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In this cohort of patients with pulmonary arterial hypertension (PAH), we investigate metabolomic associations with comprehensive right ventricular (RV) functional measurements derived from multibeat RV pressure-volume loop analysis. Our results show that tryptophan metabolism, particularly the kynurenine pathway, is linked to intrinsic RV function and PAH pathobiology. Findings also highlight the importance of arginine bioavailability in the cardiopulmonary system’s response to exercise stress. Metabolite profiles selected via unbiased analysis outperformed N-terminal prohormone of B-type natriuretic peptide (NT-proBNP) in predicting load-independent measures of RV function at rest and cardiopulmonary system performance under stress. Overall, this work suggests the potential for select metabolites to function as disease-specific biomarkers, offers insights into PAH pathobiology, and informs discovery of potentially targetable RV-centric pathways.

We have previously identified mitogen-activated protein kinase-activated protein kinase 2 (MK2) is required for caspase-3 nuclear translocation in the execution of apoptosis; however, little is known of the underlying mechanisms. Therefore, we sought to determine the role of kinase and nonkinase functions of MK2 in promoting nuclear translocation of caspase-3. We identified two non-small cell lung cancer cell lines for use in these experiments based on low MK2 expression. Wild-type, enzymatic and cellular localization mutant MK2 constructs were expressed using adenoviral infection. Cell death was evaluated by flow cytometry. In addition, cell lysates were harvested for protein analyses. Phosphorylation of caspase-3 was determined using two-dimensional gel electrophoresis followed by immunoblotting and in vitro kinase assay. Association between MK2 and caspase-3 was evaluated using proximity-based biotin ligation assays and co-immunoprecipitation. Overexpression of MK2 resulted in nuclear translocation of caspase-3 and caspase-3-mediated apoptosis. MK2 directly phosphorylates caspase-3; however, phosphorylation status of caspase-3 or MK2-dependent phosphorylation of caspase-3 did not alter caspase-3 activity. The enzymatic function of MK2 was dispensable in nuclear translocation of caspase-3. MK2 and caspase-3 associated together and a nonenzymatic function of MK2, chaperoned nuclear trafficking, is required for caspase-3-mediated apoptosis. Taken together, our results demonstrate a nonenzymatic role for MK2 in the nuclear translocation of caspase-3. Furthermore, MK2 may function as a molecular switch in regulating the transition between the cytosolic and nuclear functions of caspase-3.

MK2, known to promote caspase-3 nuclear translocation in the execution of apoptosis, is reduced in non-small cell lung cancer cells. In adenocarcinomas of patients, higher MK2 expression is associated with early-stage disease, better clinical response following chemotherapy and independently associated with improved survival.

Program translation aims to translate source code from one programming language to another. It is particularly useful in applications such as multiple-platform adaptation and legacy code migration. Traditional rule-based program translation methods usually rely on meticulous manual rule-crafting, which is costly both in terms of time and effort. Recently, neural network based methods have been developed to address this problem. However, the absence of high-quality parallel code data is one of the main bottlenecks which impedes the development of program translation models. In this paper, we introduce CoST, a new multilingual Code Snippet Translation dataset that contains parallel data from 7 commonly used programming languages. The dataset is parallel at the level of code snippets, which provides much more fine-grained alignments between different languages than the existing translation datasets. We also propose a new program translation model that leverages multilingual snippet denoising auto-encoding and Multilingual Snippet Translation (MuST) pre-training. Extensive experiments show that the multilingual snippet training is effective in improving program translation performance, especially for low-resource languages. Moreover, our training method shows good generalizability and consistently improves the translation performance of a number of baseline models. The proposed model outperforms the baselines on both snippet-level and program-level translation, and achieves state-of-the-art performance on CodeXGLUE translation task. The code, data, and appendix for this paper can be found at https://github.com/reddy-lab-code-research/MuST-CoST.

