Disparities and guideline adherence for HPV testing among patients with oropharyngeal squamous cell carcinoma, NCDB, and SEER.

BACKGROUND: Human papilloma virus testing for oropharyngeal squamous-cell carcinoma has been recommended by the National Comprehensive Cancer Network since 2012. We examine disparities, reported rates of human papillomavirus (HPV) testing, and the impact on these findings of limitations with the variable in database registries. METHODS: The HPV variable was queried for patients with oropharyngeal squamous carcinoma (OPSCC) from 2013 to 2016 in National Cancer Data Base (NCDB) and Surveillance, Epidemiology, and End Results (SEER). Multivariable regression was used to identify disparities based on sociodemographic variables. Sensitivity analyses were used to investigate limitations of the variable. RESULTS: Despite limitations in the HPV variable in the databases, there was less than 100% adherence to recommended testing, and there were significant disparities in multiple sociodemographic variables. For example, in NCDB 70% of white versus 60.4% of black patients were tested (odds ratio [OR] 0.75, confidence interval [CI] 0.66-0.85, p ≤ 0.0001); in SEER 59.8% of white and 47.6% of black patients were tested (OR 0.73, CI 0.67-0.81; p ≤ 0.0001). CONCLUSIONS: Disparities exist among patients undergoing testing for HPV-associated OPSCC and adherence to guideline recommended HPV testing has been suboptimal. In addition, the HPV variable definition, especially as it relates to p16 positivity, and use in these two registries should be improved.

Variation in use of postoperative chemoradiation following surgery for T1 and T2 oropharyngeal squamous cell carcinoma; National Cancer Database.

BACKGROUND AND OBJECTIVES: Primary surgical treatment of patients with early T-classification (T1-T2) oropharyngeal squamous cell carcinoma (OPSCC) has increased. We sought to determine how often these patients receive postoperative chemoradiation (CRT). METHODS: Patients with T1-T2 OPSCC in the National Cancer Database who underwent primary surgery were evaluated for receipt of postoperative CRT. Postoperative CRT use was examined among patients with high risk factors (positive margins and/or extracapsular spread [ECS]), intermediate risk factors (negative margins, no ECS, and either pT3-4 and/or N2-N3), and no apparent risk factors. RESULTS: Of 4833 patients with T1-T2 OPSCC who underwent primary surgery, 43% had high risk pathologic factors, of whom only 63% received postoperative CRT. Another 31% had no apparent risk factors, of whom 16% nonetheless received postoperative CRT. On multivariable analysis, in addition to tumor and demographic factors, patients treated at community hospitals were more likely to receive postoperative CRT (O.R. 1.41 C.I. 1.18-1.87, P = 0.001). CONCLUSIONS: Variation in postoperative CRT use indicates a lack of consensus and/or knowledge about its benefits and indications. Usage of postoperative CRT regardless of pathologic risk factors suggests an area where future efforts at implementation of best practices may be targeted.

Intratracheal bleomycin causes airway remodeling and airflow obstruction in mice.

In addition to parenchymal fibrosis, fibrotic remodeling of the distal airways has been reported in interstitial lung diseases. Mechanisms of airway wall remodeling, which occurs in a variety of chronic lung diseases, are not well defined and current animal models are limited. The authors quantified airway remodeling in lung sections from subjects with idiopathic pulmonary fibrosis (IPF) and controls. To investigate intratracheal bleomycin as a potential animal model for fibrotic airway remodeling, the authors evaluated lungs from C57BL/6 mice after bleomycin treatment by histologic scoring for fibrosis and peribronchial inflammation, morphometric evaluation of subepithelial connective tissue volume density, TUNEL (terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling) assay, and immunohistochemistry for transforming growth factor ß1 (TGFß1), TGFß2, and the fibroblast marker S100A4. Lung mechanics were determined at 3 weeks post bleomycin. IPF lungs had small airway remodeling with increased bronchial wall thickness compared to controls. Similarly, bleomycin-treated mice developed dose-dependent airway wall inflammation and fibrosis and greater airflow resistance after high-dose bleomycin. Increased TUNEL(+) bronchial epithelial cells and peribronchial inflammation were noted by 1 week, and expression of TGFß1 and TGFß2 and accumulation of S100A4(+) fibroblasts correlated with airway remodeling in a bleomycin dose-dependent fashion. IPF is characterized by small airway remodeling in addition to parenchymal fibrosis, a pattern also seen with intratracheal bleomycin. Bronchial remodeling from intratracheal bleomycin follows a cascade of events including epithelial cell injury, airway inflammation, profibrotic cytokine expression, fibroblast accumulation, and peribronchial fibrosis. Thus, this model can be utilized to investigate mechanisms of airway remodeling.

