVZ reside type B neural stem cells (NSCs) and type A neuroblasts. The V‐SVZ is also a primary site for very aggressive glioblastoma (GBM). Standard‐of‐care therapy for GBM consists of safe maximum resection, concurrent temozolomide (TMZ), and X‐irradiation (XRT), followed by adjuvant TMZ therapy. The question of how this therapy impacts neurogenesis is not well understood and is of fundamental importance as normal tissue tolerance is a limiting factor. Here, we studied the effects of concurrent TMZ/XRT followed by adjuvant TMZ on type B stem cells and type A neuroblasts of the V‐SVZ in C57BL/6 mice. We found that chemoradiation induced an apoptotic response in type A neuroblasts, as marked by cleavage of caspase 3, but not in NSCs, and that A cells within the V‐SVZ were repopulated given sufficient recovery time. 53BP1 foci formation and resolution was used to assess the repair of DNA double‐strand breaks. Remarkably, the repair was the same in type B and type A cells. While Bax expression was the same for type A or B cells, antiapoptotic Bcl2 and Mcl1 expression was significantly greater in NSCs. Thus, the resistance of type B NSCs to TMZ/XRT appears to be due, in part, to high basal expression of antiapoptotic proteins compared with type A cells. This preclinical research, demonstrating that murine NSCs residing in the V‐SVZ are tolerant of standard chemoradiation therapy, supports a dose escalation strategy for treatment of GBM. stem cells 2019;37:1629–1639, The brain's ventricular–subventricular zone is a site of type B neural stem cell‐mediated neurogenesis and unfortunately for very aggressive glioblastoma. Treatment for glioblastoma consists of resection, concurrent temozolomide, and X‐irradiation, followed by adjuvant temozolomide. We show that concurrent temozolomide/X‐irradiation followed by adjuvant temozolomide induces apoptosis in type A neuroblasts. In contrast, quiescent neural stem cells, which express high levels of Bcl2 and Mcl1, survive and repopulate type A neuroblasts.

Tissue-resident memory T cells (TRM) patrol nonlymphoid organs and provide superior protection against pathogens that commonly infect mucosal and barrier tissues, such as the lungs, intestine, liver, and skin. Thus, there is a need for vaccine technologies that can induce a robust, protective TRM response in these tissues. Nanoparticle (NP) vaccines offer important advantages over conventional vaccines; however, there has been minimal investigation into the design of NP-based vaccines for eliciting TRM responses. Here, we describe a pH-responsive polymeric nanoparticle vaccine for generating antigen-specific CD8+ TRM cells in the lungs. With a single intranasal dose, the NP vaccine elicited airway- and lung-resident CD8+ TRM cells and protected against respiratory virus challenge in both sublethal (vaccinia) and lethal (influenza) infection models for up to 9 weeks after immunization. In elucidating the contribution of material properties to the resulting TRM response, we found that the pH-responsive activity of the carrier was important, as a structurally analogous non-pH-responsive control carrier elicited significantly fewer lung-resident CD8+ T cells. We also demonstrated that dual-delivery of protein antigen and nucleic acid adjuvant on the same NP substantially enhanced the magnitude, functionality, and longevity of the antigen-specific CD8+ TRM response in the lungs. Compared to administration of soluble antigen and adjuvant, the NP also mediated retention of vaccine cargo in pulmonary antigen-presenting cells (APCs), enhanced APC activation, and increased production of TRM-related cytokines. Overall, these data suggest a promising vaccine platform technology for rapid generation of protective CD8+ TRM cells in the lungs.

