Enzo Life Sciences Research Articles

Pilot Study: Comparative Hsp72 Expression in Blood Plasma, Saliva, and Exosomal Fractions During Acute Heat Stress and Acclimation

Objective: To support long-term study biomonitoring during stress, we sought to establish optimized protocols for blood and saliva. Our hypotheses were that 1) stress-specific marker Hsp72 is expressed to suffcient detectable limits and responsive to rest, exercise-heat stress, and to multi-day acclimation in saliva and blood plasma and 2) Hsp72 patterns are distinct in exosome fractions vs. unprocessed whole samples. Methods: Participants (3M/3F, M: 28±4yo, VO2max 48.87±4.08mL·kg−1·min−1, F: 25±2yo, VO2max 41.17±0.74mL·kg−1·min−1) completed a 5-day heat acclimation protocol (~40°C and relative humidity ~40%, ~70% vVO2max jog to reach and maintain a rectal temperature (Trec) range of 38.5-39.5°C for 60 total min). EDTA-treated plasma was separated and a portion was used to fractionate and isolate plasma exosomes for analyses. Whole saliva was processed and fractionated to isolate saliva exosomes for analyses. Hsp72 was analyzed in samples using a high-sensitivity ELISA kit (Enzo Life Sciences). Physiological variables (heart rate (HR), Trec) were used to confirm the stress of the exercise-heat exposures. ANOVA with t-tests for comparisons were used to assess statistical significance (p<0.05). Results: All subjects acclimated, represented by decreased HR (PREHA:107±11.14bpm (M), 170.33±19.50bpm (W), p=0.01) vs. POSTHA:103.67±7.57bpm (M), 140±18.68bpm (W), p=0.04) and decreased Trec post-exercise (PREHA:38.16±0.61°C (M), 39.33±0.50°C (W), p=0.06) vs. POSTHA:37.91±0.41°C (M), 38.39±0.11°C (W), p=0.12). Men and women did differ significantly (p<0.05) with HR changes but were not significantly different (p>0.05) in regards to Trec. Despite physiological acclimation, there were no differences in Hsp72 across time points and by sex. Hsp72 expression was detected in plasma exosomes (0.82-5.18ng·mL−1 (M), 0.48-2.22ng·mL−1 (W)) and saliva exosomes (0.45-10.09ng·mL−1 (M), 0.25-7.18ng·mL−1(W)) and there were no significant differences between men and women (p>0.05) during acute exposures and acclimation, despite some differences in physiological responses. Hsp72 was significantly higher in males overall as compared to females only in saliva exosomes (3.94±3.14ng·mL−1 (M), 1.5±1.27 ng·mL−1 (F), p=0.0003). Conclusion: Subjects acclimatized physiologically without exhibiting Hsp72 changes in their blood plasma or plasma exosomes. Salivary exosomal Hsp72 may be different between men and women. Ongoing research includes validation of the exosome fraction using exosome specific protein markers in co-expression (exosome marker and Hsp72) experiments and ongoing assays of salivary biomarkers of heat stress. Funding: DoD BA200299. This is the full abstract presented at the American Physiology Summit 2024 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.

SAT270 Parity Increases Corticosterone Response To Restraint-induced Stress After Mild Blast Traumatic Brain Injury In Mice

Abstract Disclosure: S. Anisowicz: None. M. Bodemeier Loayza Careaga: None. T. Wu: None. Background: Traumatic brain injury (TBI) is a common problem worldwide, with an estimated incidence of 69 million injuries per year. In the US military, one of the most common forms of TBI during operational deployments after Sep 11, 2001 was the blast TBI, thought to be responsible for over 80% of TBI sustained in theater. One consequence of TBI is dysregulation of the hypothalamus-pituitary-adrenal (HPA) axis. HPA axis dysregulation is linked to increased risk for psychiatric disorders. Furthermore, prior research has shown that TBI may dysregulate the HPA axis in a sex-dependent manner. Throughout a female’s life span, there are several sentinel events of hormonal consequence, including pubarche, menopause, and parity, which are known to be times of changes in risks for psychiatric disorders. Objectives: The role of parity on TBI response has not yet been studied, though parity has been shown to decrease fear-related behaviors and decreased corticosterone load in multiparous mice when compared to nulliparous (NP) controls. Our objective is to determine if parity alters corticosterone (CORT) response to restraint, a mild psychological stressor, in mice exposed to mild blast TBI (mbTBI). Methods: One hundred thirteen NP or primiparous (PP) C57Bl6/J mice were randomly assigned to receive sham or mbTBI. Among those that received mbTBI (19.2 ± 0.12 psi), 32 were NP and 24 were PP. Among sham mice, 32 were NP and 26 were PP. All mice received isoflurane anesthesia prior to sham or mbTBI treatment. At 7-8 days after mbTBI, half the mice within each treatment group were subjected to a mild 20-min restraint before being anesthetized by CO2 followed by collection of trunk blood. Serum was harvested and analyzed for CORT concentrations by ELISA (Enzo Life Sciences). Results: All mice that received mbTBI had a longer (p<0.05) righting time compared to those that did not. There was no main effect of parity in the righting time (p>0.1). PP mice who experienced mbTBI demonstrated a greater (p<0.05) CORT response to restraint (157.3 ± 21 pg/mL) when compared to NP mice (95.9 ± 7.2 pg/mL). CORT response to restraint did not differ (p>0.1) between NP and PP mice who did not experience mbTBI (96.1 ± 7.6 vs 109.2 ± 16 pg/mL). Conclusions: Our data indicates that mbTBI alters HPA axis reactivity in PP mice compared to NP mice, increasing the CORT response to restraint induced stress. Parity alone did not alter CORT response to restraint stress or baseline CORT levels. The results of our finding highlight the importance of parity as a factor in an individual’s lifespan. Presentation: Saturday, June 17, 2023