Pulmonary arterial hypertension (PAH) is a deadly disease characterized by remodeling of the pulmonary vasculature including vascular medial layer hypertrophy and formation of vaso-occlusive lesions, both of which contain pulmonary arterial smooth muscle cells (PASMCs). We have demonstrated that PASMCs from a well-established rat model of disease have increased expression of the membrane protein aquaporin 1 (AQP1) which is associated with increased cell migration, proliferation, and resistance to apoptosis. These pathologic behaviors are mitigated by decreasing AQP1 expression. Understanding the mechanism(s) driving increased AQP1 levels could lead to novel therapeutic targets that treat and potentially reverse disease. Upregulation of AQP1 is a hypoxia inducible factor-1 – related event, while degradation of the protein occurs via ubiquination and the proteasome. Via in silico analysis, we identified two possible caspase-3 cleavage sites followed by a distal ubiquination site near the 3’ end of the AQP1 cytosolic tail. We hypothesized that caspase-3 cleavage results in loss of the AQP1 ubiquination site, allowing for persistence of the cell membrane protein and its pathologic properties. Methods: A proximity-based BioID assay was performed in human embryonic kidney (HEK 293) cells as they do not express native AQP1, using the AQP1 construct alone or AQP1 fused to biotin ligase. Cells were transfected, incubated with cell-permeable biotin (50 μM), lysed, and biotinylated proteins precipitated and immunoblotted for caspase-3. Next the assay was repeated using adenoviral constructs with caspase-3 fused to biotin ligase at the c-terminal end (CTcas) or n-terminal end (NTcas) to ensure biotin ligase did not interfere with enzyme activation and/or activity. Primary PASMCs isolated from distal pulmonary arteries of rats were obtained via removal of the endothelium and enzymatic digestion of the tissue to obtain a pure smooth muscle population. PASMCs were infected with the BioID caspase constructs or wild-type caspase (WTcas) as a control, and precipitated biotinylated proteins were immunoblotted for AQP1. Lastly, PASMCs were cultured, pre-treated with the irreversible caspase-3 inhibitor DEVD or vehicle (30 min) then treated with PBS or the caspase-3 stimulus hydrogen peroxide (500 μM for 24 h). Cells were collected, lysed and immunoblotted for AQP1. Results: The BioID assays demonstrated an interaction between AQP1 and caspase-3 in live cells. Primary PASMCs treated with hydrogen peroxide demonstrate more AQP1 than controls, an effect which is lost when caspase-3 activity is blocked. Conclusions: These data suggest a novel mechanism to control AQP1 protein abundance in PASMCs. Further studies to elucidate the caspase-3 cleavage site necessary for this interaction are underway. Similar studies using PASMCs from animal models of PAH disease are warranted to further validate this potential novel therapeutic target. T 32 HL 007534; F32 HL165766-01 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Introduction: Pulmonary arterial obstruction due to acute thromboembolism causes local reduction or cessation of blood flow. Under normal conditions, pulmonary blood flow exerts shear stress on endothelial cells, known to play an important role in maintaining vascular homeostasis. We hypothesized that pulmonary microvascular endothelial cells exposed to abrupt reductions in shear stress would demonstrate attenuated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and phosphorylation of downstream effectors Akt and endothelial nitric oxide synthase (eNOS). Methods: We tested our hypothesis in primary cultures of human lung microvascular endothelial cells (hLMVECs). We cultured early passage (p≤8) hLMVECs from 5 unique donor lots in standard 6-well culture plates and exposed cells to shear stress using an orbital shaker. Using a fixed depth (volume) and viscosity of tissue culture media, we adjusted the magnitude of shear stress exerted on hLMVECs by changing the angular velocity of orbital rotation. We acclimatized hLMVECs to physiologically-relevant shear stress (~12 dyn/cm2) for 24 hours, then exposed cells to low shear stress (~3 dyn/cm2) or no shear stress (0 dyn/cm2) for 30 minutes. Control cells were maintained at 12 dyn/cm2 of shear stress. In some experiments, PI3K activity was inhibited throughout the acclimatization and shear stress manipulation period by including LY294002 (10 μM) or vehicle (DMSO, 0.1% v/v) in culture media. We prepared whole cell lysates and used western blot to evaluate the expression and phosphorylation of Akt (ser473) and eNOS (ser1177). Results: We found that phosphorylation of Akt, but not Akt expression, decreased significantly (p2) or removed (0 dyn/cm2). Inhibition of PI3K activity with LY294002 further diminished shear-mediated changes in Akt phosphorylation in all 3 groups. Conversely, eNOS phosphorylation was not modified by acute reductions in shear stress, but eNOS expression was markedly attenuated by PI3K inhibition under all conditions. Conclusions: Our results indicate that phosphorylation of Akt, but not its downstream target eNOS, is rapidly attenuated by abrupt reductions in shear stress in hLMVECs. However, shear stress appears to regulate eNOS expression in a PI3K-dependent manner. Taken together, these findings suggest that acute disruption of flow, as seen in pulmonary embolism, may disrupt human lung microvascular endothelial cell homeostasis and nitric oxide bioavailability via multiple PI3K-dependent signaling pathways. This work is supported by HL126514 and HL159906. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Pulmonary arterial hypertension (PAH) is characterized by narrowing of the pulmonary arteries, endothelial cell dysfunction, and an increased vascular cell proliferation. Increased proliferation is also accompanied by glycolytic shift, where glucose is shunted towards anaerobic respiration and production of critical macromolecules. This decreases the available glucose that can be used as a fuel in the tricarboxylic acid (TCA) cycle. To compensate for the lack of TCA cycle metabolites, there is an increase in the use of non-glucose fuels - such as amino acids and fatty acids. However, the effect of this shift on other metabolic pathways is still unknown. In this study, we investigated the levels of metabolites across the pulmonary circulation to define the metabolic shift that cells undergo during PAH. Material & Methods: We carried out metabolomic analysis on serum samples taken from the right and left ventricles (RV and LV) of rats with PAH (SU5416/Hypoxia model: SuHx) and normoxic controls. We also isolated MVECs from each group and performed both targeted and untargeted metabolomics on the cell lysates. Results: Induction of PAH resulted in increased pulmonary artery pressures and RV hypertrophy. Metabolomics of the RV serum were different in PAH versus control rats. This was driven by lower metabolite levels of several amino acids, including glutamine, phenylalanine, and valine. Normoxia- and SuHx-MVEC lysates samples were also distinct, with increased levels of lactic acid contributing significantly to the differences between animals with PAH and controls. Importantly, and in contrast to the serum, where glutamine levels were decreased, we observed increased levels of intracellular glutamine in our analysis. Similarly, intracellular levels of amino acids like phenylalanine, tyrosine and leucine were increased in these lysates. This also contrasted with the serum, where levels of these amino acids were decreased. Interestingly, LV levels of amino acids like valine, glutamine and lysine were similar in normoxic and SuHx samples. However, we did observe increased citrulline levels in SuHx LV serum samples. Comparing the LV/RV ratio in our samples revealed that increased oxidized glutathione was a major determinant of the difference between the SuHx and control groups. Conclusion: Our data suggests that decreased RV serum levels of amino acids in SuHx rats may be driven by increased consumption in the pulmonary vasculature. Given that glutamine levels in the RV are decreased, while being increased intracellularly in MVECs, we hypothesize that glutamine consumption in the lung microvasculature may be driven in part, to produce reduced glutathione. This is also supported by the oxidized glutathione gradient across the pulmonary vasculature in our experiments. These results provide the basis for a mechanistic study aimed at understanding how interrupting glutathione and/or branched-chain amino acid metabolism in MVECs contributes to PAH. NHLBI grant K08HL145132 (J.S.), NHLBI grant R01HL148112 01 (L.S), NHLBI grant R01HL151530 (K.S) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