Endotoxin shock is induced by LPS, one of the most potent virulence factors of the Gram-negative bacteria that cause sepsis. It remains unknown if either proinflammatory stress-responsive transcription factors (SRTFs), ferried to nucleus by importin α5, or lipid-regulating sterol regulatory element binding proteins (SREBPs), transported to the nucleus by importin β1, mediate endotoxin shock. A novel cell-penetrating peptide targeting importin α5 while sparing importin β1 protected 80% of animals from death in response to a high dose of LPS. This peptide suppresses inflammatory mediators, liver glycogen depletion, endothelial injury, neutrophil trafficking, and apoptosis caused by LPS. In d-galactosamine–pretreated mice challenged by 700-times lower dose of LPS, rapid death through massive apoptosis and hemorrhagic necrosis of the liver was also averted by the importin α5–selective peptide. Thus, using a new tool for selective suppression of nuclear transport, we demonstrate that SRTFs, rather than SREBPs, mediate endotoxin shock.

Proliferating tricholemmal tumors (PTTs) are rare benign neoplasms that arise from the outer sheath of a hair follicle. Occasionally, these PTTs undergo malignant transformation to become malignant proliferating tricholemmal tumors (MPTTs). Little is known about the molecular alterations, malignant progression, and management of MPTTs. Here, we describe the case of a 58-year-old female that had a widely metastatic MPTT that harbored an activating PIK3CA mutation and was sensitive to the PI3K inhibitor, alpelisib (BYL719). We review the available literature on metastatic MPTT, detail the patient’s course, and present a whole genome analysis of this rare tumor.

Invertebrates are often overlooked as laboratory animals, yet they are commonly used in toxicology, developmental, cellular and molecular biology, and radiation studies with euthanasia as an endpoint. Little is known regarding appropriate euthanasia methods for invertebrate species, particularly for Artemia. Here, we evaluated the AVMA-recommended 2-step method of euthanasia in brine shrimp (Artemia franciscana). Artemia were exposed first to anesthetic solutions of 60% alcohol, 2.5 mg/L eugenol, or 4 g/L tricaine methanesulfonate (TMS) and then were transferred to euthanasia solutions of 70% alcohol, 95% alcohol, or 10% neutral buffered formalin. We examined time to anesthesia, behavioral response to anesthesia, anesthesia recovery, and time to euthanasia. Our results show that 2.5 mg/L eugenol and 4 g/L TMS inconsistently achieved anesthesia. Although 60% alcohol produced anesthesia, the time to anesthesia varied among replicate groups, and exposure resulted in an increase in abnormal behavior. We therefore do not recommend any of the tested anesthetic solutions for use in Artemia. Although all 3 euthanasia solutions were effective, more research is needed to provide recommendations regarding euthanasia for this species.

Hyperlipidemia, the hallmark of Metabolic Syndrome that afflicts millions of people worldwide, exacerbates life-threatening infections. We present a new evidence for the mechanism of hyperlipidemic hypersensitivity to microbial inflammation caused by pathogen-derived inducer, LPS. We demonstrate that hyperlipidemic animals succumbed to a non-lethal dose of LPS whereas normolipidemic controls survived. Strikingly, survival of hyperlipidemic animals was restored when the nuclear import of stress-responsive transcription factors (SRTFs), Sterol Regulatory Element-Binding Proteins (SREBPs), and Carbohydrate-Responsive Element-Binding Proteins (ChREBPs) was impeded by targeting the nuclear transport checkpoint with cell-penetrating, biselective nuclear transport modifier (NTM) peptide. Furthermore, the burst of proinflammatory cytokines and chemokines, microvascular endothelial injury in the liver, lungs, heart, and kidneys, and trafficking of inflammatory cells were also suppressed. To dissect the role of nuclear transport signaling pathways we designed and developed importin-selective NTM peptides. Selective targeting of the importin α5, ferrying SRTFs and ChREBPs, protected 70–100% hyperlipidemic animals. Targeting importin β1, that transports SREBPs, was only effective after 3-week treatment that lowered blood triglycerides, cholesterol, glucose, and averted fatty liver. Thus, the mechanism of hyperlipidemic hypersensitivity to lethal microbial inflammation depends on metabolic and proinflammatory transcription factors mobilization, which can be counteracted by targeting the nuclear transport checkpoint.