Sudanese Toombak smokeless tobacco users harbour significantly altered long-term cortisol body production.

The Sudanese, in particular its male population, are known to utilise a smokeless tobacco product (Toombak) which is placed in the oral cavity and can be replaced several times a day. Toombak has been shown to harm human health and is highly addictive. The effect on body cortisol response over a retrospective period in users of this product has not been previously explored. In addition, psycho-dependency scores of Toombak users have not been analysed. In this study, 37 male subjects, age 18-45years were recruited, of which 18 were non-users of Toombak and 19 were Toombak users. One hair sample was collected from each user and non-user of Toombak. Each hair sample (n=37) was placed in a pre-prepared long piece of foil with two labels on either side marked: 'scalp-side' and 'distant-side'. Cortisol was extracted by mincing 10mg of 'scalp-side' hair, not exceeding 3cm, with methanol addition, incubation, and sonication. Cortisol was measured using the enzyme-linked immunosorbent assay kit (Enzo Life Sciences, UK). The amount of hair cortisol in the samples was determined using spectrophotometry at wavelength 405nm measured in pg/ml and visualised with a four parametric logistic curve. Toombak users were further asked to complete the Fagerstrom Test for Nicotine Dependence-Smokeless Tobacco questionnaire (FTND-ST) comprising of six questions. Scores of>5 indicated a significant dependence, while a score of<4 marked low to moderate dependence. The mean concentration of hair cortisol in Toombak users (9.7pg/ml) was significantly lower (p=0.023) compared to non-users (19.4pg/ml), with total concentrations ranging from 2.1 to 55.6pg/ml. FTND-ST scores ranged from 4 to 9, with high levels of psycho-dependency (score>5) and nicotine tolerance found in 85% of Toombak users. Cortisol body release in Sudanese smokeless tobacco users was found to be significantly altered. While low cortisol levels do lead to anxiolytic effects, in the long-term, this can allow for increased susceptibility to low cortisol-associated diseases.

MO403: The Association Between Urinary 11-Dehydrothromboxane B2 and Laboratory Parameters in Aspirin-Treated Patients With Cardiorenal Syndrome

Abstract BACKGROUND AND AIMS Evaluation of urinary levels of the 11-dehydrothromboxane B2 is one of the methods for assessing platelet reactivity while taking aspirin. High levels of 11-dehydrothromboxane B2 in urine indicate continued thromboxane generation which is associated with an increased risk of atherothrombotic cardiovascular events. The present study aimed to evaluate the association between 11-dehydrothromboxane B2 in urine and laboratory parameters in aspirin-treated patients with the cardiorenal syndrome in subgroups from quartile with the highest and the lowest levels of thromboxane metabolite. METHOD A total of 82 patients with stable coronary artery disease (CAD) and CKD (stages 2–4) were included in the study. Urinary levels of 11-dehydrothromboxane B2 (a stable metabolite of thromboxane A2) were assessed using ELISA kit by Enzo Life Sciences (Switzerland). Statistical analysis was performed using the chi-squared test and Spearman's rank correlation coefficient. RESULTS In the subgroup of patients from the quartile with the highest levels of urinary 11-dehydrothromboxane B2, a history of ischemic stroke (P = 0.032) and a history of myocardial infarction (P = 0.048) were more common. There were significant correlations between urinary 11-dehydrothromboxane B2 and platelet count (rs = 0.457, P = 0.043), platelet crit (rs = 0.481, P = 0.037), pulmonary artery systolic pressure (rs = 0.847, P = 0.016) and left ventricular ejection fraction (rs = −0.719, P = 0.045) in the same subgroup of patients. In the subgroup of patients from the quartile with the lowest levels of 11-dehydrothromboxane B2 in urine statistically significant correlations between thromboxane metabolite and fasting plasma glucose (rs = 0.581, P = 0.007) and hematocrit (rs = 0.441, P = 0.003) were found. CONCLUSION Our study identified the relationships between the urinary concentration of 11-dehydrothromboxane B2 and laboratory and echocardiography parameters in aspirin-treated patients with cardiorenal syndrome. Accordingly, the received results represent the varying response to aspirin in patients with stable CAD and CKD. Additional research is needed to clarify the impact of platelet indices and the association between platelet reactivity while taking aspirin and pulmonary hypertension on behalf of the possibility of personalization of antiplatelet therapy.