With the advent of next-generation sequencing technologies, there has been a dramatic increase in the availability of paired clinical and transcriptomic data in a variety of disease states. For basic science researchers, this has provided a valuable opportunity for querying the impact of the transcript levels of a gene on disease survival in humans. However, there are a multitude of methodological and technical considerations to evaluate before embarking on these analyses. Herein, we provide a brief description of statistical considerations involved in these analyses, geared toward basic scientists who may not necessarily routinely use such statistical models as part of their studies.

Pulmonary hypertension (PH) is a debilitating condition characterized by increased pulmonary arterial pressures and remodeling of pulmonary arteries, leading to right heart failure. Women have a higher prevalence of PH, whereas men have more severe disease and poorer outcomes. Animal models also show female-predominant disease. Despite the known sex differences in PH, little is known about how pathogenesis differs between the sexes. There is growing evidence of mitochondrial dysfunction, as well as altered mitophagy in PH. We hypothesized that sexual dimorphism contributes to mitochondrial dysfunction and altered mitophagy in PH. Using mouse lung endothelial cells, we exposed both wild-type and

Oral cavity squamous cell carcinoma (OCSCC) is a devastating disease, causing substantial morbidity and mortality. While many OCSCCs arise from an existing dysplastic lesion, not all oral premalignant lesions progress to OCSCC. Current methods for oral premalignancy and OCSCC diagnosis (visual and tactile exam followed by tissue biopsy and histologic evaluation) cannot discriminate between benign inflammatory changes and high-risk premalignant lesions that require interventions, underscoring the need for molecular-based biomarkers. The multi-step cancer progression from normal epithelium to premalignant lesion and invasive SCC is driven by the accumulation of genetic alterations, including changes in mitochondrial DNA (mtDNA). Due to the lack of protective histones and limited repair mechanisms, mtDNA is susceptible to damage by environmental carcinogens and reactive oxygen species, a byproduct of the oxidative phosphorylation system. As a result, mutation rate in mtDNA is ~10 times higher than in nuclear DNA, and may greatly facilitate the risk of mitochondrial dysfunction. Previous studies underscore that acquisition of somatic mtDNA mutations directly involved in tumorigenesis, and not merely epiphenomena. However, the impact of these studies is limited by an incomplete understanding of mitochondrial genomic alterations in the transition of preneoplastic lesions to invasive disease. In this study we used a unique cohort of 27 patients with matched longitudinally collected samples (histologically normal mucosa, dysplastic lesion, and SCC) coupled with novel ultra-deep mitochondrial sequencing (mtDNA-Seq) method to assess the mtDNA mutational landscape throughout the continuum of OCSCC progression. Using a custom bioinformatics workflow, somatic mutations were detected in a subset of the premalignant lesions, with overall higher mutational load observed in OCSCC specimens. While sequencing revealed a large degree of inter-patient heterogeneity, a panel of non-synonymous aberrations were present in both premalignant and invasive neoplasms. The majority of shared mutations showed an increase in fractional abundance in tumors, compared to the precursor lesions (suggesting a spatial expansion of these clones as they progressed histologically), and were enriched for coding mutations in complex-I subunits (critical region for ATP production), which is associated with an oncogenic phenotype. Additionally, mtDNA content increased in OCSCC tumor in a subset of patients, suggesting a cell compensation for defective oxidative phosphorylation and lower ATP production per mitochondria. Here we report the first comprehensive characterization of mitochondrial mutational landscape in dysplastic and invasive SCC lesions, and reveal key molecular events associated with the transition from non-invasive to invasive state. Citation Format: Alka Singh, Ashwin Lakshman Koppayi, Ping Wu, Mark Lingen, Vasudha Mishra, Alexander Pearson, Ari Rosenberg, Nishant Agrawal, Karthik Suresh, Evgeny Izumchenko. Ultra-deep sequencing of mitochondrial genome to explore the dynamic mutational changes associated with oral cavity squamous cell carcinoma progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4845.