T regulatory cells (Treg) restrain both the innate and adaptive immune systems to maintain homeostasis. Allergic airway inflammation, characterized by a type 2 (Th2) response that results from a breakdown of tolerance to innocuous environmental antigens, is negatively regulated by Treg. We previously reported that prostaglandin I2 (PGI2) promoted immune tolerance in models of allergic inflammation; however, the effect of PGI2 on Treg function was not investigated. Treg from mice deficient in the PGI2 receptor IP (IP KO) had impaired suppressive capabilities during allergic airway inflammatory responses compared to mice with PGI2 signaling was intact. IP KO Treg had significantly enhanced expression of immunoglobulin-like transcript 3 (ILT3) compared to wild-type Treg, which may contribute to the impairment of the IP KO Treg's ability to suppress Th2 responses. Using fate-mapping mice, we reported that PGI2 signaling prevents Treg reprogramming toward a pathogenic phenotype. PGI2 analogs promoted the differentiation of naive T cells to Treg in both mice and humans via repression of β-catenin signaling. Finally, a missense variant in IP in humans was strongly associated with chronic obstructive asthma. Together, these data support that PGI2 signaling licenses Treg suppressive function and that PGI2 is a therapeutic target to enhance Treg function.

p73 and p63 are members of the p53 family that exhibit overlapping and distinct functions in development and homeostasis. The evaluation of p73 and p63 isoform expression across human tissue can provide greater insight to the functional interactions between family members. We determined the mRNA isoform expression patterns of TP73 and TP63 across a panel of 36 human tissues and protein expression within the highest-expressing tissues. TP73 and TP63 expression significantly correlated across tissues. In tissues with concurrent mRNA expression, nuclear co-expression of both proteins was observed in a majority of cells. Using GTEx data, we quantified p73 and p63 isoform expression in human tissue and identified that the α-isoforms of TP73 and TP63 were the predominant isoform expressed in nearly all tissues. Further, we identified a previously unreported p73 mRNA product encoded by exons 4 to 14. In sum, these data provide the most comprehensive tissue-specific atlas of p73 and p63 protein and mRNA expression patterns in human and murine samples, indicating coordinate expression of these transcription factors in the majority of tissues in which they are expressed.

Altres ajuts: Cancer Center (CORE) Support grant CA 21765, i l'American Lebanese Syrian Associated Charities Altres ajuts: NIH/GM45737 Choline cytidylyltransferase (CCT) is the rate-limiting enzyme in the phosphatidylcholine biosynthetic pathway. Here, we demonstrate that CCTα-mediated phosphatidylcholine synthesis is required to maintain normal Golgi structure and function as well as cytokine secretion from the Golgi complex. CCTα is localized to the trans-Golgi region and its expression is increased in lipopolysaccharide (LPS)-stimulated wild-type macrophages. Although LPS triggers transient reorganization of Golgi morphology in wild-type macrophages, similar structural alterations persist in CCTα-deficient cells. Pro-tumor necrosis factor α and interleukin-6 remain lodged in the secretory compartment of CCTα-deficient macrophages after LPS stimulation. However, the lysosomal-mediated secretion pathways for interleukin-1β secretion and constitutive apolipoprotein E secretion are unaltered. Exogenous lysophosphatidylcholine restores LPS-stimulated secretion from CCTα-deficient cells, and elevated diacylglycerol levels alone do not impede secretion of pro-tumor necrosis factor α or interleukin-6. These results identify CCTα as a key component in membrane biogenesis during LPS-stimulated cytokine secretion from the Golgi complex.