Low-level laser therapy activates NF-kB via generation of reactive oxygen species in mouse embryonic fibroblasts.

BACKGROUND: Despite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying photobiomodulation at a cellular level remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we isolated murine embryonic fibroblasts (MEF) from transgenic NF-kB luciferase reporter mice and studied their response to 810 nm laser radiation. Significant activation of NF-kB was observed at fluences higher than 0.003 J/cm(2) and was confirmed by Western blot analysis. NF-kB was activated earlier (1 hour) by LLLT compared to conventional lipopolysaccharide treatment. We also observed that LLLT induced intracellular reactive oxygen species (ROS) production similar to mitochondrial inhibitors, such as antimycin A, rotenone and paraquat. Furthermore, we observed similar NF-kB activation with these mitochondrial inhibitors. These results, together with inhibition of laser induced NF-kB activation by antioxidants, suggests that ROS play an important role in the laser induced NF-kB signaling pathways. However, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that LLLT also upregulates mitochondrial respiration. CONCLUSION: We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive NFkB signaling via generation of ROS. Expression of anti-apoptosis and pro-survival genes responsive to NFkB could explain many clinical effects of LLLT.

Reducing the activity and secretion of microbial antioxidants enhances the immunogenicity of BCG.

BACKGROUND: In early clinical studies, the live tuberculosis vaccine Mycobacterium bovis BCG exhibited 80% protective efficacy against pulmonary tuberculosis (TB). Although BCG still exhibits reliable protection against TB meningitis and miliary TB in early childhood it has become less reliable in protecting against pulmonary TB. During decades of in vitro cultivation BCG not only lost some genes due to deletions of regions of the chromosome but also underwent gene duplication and other mutations resulting in increased antioxidant production. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether microbial antioxidants influence vaccine immunogenicity, we eliminated duplicated alleles encoding the oxidative stress sigma factor SigH in BCG Tice and reduced the activity and secretion of iron co-factored superoxide dismutase. We then used assays of gene expression and flow cytometry with intracellular cytokine staining to compare BCG-specific immune responses in mice after vaccination with BCG Tice or the modified BCG vaccine. Compared to BCG, the modified vaccine induced greater IL-12p40, RANTES, and IL-21 mRNA in the spleens of mice at three days post-immunization, more cytokine-producing CD8+ lymphocytes at the peak of the primary immune response, and more IL-2-producing CD4+ lymphocytes during the memory phase. The modified vaccine also induced stronger secondary CD4+ lymphocyte responses and greater clearance of challenge bacilli. CONCLUSIONS/SIGNIFICANCE: We conclude that antioxidants produced by BCG suppress host immune responses. These findings challenge the hypothesis that the failure of extensively cultivated BCG vaccines to prevent pulmonary tuberculosis is due to over-attenuation and suggest instead a new model in which BCG evolved to produce more immunity-suppressing antioxidants. By targeting these antioxidants it may be possible to restore BCG's ability to protect against pulmonary TB.