Immune-related adverse events (irAEs) associated with immune checkpoint inhibitor (ICI) therapy may vary substantially in their clinical presentation, including natural history, outcomes to treatment, and patterns. The application of clinical guidelines for irAE management can be challenging for practitioners due to a lack of common or consistently applied terminology. Furthermore, given the growing body of clinical experience and published data on irAEs, there is a greater appreciation for the heterogeneous natural histories, responses to treatment, and patterns of these toxicities, which is not currently reflected in irAE guidelines. Furthermore, there are no prospective trial data to inform the management of the distinct presentations of irAEs. Recognizing a need for uniform terminology for the natural history, response to treatment, and patterns of irAEs, the Society for Immunotherapy of Cancer (SITC) convened a consensus panel composed of leading international experts from academic medicine, industry, and regulatory agencies. Using a modified Delphi consensus process, the expert panel developed clinical definitions for irAE terminology used in the literature, encompassing terms related to irAE natural history (ie, re-emergent, chronic active, chronic inactive, delayed/late onset), response to treatment (ie, steroid unresponsive, steroid dependent), and patterns (ie, multisystem irAEs). SITC developed these definitions to support the adoption of a standardized vocabulary for irAEs, which will have implications for the uniform application of irAE clinical practice guidelines and to enable future irAE clinical trials.

This commentary outlines the strategy employed by a multidisciplinary immune-related toxicity team to evaluate patients who may be at high risk for the development of immune-related toxicity, in particular, those with preexisting lung conditions and subsequent immune-related pneumonitis.

Lung cancer is the leading cause of cancer-related deaths worldwide. As new therapies are developed, it is important to understand the pulmonary toxicities associated with systemic lung cancer therapies. Cytotoxic chemotherapy regimens for NSCLC often include taxanes. Pulmonary toxicity from taxanes presents as an ILD-type reaction characterized by increasing dyspnoea, dry cough, fever and bilateral pulmonary interstitial infiltrates. The incidence of taxane-induced pneumonitis is rare, and many patients respond to steroid therapy; however, fatal cases have been reported. Patients with NSCLC are routinely tested for the presence of tumour oncogenes to determine their candidacy for targeted therapies, such as TKI. EGFR-TKI can cause pneumonitis characterized by progressive dyspnoea and hypoxia. EGFR-TKI-associated ILD rarely presents as an AIP with rapidly progressive respiratory failure and high mortality rates. The most recent development in lung cancer therapy has been the discovery of immune checkpoint inhibitor (ICI). ICI pneumonitis has been increasingly recognized as a common complication of ICI therapy, with reported incidence as high as 19% in some clinical settings. Early-grade ICI pneumonitis may be asymptomatic; however, high-grade ICI pneumonitis can result in progressive dyspnoea, hypoxia and respiratory failure. ICI pneumonitis is unique in that only half of the patients will improve with steroid treatment, and mortality rates are high. As treatment of NSCLC evolves, providers must be able to recognize and respond to the development of drug-induced pulmonary toxicities.

Pulmonary arterial hypertension (PAH) is an incurable disease characterized by disordered and dysfunctional angiogenesis leading to small-vessel loss and an obliterative vasculopathy. The pathogenesis of PAH is not fully understood, but multiple studies have demonstrated links between elevated angiostatic factors, disease severity, and adverse clinical outcomes. ES (endostatin), one such circulating angiostatic peptide, is the cleavage product of the proteoglycan COL18A1 (collagen α1[XVIII] chain). Elevated serum ES is associated with increased mortality and disease severity in PAH. A nonsynonymous variant of ES (aspartic acid-to-asparagine substitution at amino acid 104; p.D104N) is associated with differences in PAH survival. Although COL18A1/ES expression is markedly increased in remodeled pulmonary vessels in PAH, the impact of ES on pulmonary endothelial cell (PEC) biology and molecular contributions to PAH severity remain undetermined. In the present study, we characterized the effects of exogenous ES on human PEC biology and signaling. We demonstrated that ES inhibits PEC migration, proliferation, and cell survival, with significant differences between human variants, indicating that they are functional genetic variants. ES promotes proteasome-mediated degradation of the transcriptional repressor ID1, increasing expression and release of TSP-1 (thrombospondin 1). ES inhibits PEC migration via an ID1/TSP-1/CD36-dependent pathway, in contrast to proliferation and apoptosis, which require both CD36 and CD47. Collectively, the data implicate ES as a novel negative regulator of ID1 and an upstream propagator of an angiostatic signal cascade converging on CD36 and CD47, providing insight into the cellular and molecular effects of a functional genetic variant linked to altered outcomes in PAH.