The programmed death-ligand 1 (PD-L1) immune checkpoint inhibitors, atezolizumab and durvalumab, have received regulatory approval for the first-line treatment of patients with extensive-stage SCLC. Nevertheless, when used in combination with platinum-based chemotherapy, these PD-L1 inhibitors only improve overall survival by 2 to 3 months. This may be due to the observation that less than 20% of SCLC tumors express PD-L1 at greater than 1%. Evaluating the composition and abundance of checkpoint molecules in SCLC may identify molecules beyond PD-L1 that are amenable to therapeutic targeting.We analyzed RNA-sequencing data from SCLC cell lines (n = 108) and primary tumor specimens (n = 81) for expression of 39 functionally validated inhibitory checkpoint ligands. Furthermore, we generated tissue microarrays containing SCLC cell lines and patient with SCLC specimens to confirm expression of these molecules by immunohistochemistry. We annotated patient outcomes data, including treatment response and overall survival.The checkpoint protein B7-H6 (NCR3LG1) exhibited increased protein expression relative to PD-L1 in cell lines and tumors (p0.05). Higher B7-H6 protein expression correlated with longer progression-free survival (p = 0.0368) and increased total immune infiltrates (CD45+) in patients. Furthermore, increased B7-H6 gene expression in SCLC tumors correlated with a decreased activated natural killer cell gene signature, suggesting a complex interplay between B7-H6 expression and immune signature in SCLC.We investigated 39 inhibitory checkpoint molecules in SCLC and found that B7-H6 is highly expressed and associated with progression-free survival. In addition, 26 of 39 immune checkpoint proteins in SCLC tumors were more abundantly expressed than PD-L1, indicating an urgent need to investigate additional checkpoint targets for therapy in addition to PD-L1.

Summary Patient-derived cancer organoids hold great potential to accurately model and predict therapeutic responses. Efficient organoid isolation methods that minimize post-collection manipulation of tissues would improve adaptability, accuracy, and applicability to both experimental and real-time clinical settings. Here we present a simple and minimally invasive fine-needle aspiration (FNA)-based organoid culture technique using a variety of tumor types including gastrointestinal, thyroid, melanoma, and kidney. This method isolates organoids directly from patients at the bedside or from resected tissues, requiring minimal tissue processing while preserving the histologic growth patterns and infiltrating immune cells. Finally, we illustrate diverse downstream applications of this technique including in vitro high-throughput chemotherapeutic screens, in situ immune cell characterization, and in vivo patient-derived xenografts. Thus, routine clinical FNA-based collection techniques represent an unappreciated substantial source of material that can be exploited to generate tumor organoids from a variety of tumor types for both discovery and clinical applications., Graphical Abstract, Highlights • Fine-needle aspiration (FNA) is safe, minimally invasive, and widely used clinically • FNA is a source of material for organoid culture and personalized medicine • This technique requires minimal processing, preserving histology, and immune cells • Downstream applications: high-throughput screens, immune analysis, and xenografts, Clinical Medicine; Tissue Engineering; Cancer

Immune checkpoint inhibitors (ICIs) are used for the treatment of numerous cancers, but risks associated with ICI-therapy during the COVID-19 pandemic are poorly understood. We report a case of acute lung injury in a lung cancer patient initially treated for ICI-pneumonitis and later found to have concurrent SARS-CoV-2 infection. Post-mortem analyses revealed diffuse alveolar damage in both the acute and organizing phases, with a predominantly CD68+ inflammatory infiltrate. Serum was positive for anti-SARS-CoV-2 IgG, suggesting that viral infection predated administration of ICI-therapy and may have contributed to a more fulminant clinical presentation. These data suggest the need for routine SARS-CoV-2 testing in cancer patients, where clinical and radiographic evaluations may be non-specific.