Electrophilic cyclopentenone neuroprostanes are anti-inflammatory mediators formed from the peroxidation of the omega-3 polyunsaturated fatty acid docosahexaenoic acid.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) possesses potent anti-inflammatory properties and has shown therapeutic benefit in numerous inflammatory diseases. However, the molecular mechanisms of these anti-inflammatory properties are poorly understood. DHA is highly susceptible to peroxidation, which yields an array of potentially bioactive lipid species. One class of compounds are cyclopentenone neuroprostanes (A(4)/J(4)-NPs), which are highly reactive and similar in structure to anti-inflammatory cyclopentenone prostaglandins. Here we show that a synthetic A(4)/J(4)-NP, 14-A(4)-NP (A(4)-NP), potently suppresses lipopolysaccharideinduced expression of inducible nitric-oxide synthase and cyclooxygenase-2 in macrophages. Furthermore, A(4)-NP blocks lipopolysaccharide-induced NF-kappaB activation via inhibition of Ikappa kinase-mediated phosphorylation of IkappaBalpha. Mutation on Ikappa kinase beta cysteine 179 markedly diminishes the effect of A(4)-NP, suggesting that A(4)-NP acts via thiol modification at this residue. Accordingly, the effects of A(4)-NP are independent of peroxisome proliferator-activated receptor-gamma and are dependent on an intact reactive cyclopentenone ring. Interestingly, free radical-mediated oxidation of DHA greatly enhances its anti-inflammatory potency, an effect that closely parallels the formation of A(4)/J(4)-NPs. Furthermore, chemical reduction or conjugation to glutathione, both of which eliminate the bioactivity of A(4)-NP, also abrogate the anti-inflammatory effects of oxidized DHA. Thus, we have demonstrated that A(4)/J(4)-NPs, formed via the oxidation of DHA, are potent inhibitors of NF-kappaB signaling and may contribute to the anti-inflammatory actions of DHA. These findings have implications for understanding the anti-inflammatory properties of omega-3 fatty acids, and elucidate novel interactions between lipid peroxidation products and inflammation.

Yin Yang 1 enhances cyclooxygenase-2 gene expression in macrophages.

Expression of cyclooxygenase-2 (COX-2) is associated with the pathogenesis of inflammation and various cancers, including lung cancer. Yin Yang 1 (YY1) is a zinc-finger transcription factor that interacts with histone acetyltransferases and deacetylases for its transcriptional activity and also is involved in inflammation and tumorigenesis. We investigated whether YY1 regulates COX-2 expression. We located a possible YY1 binding site proximal to the transcription initiation site of the COX-2 promoter. Electrophoretic mobility shift assays show that YY1 bound to the putative YY1 site in vitro. To show biological relevance, we performed chromatin immunoprecipitation assays showing that lipopolysaccharide (LPS) treatment induced YY1 binding to the cognate site in the endogenous COX-2 promoter. Overexpression of YY1 in macrophages treated with either LPS or live Pseudomonas aeruginosa increased COX-2 transcriptional activity. Furthermore, YY1 enhanced COX-2 protein expression and prostaglandin D(2) production elicited by LPS treatment. Mechanistically, we observed that LPS treatment resulted in disruption of an interaction between YY1 and p300, a histone acetyltransferase, but did not affect the interaction between YY1 and histone deacetylase 1/2. These data suggest that in response to LPS, YY1 dissociates from p300 and binds to the COX-2 promoter, contributing to COX-2 expression in an inflammatory milieu.

Activation of nuclear factor kappa B and severe hepatic necrosis may mediate systemic inflammation in choline-deficient/ethionine-supplemented diet-induced pancreatitis.

OBJECTIVES: We hypothesized that hepatic injury is associated with severe acute pancreatitis (SAP) and may result in lung injury through nuclear factor kappa B (NF-kappaB)-dependent inflammatory mediators. The study characterizes the timing and determines the involvement of selected cytokines and chemokines in the pathogenesis of hepatocellular injury associated with SAP. METHODS: The SAP was induced in C57BL/6 mice by feeding a choline-deficient/ethionine-supplemented diet. The mice were killed at 12-hour intervals for 96 hours. Terminal deoxynucleotidyl transferase-mediated nick-end labeling staining was used to determine the extent of hepatic apoptosis. The NF-kappaB activation in nuclear protein extracts from liver tissue was measured using a sensitive RelA enzyme-linked immunoadsorbent assay. Tumor necrosis factor alpha, interleukin 6, macrophage inflammatory protein (MIP) 2, and keratinocyte-derived chemokine (KC) levels in homogenates of liver and lung tissues were measured by enzyme-linked immunoadsorbent assay. The SAP-associated neutrophil lung inflammation was measured as tissue myeloperoxidase activity. RESULTS: The SAP and subsequent liver injury were confirmed by histological analysis and rises in plasma amylase and transaminase levels. Severe hepatocellular apoptosis was detected at 36 and 48 hours after the diet initiation by terminal deoxynucleotidyl transferase-mediated nick-end labeling staining (P < 0.05) and subsequently progressed to hepatic necrosis. Liver NF-kappaB activation was detected at 36 hours (P < 0.05) and followed by a sharp increase in hepatocellular levels of interleukin 6, MIP-2, and KC at 72 hours and thereafter (P < 0.05). Levels of MIP-2 and KC in lung tissue were also elevated at 72 hours (P < 0.05) and closely correlated with increased myeloperoxidase activity and increased inflammatory cell infiltrate in the lung. CONCLUSIONS: Choline-deficient/ethionine-supplemented diet-induced SAP is accompanied with hepatocellular apoptosis and eventual necrosis. This injury is associated with the hepatic NF-kappaB activation leading to the production of NF-kappaB-dependent cytokines and chemokines in the liver, which may mediate the lung injury.