Mutations in mitochondrial DNA (mtDNA) have been linked to risk, progression, and treatment response of head and neck squamous cell carcinoma (HNSCC). Due to their clonal nature and high copy number, mitochondrial mutations could serve as powerful molecular markers for detection of cancer cells in bodily fluids, surgical margins, biopsies and lymph node (LN) metastasis, especially at sites where tumor involvement is not histologically apparent. Despite a pressing need for high-throughput, cost-effective mtDNA mutation profiling system, current methods for library preparation are still imperfect for detection of low prevalence heteroplasmic mutations. To this end, we have designed an ultra-deep amplicon-based sequencing library preparation approach that covers the entire mitochondrial genome. We sequenced mtDNA in 28 HNSCCs, matched LNs, surgical margins and bodily fluids, and applied multiregional sequencing approach on 14 primary tumors. Our results demonstrate that this quick, sensitive and cost-efficient method allows obtaining a snapshot on the mitochondrial heterogeneity, and can be used for detection of low frequency tumor-associated mtDNA mutations in LNs, sputum and serum specimens. These findings provide the foundation for using mitochondrial sequencing for risk assessment, early detection, and tumor surveillance.

Non-small cell lung cancers demonstrate intrinsic resistance to cell death even in response to chemotherapy. Previous work suggested that defective nuclear translocation of active caspase 3 may play a role in resistance to cell death. Separately, our group has identified that mitogen activated protein kinase activated protein kinase 2 (MK2) is required for nuclear translocation of active caspase 3 in the execution of apoptosis. This study demonstrates a relatively low expression of MK2 in non-small cell lung carcinoma cell lines compared to small cell carcinoma cell lines. Further, overexpression of MK2 in non-small cell lung carcinoma cell lines results in increased caspase 3 activity and caspase 3 mediated cell death. Higher MK2 transcript levels were observed in patients with earlier-stage non-small cell lung cancer. Higher expression of MK2 is associated with better survival in patients with early stage non-small cell lung cancer across two independent clinical datasets. Using data sets spanning multiple cancer types, we observed improved survival with higher MK2 expression was unique to lung adenocarcinoma. Mechanistically, MK2 promotes nuclear translocation of caspase 3 leading to PARP1 cleavage and execution of cell death. While MK2 can directly phosphorylate caspase 3, neither phosphorylation status of caspase 3 nor the kinase activity of MK2 impacts caspase 3 activation, nuclear translocation and execution of cell death. Rather, a non-kinase function of MK2, specifically trafficking via its nuclear localization sequence, is required for caspase 3 mediated cell death. In summary this study highlights the importance of a non-enzymatic function of MK2 in the execution of apoptosis, which may be leveraged in the adjunctive treatment of NSCLC or other conditions where regulation of apoptosis is crucial.

In patients with non–small-cell lung cancer treated with anti–PD-1/PD-L1 agents, no specific radiation parameter was significantly associated with immune-related (IR) pneumonitis. We identify on subset analysis of patients who developed IR pneumonitis and received chest radiation, patients were numerically more likely to have received chest radiation with curative intent than with palliative intent (89% vs. 11 that approached statistical significance. PURPOSE: To investigate the relationship between radiotherapy (RT), in particular chest RT, and development of immune-related (IR) pneumonitis in non–small-cell lung cancer (NSCLC) patients treated with anti–programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-LI). PATIENTS AND METHODS: Between June 2011 and July 2017, NSCLC patients treated with anti–PD-1/PD-L1 at a tertiary-care academic cancer center were identified. Patient, treatment, prior RT (intent, technique, timing, courses), and IR pneumonitis details were collected. Treating investigators diagnosed IR pneumonitis clinically. Diagnostic IR pneumonitis scans were overlaid with available chest RT plans to describe IR pneumonitis in relation to prior chest RT. We evaluated associations between patient, treatment, RT details, and development of IR pneumonitis by Fisher exact and Wilcoxon rank-sum tests. RESULTS: Of the 188 NSCLC patients we identified, median follow-up was 6.78 (range, 0.30–79.3) months and median age 66 (range, 39–91) years; 54% (n = 102) were male; and 42% (n = 79) had stage I-III NSCLC at initial diagnosis. Patients received anti–PD-1/PD-L1 monotherapy (n = 127, 68%) or PD-I/PD-LI -based combinations (n = 61, 32%). In the entire cohort, 70% (132/188) received any RT, 53% (100/188) chest RT, and 37% (70/188) curative-intent chest RT. Any grade IR pneumonitis occurred in 19% (36/188; 95% confidence interval, 13.8–25.6). Of those who developed IR pneumonitis and received chest RT (n = 19), patients were more likely to have received curative-intent versus palliative-intent chest RT (17/19, 89%, vs. 2/19, 11%; P = .051). Predominant IR pneumonitis appearances were ground-glass opacities outside high-dose chest RT regions. CONCLUSION: No RT parameter was significantly associated with IR pneumonitis. On subset analysis of patients who developed IR pneumonitis and who had received prior chest RT, IR pneumonitis was more common in patients who received curative-intent chest RT. Attention should be paid to NSCLC patients receiving curative-intent RT followed by anti –PD-I/PD-LI agents.