Antimicrobial resistance is increasing in pathogenic bacteria. Yet, the effect of antibiotic exposure on resistant bacteria has been underexplored and may affect pathogenesis. Here we describe the discovery that propagation of the human pathogen Acinetobacter baumannii in an aminoglycoside antibiotic results in alterations to the bacterium that interact with lung innate immunity resulting in enhanced bacterial clearance. Co-inoculation of mice with A. baumannii grown in the presence and absence of the aminoglycoside, kanamycin, induces enhanced clearance of a non-kanamycin-propagated strain. This finding can be replicated when kanamycin-propagated A. baumannii is killed prior to co-inoculation of mice, indicating the enhanced bacterial clearance results from interactions with innate host defenses in the lung. Infection with kanamycin-propagated A. baumannii alters the kinetics of phagocyte recruitment to the lung and reduces pro- and anti-inflammatory cytokine and chemokine production in the lung and blood. This culminates in reduced histopathologic evidence of lung injury during infection despite enhanced bacterial clearance. Further, the antibacterial response induced by killed aminoglycoside-propagated A. baumannii enhances the clearance of multiple clinically relevant Gram-negative pathogens from the lungs of infected mice. Together, these findings exemplify cooperation between antibiotics and the host immune system that affords protection against multiple antibiotic-resistant bacterial pathogens. Further, these findings highlight the potential for the development of a broad-spectrum therapeutic that exploits a similar mechanism to that described here and acts as an innate immunity modulator., Author summary Preserving the ability to treat infectious diseases with antibiotics in the face of the rapid proliferation of drug-resistant bacterial pathogens is among the greatest challenges facing medicine. Efforts to combat antimicrobial resistance may include strategies to maximize the utility of existing antibiotics while also identifying new therapeutic targets to treat bacterial infections. Acinetobacter baumannii is a human pathogen and strains of A. baumannii have acquired multi- and pan-antibiotic resistance. Here, we demonstrate that A. baumannii that is resistant to the aminoglycoside class of antibiotics is rapidly cleared from the lungs of mice when exposed to aminoglycoside antibiotics. Exposure to aminoglycosides induces changes in A. baumannii that interact with mouse antibacterial defenses, leading to rapid clearance of the infection. Further, killed aminoglycoside-exposed A. baumannii interacts with innate immunity in the lung to enhance the clearance of other pathogenic bacteria. These findings indicate that pneumonia caused by aminoglycoside-resistant A. baumannii may be effectively treated with aminoglycoside antibiotics and also suggests that the host immune response can be targeted to enhance the clearance of bacterial infections.

SUMMARY Acinetobacter baumannii is a leading cause of ventilator-associated pneumonia and a critical threat due to multidrug resistance. The A. baumannii outer membrane is an asymmetric lipid bilayer composed of inner leaflet glycerophospholipids and outer leaflet lipooligosaccharides. Deleting mlaF of the maintenance of lipid asymmetry (Mla) system causes A. baumannii to become more susceptible to pulmonary surfactants and antibiotics and decreases bacterial survival in the lungs of mice. Spontaneous suppressor mutants isolated from infected mice contain an ISAba11 insertion upstream of the ispB initiation codon, an essential isoprenoid biosynthesis gene. The insertion restores antimicrobial resistance and virulence to ΔmlaF. The suppressor strain increases lipooligosaccharides, suggesting that the mechanism involves balancing the glycerophospholipids/lipooligosaccharides ratio on the bacterial surface. An identical insertion exists in an extensively drug-resistant A. baumannii isolate, demonstrating its clinical relevance. These data show that the stresses bacteria encounter during infection select for genomic rearrangements that increase resistance to antimicrobials., Graphical Abstract, In Brief The outer membrane is a critical barrier to resist host and antibiotic stresses for Gram-negative bacteria. Palmer et al. identify an Acinetobacter baumannii suppressor mutation that restores cell size, resistance to membrane stress and antibiotics, and virulence to a ΔmlaF mutant.