Release of free F2-isoprostanes from esterified phospholipids is catalyzed by intracellular and plasma platelet-activating factor acetylhydrolases.

F2-isoprostanes are produced in vivo by nonenzymatic peroxidation of arachidonic acid esterified in phospholipids. Increased urinary and plasma F2-isoprostane levels are associated with a number of human diseases. These metabolites are regarded as excellent markers of oxidant stress in vivo. Isoprostanes are initially generated in situ, i.e. when the arachidonate precursor is esterified in phospholipids, and they are subsequently released in free form. Although the mechanism(s) responsible for the release of free isoprostanes after in situ generation in membrane phospholipids is, for the most part, unknown, this process is likely mediated by phospholipase A2 activity(ies). Here we reported that human plasma contains an enzymatic activity that catalyzes this reaction. The activity associates with high density and low density lipoprotein and comigrates with platelet-activating factor (PAF) acetylhydrolase on KBr density gradients. Plasma samples from subjects deficient in PAF acetylhydrolase do not release F2-isoprostanes from esterified precursors. The intracellular PAF acetylhydrolase II, which shares homology to the plasma enzyme, also catalyzes this reaction. We found that both the intracellular and plasma PAF acetylhydrolases have high affinity for esterified F2-isoprostanes. However, the rate of esterified F2-isoprostane hydrolysis is much slower compared with the rate of hydrolysis of other substrates utilized by these enzymes. Studies using PAF acetylhydrolase transgenic mice indicated that these animals have a higher capacity to release F2-isoprostanes compared with nontransgenic littermates. Our results suggested that PAF acetylhydrolases play key roles in the hydrolysis of F2-isoprostanes esterified on phospholipids in vivo.

Systemic nf-kappaB activation in a transgenic mouse model of acute pancreatitis.

BACKGROUND: Transcription factor NF-kappaB has been implicated in numerous human inflammatory diseases. Acute pancreatitis can result in remote tissue injury, but the involved mechanisms are unknown. This study evaluates the role of systemic NF-kappaB activation in the pathogenesis of lung inflammation in a transgenic pancreatitis model. MATERIALS AND METHODS: Using transgenic mice expressing photinus luciferase controlled by an NF-kappaB-dependent promoter, luciferase activity was measured in pancreas, liver, and lung tissues as a surrogate marker of NF-kappaB activity. Luciferase activity was measured by in vivo bioluminescence and correlated to an in vitro luciferase assay of organ homogenates. Following measurement of luciferase activity in uninjured animals, these animals were fed a choline-deficient, ethionine supplemented diet for 48 h to induce pancreatitis, and luciferase activity was then measured at 48, 60, 72, and 96 h. Lung inflammation was determined by total nucleated cell counts in bronchoalveolar lavage (BAL) fluid. RESULTS: Bioluminescence detected increased luciferase activity over the upper abdominal region at 48 and 60 h (P < 0.05), and over the thorax at 60 and 72 h (P < 0.05). Luciferase assays showed significantly increased luciferase activity in both liver and lung tissue at 48 (liver = P < 0.005, lung = P < 0.05) and 60 h (liver = P < 0.05, lung = P < 0.05) compared to activity in uninjured controls. Total nucleated cell counts in BAL fluid were significantly increased at 72 h (P < 0.05) compared with controls. CONCLUSION: In this model, NF-kappaB binding activity is increased in the liver and lung. These data suggest that the liver modulates pancreatitis-induced systemic inflammatory response syndrome (SIRS) and suggest strategies to reduce multisystem injury.