Background: Immune checkpoint inhibitors (ICIs) may cause immune-related adverse events (irAEs). Methods to obtain real-time multidisciplinary input for irAEs that require subspecialist care are unknown. This study aimed to determine whether a virtual multidisciplinary immune-related toxicity (IR-tox) team of oncology and medicine subspecialists would be feasible to implement, be used by oncology providers, and identify patients for whom multidisciplinary input is sought. Patients and Methods: Patients treated with ICIs and referred to the IR-tox team in August 2017 through March 2018 were identified. Feasibility was defined as receipt of electronic referrals and provision of recommendations within 24 hours of referral. Use was defined as the proportion of referring providers who used the team’s recommendations, which was determined through a postpilot survey. Demographics and tumor, treatment, and referral data were collected. Patient features and irAE associations were analyzed. Results: The IR-tox team was found to be feasible and used: 117 referrals from 102 patients were received in 8 months, all providers received recommendations within 24 hours, 100% of surveyed providers used the recommendations, and 74% changed patient management based on IR-tox team recommendations. Referrals were for suspected irAEs (n=106; 91%) and suitability to treat with ICIs (n=11; 10%). In referred patients, median age was 64 years, 54% were men, 13% had prior autoimmunity, and 46% received ICI combinations versus monotherapy (54%). The most commonly referred toxicities were pneumonitis (23%), arthritis (16%), and dermatitis (15%); 15% of patients had multisystem toxicities. Multiple referrals were more common in those treated with combination ICIs (odds ratio [OR], 6.0; P=.035) or with multisystem toxicities (OR, 8.1; P=.005). The IR-tox team provided a new multidisciplinary forum to assist providers in diagnosing and managing complex irAEs. This model identifies educational and service needs, and patients with irAEs for whom multidisciplinary care is most sought. Conclusions: A virtual multidisciplinary toxicity team for irAEs was a feasible and used service, and facilitated toxicity identification and management.

Noncanonical roles for caspase-3 are emerging in the fields of cancer and developmental biology. However, little is known of nonapoptotic functions of caspase-3 in most cell types. We have recently demonstrated a disassociation between caspase-3 activation and execution of apoptosis with accompanying cytoplasmic caspase-3 sequestration and preserved endothelial barrier function. Therefore, we tested the hypothesis that nonapoptotic caspase-3 activation promotes endothelial barrier integrity. Human lung microvascular endothelial cells were exposed to thrombin, a nonapoptotic stimulus, and endothelial barrier function was assessed using electric cell-substrate impedance sensing. Actin cytoskeletal rearrangement and paracellular gap formation were assessed using phalloidin staining. Cell stiffness was evaluated using magnetic twisting cytometry. In addition, cell lysates were harvested for protein analyses. Caspase-3 was inhibited pharmacologically with pan-caspase and a caspase-3-specific inhibitor. Molecular inhibition of caspase-3 was achieved using RNA interference. Cells exposed to thrombin exhibited a cytoplasmic activation of caspase-3 with transient and nonapoptotic decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery. Inhibition of caspases led to a more pronounced and rapid drop in thrombin-induced endothelial barrier function, accompanied by increased endothelial cell stiffness and paracellular gaps. Caspase-3-specific inhibition and caspase-3 knockdown both resulted in more pronounced thrombin-induced endothelial barrier disruption. Taken together, our results suggest cytoplasmic caspase-3 has nonapoptotic functions in human endothelium and can promote endothelial barrier integrity.

With increasing use of immune checkpoint inhibitors (ICIs) for advanced NSCLC, there is increasing recognition of immune-related adverse events associated with ICI use. We recently reported increased incidence of checkpoint inhibitor pneumonitis (CIP) in ICI-treated NSCLC patients. Since development of immune-related adverse events in other organ systems has been associated with either no change or even improvement in tumor response/cancer outcomes, we sought to better understand the impact of CIP development on overall survival in ICI-treated NSCLC patients. Using baseline and follow-up data collected on a cohort of 205 ICI-treated NSCLC patients, we used a multi-state modeling approach to understand the effect of developing CIP on the risk of death. We observed time-dependent changes in risk of developing and recovery from CIP, with an increased risk of both developing and recovering from CIP in the first year after initiating ICI. We found that developing CIP independently increased the risk of transitioning to death in both adjusted and unadjusted models. In the multivariate model, we found that the increase in mortality associated with CIP was only seen in patients with adenocarcinoma tumor histology. Collectively, these findings suggest that in NSCLC, development of CIP worsens survival in patients receiving immunotherapy.

Immune checkpoint inhibitors (ICIs) are newer, immunotherapy-based drugs that have been shown to improve survival in advanced non-small cell lung cancer (NSCLC). Unlike traditional chemotherapeutic agents, ICIs work by boosting the body's natural tumor killing response. However, this unique mechanism of action has also led to the recognition of class-specific side effects. Labeled immune-related adverse events, these toxicities can affect multiple organ systems including the lungs. Immune-mediated lung injury because of ICI use, termed checkpoint inhibitor pneumonitis (CIP), occurs in about 3% to 5% of patients receiving ICIs; however, the real-world incidence of this entity may be higher, especially now that ICIs are being used in nonclinical trial settings. In this review, we briefly introduce the biology of ICIs and the indications for ICI use in NSCLC and then discuss the epidemiology and clinical and radiologic manifestations of CIP. Next, we discuss management strategies for CIP, including the current consensus on management of steroid-refractory CIP. Given the nascent nature of this field, we highlight areas of uncertainty and emerging research questions in the burgeoning field of checkpoint inhibitor pulmonary toxicity.