The cyclooxygenase (COX) metabolic pathway regulates immune responses and inflammation. The effect of the COX pathway on innate pulmonary inflammation induced by protease-containing fungal allergens such as Alternaria alternata is not fully defined. In this study, we tested the hypothesis that COX inhibition augments Alternaria-induced pulmonary group 2 innate lymphoid cell (ILC2) responses and IL-33 release. Mice were treated with the COX inhibitor indomethacin, flurbiprofen, or vehicle and challenged intranasally with Alternaria extract for four consecutive days to induce innate lung inflammation. We found that indomethacin and flurbiprofen significantly increased the numbers of ILC2, and IL-5 and IL-13 expression by ILC2 in the lung. Indomethacin also increased ILC2 proliferation, the percentages of eosinophils and mucus production in the lung. Both indomethacin and flurbiprofen augmented the release of IL-33 in bronchoalveolar lavage fluid after Alternaria challenge, suggesting that more IL-33 was available for ILC2 activation and that a COX product(s) inhibited IL-33 release. This is supported by the in vitro finding that the COX product PGE(2) and the PGI(2) analogs cicaprost decreased Alternaria extract-induced IL-33 release by human bronchial epithelial cells. Although contrasting effects of PGD(2), PGE(2), and PGI(2) on ILC2 responses have been previously reported, the overall effect of the COX pathway on ILC2 function is inhibitory in Alternaria-induced innate airway inflammation. (Word count: Abstract 211; Manuscript: 5109)

Summary We report that p73 is expressed in ovarian granulosa cells and that loss of p73 leads to attenuated follicle development, ovulation, and corpus luteum formation, resulting in decreased levels of circulating progesterone and defects in mammary gland branching. Ectopic progesterone in p73-deficient mice completely rescued the mammary branching and partially rescued the ovarian follicle development defects. Performing RNA sequencing (RNA-seq) on transcripts from murine wild-type and p73-deficient antral follicles, we discovered differentially expressed genes that regulate biological adhesion programs. Through modulation of p73 expression in murine granulosa cells and transformed cell lines, followed by RNA-seq and chromatin immunoprecipitation sequencing, we discovered p73-dependent regulation of a gene set necessary for cell adhesion and migration and components of the focimatrix (focal intra-epithelial matrix), a basal lamina between granulosa cells that promotes follicle maturation. In summary, p73 is essential for ovarian folliculogenesis and functions as a key regulator of a gene network involved in cell-to-cell adhesion and migration., Graphical Abstract, Highlights • p73 is required for murine ovarian folliculogenesis and proper corpus luteum formation • p73 loss leads to defects in progesterone signaling and mammary gland branching • In murine ovaries, p73 is expressed specifically in granulosa cells • p73 regulates components of the granulosa cell focimatrix and migration, Molecular Network; Functional Aspects of Cell Biology; Developmental Biology; Omics

Female nude mice (J:NU-Foxn1nu; age, 6 wk) were injected with 1 million MCF7 human breast cancer cells in the fourth mammary fat pads and received a 21-d sustained-release estrogen pellet (0.25 mg) subcutaneously in the dorsum of the neck. All mice were maintained in sterile housing and provided sterile water and irradiated rodent chow. Approximately 6 wk after implantation, 4 of the 30 mice showed clinical signs of depression and dehydration. The 2 animals most severely affected were euthanized and presented for necropsy. The urinary bladders of these animals were distended with variable sized white, opaque uroliths. Urinalysis revealed coccal bacteria, erythrocytes, neutrophils and struvite crystals. Urine cultures from both necropsied animals grew heavy, pure growths of Staphylococcus xylosus. The organism was sensitive to all antibiotics tested except erythromycin (intermediate). Analysis of the uroliths revealed 100% struvite composition. Remaining mice in the study were evaluated clinically for hydration status, the ability to urinate, and the presence of palpable stones in the urinary bladder; one additional mouse had a firm, nonpainful bladder (urolithiasis suspected). Given the sensitivity of the organisms cultured from urine samples, the remaining mice were placed on enrofloxacin in the drinking water (0.5 mg/mL). All remaining mice completed the study without further morbidity or mortality. Previous studies have reported the association of estrogen supplementation with urinary bladder pathology, including infection and urolithiasis. Here we present a case of urolithiasis and cystitis in nude mice receiving estrogen supplementation that was associated with Staphylococcus xylosus, which previously was unreported in this context. When assessing these nude mice for urolithiasis, we found that visualizing the stones through the body wall, bladder palpation, and bladder expression were helpful in identifying affected mice.

Euthanasia followed by histological preparation is a common endpoint for invertebrate species utilized in research. These species present unique challenges to the histology laboratory because there...