Current treatments for pneumonia (PNA) are focused on the pathogens. Mortality from PNA-induced acute lung injury (PNA-ALI) remains high, underscoring the need for additional therapeutic targets. Clinical and experimental evidence exists for potential sex differences in PNA survival, with males having higher mortality. In a model of severe pneumococcal PNA, when compared with male mice, age-matched female mice exhibited enhanced resolution characterized by decreased alveolar and lung inflammation and increased numbers of Tregs. Recognizing the critical role of Tregs in lung injury resolution, we evaluated whether improved outcomes in female mice were due to estradiol (E2) effects on Treg biology. E2 promoted a Treg-suppressive phenotype in vitro and resolution of PNA in vivo. Systemic rescue administration of E2 promoted resolution of PNA in male mice independent of lung bacterial clearance. E2 augmented Treg expression of Foxp3, CD25, and GATA3, an effect that required ERβ, and not ERα, signaling. Importantly, the in vivo therapeutic effects of E2 were lost in Treg-depleted mice (Foxp3DTR mice). Adoptive transfer of ex vivo E2-treated Tregs rescued Streptococcus pneumoniae–induce PNA-ALI, a salutary effect that required Treg ERβ expression. E2/ERβ was required for Tregs to control macrophage proinflammatory responses. Our findings support the therapeutic role for E2 in promoting resolution of lung inflammation after PNA via ERβ Tregs.

Chemical agents released into the environment can induce oxidative stress in organisms, which is detrimental for health. Although environmental exposures typically incorporate multiple chemicals, organismal studies on oxidative stress derived from chemical agents commonly study exposures to individual compounds. In this work, we explore how chemical mixtures drive the oxidative stress response under various conditions in the nematode C. elegans, by quantitatively assessing levels of gst-4 expression. Our results indicate that naphthoquinone mixtures drive responses differently than individual components, and that altering environmental conditions, such as increased heat and reduced food availability, result in dramatically different oxidative stress responses mounted by C. elegans. When exposed to heat, the oxidative stress response is diminished. Notably, when exposed to limited food, the oxidative stress response specific to juglone is significantly heightened, while identified antagonistic interactions between some naphthoquinone components in mixtures are abolished. This implies that organismal responses to xenobiotics is confounded by environment and stressor interactions. Given the high number of variables under study, and their potential combinations, a simplex centroid design was used to capture such non-trivial response over the design space. This makes the case for the adoption of Design of Experiments approaches as they can greatly expand the experimental space probed in noisy biological readouts, and in combinatorial experiments. Our results also reveal gaps in our current knowledge of the organismal oxidative stress response, which can be addressed by employing sophisticated design of experiments approaches to identify significant interactions.

Background/Hypothesis: Pulmonary Arterial Hypertension (PAH) is a life-threatening disease characterized by loss of pulmonary microvessels and vascular remodeling. Loss of function BMPR2 mutations contribute to pulmonary endothelial cell (EC) apoptosis and oxidative stress, but their reduced penetrance suggests need for additional modifiers. Carboxylesterase1 (CES1) is endoplasmic reticulum (ER) enzyme responsible for detoxification, proteostasis, and redox balance. Similar to BMPR2 mutations, we found that loss of CES1 is associated with oxidative stress and apoptosis. In this study, we explore a plausible link between BMPR2 pathway and CES1 regulation in promoting EC survival and angiogenesis. Methods: PECs & lung tissue from healthy donors and PAH patients were obtained from PHBI. To induce oxidative stress, we used H 2 O 2 (100 μM) & methamphetamine HCl (METH, 0.5-2mM). Both siRNA and pharmacological approaches were used to inhibit BMPR2, Nrf2, and CES1 expression. Caspase and Matrigel assays were used to assess PEC survival and tube formation, respectively. Results: RNAseq of BMPR2-mutant PECs showed significantly less CES1 expression, which correlated with reduced protein expression in PEC lysates and within lung vascular lesions. In healthy PECs, BMP9 stimulation led to increase in CES1 expression that was absent post BMPR2 knockdown. CES1 gene transcription was by BMPR2-dependent activation of Nrf2, a transcription factor responsible for antioxidant gene expression and mitochondrial biogenesis. Inhibition of Nrf2 activation by ML385 (5μM) abrogates BMP9 induced CES1 mRNA levels similar to BMPR2 knockdown. The connection between BMPR2 and CES1 was further strengthened by CES1 knockdown studies in PECs that demonstrated reduction in BMPR2 protein synthesis associated with ER stress and reduced autophagy. Finally, lung examination in CRISPR generated CES1 +/- mice demonstrated increased microvascular muscularization at normoxia compared to wild type mice. Conclusion: BMPR2 and CES1 are part of common signaling pathway that protects PECs against oxidative stress and mitochondrial damage through a positive feedback loop. Interventions that restore CES1 activity could rescue BMPR2 signaling and serve as novel PAH therapeutics.

Background: Chronic obstructive pulmonary disease (COPD) is characterized by recurrent exacerbations. Macrophages play a critical role in immune response and tissue repair in COPD. Airway macrophages (AM) are exposed to environmental exposures which are retained in the cytoplasmic material. Both biomass and particulate matter have been linked to higher AM black carbon. It is unknown if AM black carbon is associated with COPD morbidity and macrophage phenotype. Methods: Former smokers with COPD were enrolled and sputum induction was performed to obtain airway macrophages. Macrophages underwent black carbon quantification and flow cytometry phenotyping. Health information was obtained the same day as sputum induction and prospective exacerbations were assessed by monthly telephone calls. Results: We studied 30 former smokers with COPD who had a mean age of 67 years and mean forced expiratory volume in 1 second (FEV(1))% predicted of 60.8%. Higher AM black carbon content was associated with increased total exacerbations and severe exacerbations and reduced CD80 expression. Conclusion: AM black carbon association with respiratory morbidity is largely unexplored and this is the first study to identify association with prospective exacerbations. Macrophages expressed reduced CD80, a surface marker providing costimulatory signals required for development of antigen-specific immune responses. Our findings suggest that reduced CD80 expression is the pathophysiologic mechanism for the association of AM black carbon content and increased exacerbations. Therefore, beyond solely serving as a marker for increased exposures, AM black carbon content may be a predictor of future exacerbations given a macrophage less equipped to respond to an acute infectious exposure.

BackgroundPneumonitis from immune checkpoint inhibitors (ICI) is a potentially fatal immune-related adverse event (irAE) from antiprogrammed death 1/programmed death ligand 1 immunotherapy. Most cases of ICI pneumonitis improve or resolve with 4–6 weeks of corticosteroid therapy. Herein, we report the incidence, clinicopathological features and management of patients with non-small cell lung cancer (NSCLC) and melanoma who developed chronic ICI pneumonitis that warrants ≥12 weeks of immunosuppression.MethodsPatients with ICI pneumonitis were identified from institutional databases of ICI-treated patients with advanced melanoma and NSCLC between January 2011 and July 2018. ICI pneumonitis was defined as clinical/radiographic evidence of lung inflammation without alternative diagnoses, adjudicated by a multidisciplinary team. Chronic ICI pneumonitis was defined as pneumonitis that persists or worsens with steroid tapering, and necessitates ≥12 weeks of immunosuppression, after ICI discontinuation. Serial chest CT was used to assess radiological features, and tumor response by Response EvaluationCriteria for Solid Tumors V.1.1. Bronchoalveolar lavage fluid (BALF) samples were assessed by cell differential. Lung biopsy samples were evaluated by H&E staining and multiplex immunofluorescence (mIF), where available.ResultsAmong 299 patients, 44 developed ICI pneumonitis (NSCLC: 5/205; melanoma: 1/94), and of these, 6 experienced chronic ICI pneumonitis. The overall incidence of chronic ICI pneumonitis was thus 2%. Of those who developed chronic ICI pneumonitis: the majority had NSCLC (5/6), all sustained disease control from ICIs, and none had other concurrent irAEs. Timing of chronic ICI pneumonitis development was variable (range: 0–50 months), and occurred at a median of 12 months post ICI start. Recrudescence of ICI pneumonitis occurred at a median of 6 weeks after initial steroid start (range: 3–12 weeks), with all patients requiring steroid reintroduction when tapered to ≤10 mg prednisone/equivalent. The median total duration of steroids was 37 weeks (range: 16–43+weeks). Re-emergence of radiographic ICI pneumonitis occurred in the same locations on chest CT, in most cases (5/6). All patients who developed chronic ICI pneumonitis had BALF lymphocytosis on cell differential and organising pneumonia on lung biopsy at initial ICI pneumonitis presentation, with persistent BALF lymphocytosis and brisk CD8+ infiltration on mIF at pneumonitis re-emergence during steroid taper.ConclusionsA subset of patients who develop pneumonitis from ICIs will develop chronic ICI pneumonitis, that warrants long-term immunosuppression of ≥12 weeks, and has distinct clinicopathological features.

We report the synthesis of a TiO2 hybrid structure, consisting of a combination of nanorods and nanoparticles, subsequently treated with hydrazine to enhance the performance of photoelectrochemical (PEC) hydrogen (H2) generation. The optimized TiO2 hybrid photoanode sensitized with Quantum dots (QDs), yields a saturated photocurrent density of 4.25 mA cm−2 (at 0.8 V vs RHE), which is 172% higher than that of the reference sample. The optimized hybrid photoanode treated with hydrazine exhibits an additional 28% increase in the saturated photocurrent density, reaching 5.43 mA cm−2 with CdS QDs, and 8.12 mA cm−2 with CdS/CdSe QDs (at 0.8 V vs RHE), while maintaining 80% of the initial value of photocurrent density, after 2 h of continuous one sun illumination (AM 1.5 G, 100 mW cm−2). We used Density Functional Theory with Hubbard energy correction (DFT + U) calculations to describe the mechanism that underpins this significant improvement. DFT + U results highlighted that the concentration of the hydrazine treatment plays a crucial role and affects the sites (surface or interstitial) where nitrogen may be present. This eventually affects the recombination centers within the hybrid photoanode. Thus, the results of this work define a promising strategy to optimize the morphology of the hybrid photoanodes via hydrazine surface engineering to fabricate efficient and stable PEC water splitting devices for H2 generation.