Abstract P084: DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with EGFR inhibitors osimertinib and afatinib in NSCLC preclinical models

Abstract Background: Activating mutations in EGFR have been reported in ~30% of patients with non-small cell lung cancer (NSCLC). Three generations of small molecule EGFR kinase inhibitors have been approved by the FDA to treat these patients, however, multiple mechanisms of resistance cause cancer progression. In addition to drug-resistant mutations that arise and re-activate EGFR, other signaling pathways can be activated to cause resistance. Although EGFR inhibitors such as osimertinib and afatinib (also a pan-ErbB inhibitor) have demonstrated clear clinical benefits, patients inevitably develop resistance. Herein we demonstrate mutant EGFR NSCLCs activate autophagy upon treatment with EGFR inhibitors as a drug resistance mechanism. Therefore, a combination of an EGFR inhibitor with an autophagy inhibitor has the potential to deepen and prolong responses and improve patient outcomes. Materials and Methods: Human cancer cells with EGFR mutations were cultured using recommended complete medium. Inhibition of ULK1/2 was measured through standard biochemical assays and cellular readouts including NanoBRET and ELISA-based ATG13 phosphorylation assays. Autophagosome formation was measured using the CytoID assay (Enzo Life Sciences). For in vivo studies, NCI-H1975 cells that harbor an EGFR T790M resistance mutation were inoculated into BALB/c nude mice. Statistical analyses for the in vivo studies were performed using the Student’s t-test. Results: Erlotinib, gefitinib, osimertinib, and afatinib activated autophagy 3–4-fold over basal levels in the HCC827 cell line (EGFR exon 19 deletion) as measured by increases in phosphorylated ATG13, a cellular substrate of the autophagy-initiating kinases ULK1/2. DCC-3116, an investigational potent and selective dual inhibitor of ULK1 (IC50 6 nM) and ULK2 (9 nM) in cellular assays, inhibited both EGFR-induced and basal phosphorylation of ATG13 with IC50 values of 61–66 nM. Treatment of the NCI-H1975 EGFR mutated (L858R/T790M) NSCLC cell line with osimertinib or afatinib induced autophagy 3-fold over basal levels. DCC-3116 potently inhibited osimertinib and afatinib induced phosphorylation of ATG13 with IC50 values of 91 nM and 71 nM, respectively, and inhibited the increase in autophagosomes induced by these agents. Importantly, these in vitro effects translated to in vivo efficacy. The combination of DCC-3116 with osimertinib or afatinib resulted in significantly greater tumor responses than single agent treatments in the NCI-H1975 NSCLC xenograft model (combination vs. osimertinib p = 0.0005; combination vs. afatinib p = 0.0001; osimertinib combination vs vehicle p &amp;lt; 0.0001; afatinib combination vs. vehicle p &amp;lt; 0.0001). These data provide a strong rationale to study the combination of the ULK inhibitor DCC-3116 with EGFR inhibitors such as osimertinib and afatinib in cancer patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors with a documented RAS or RAF mutation (NCT04892017). Citation Format: Madhumita Bogdan, Mary J. Timson, Hikmat Al-Hashimi, Yu Zhan, Bryan D. Smith, Daniel L. Flynn. DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with EGFR inhibitors osimertinib and afatinib in NSCLC preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P084.

Sickle Cell Hemoglobin Induces Autophagy in Human Macrophages

BACKGROUND: Autophagy plays an important role in multiple cell processes including elimination of misfolded proteins and damaged organelles, clearance of intracellular microbes and regulation of innate immunity. Autophagic activity is significantly up regulated in diseases characterized by proteins aggregation such as Alzheimer's, Huntington's, and Parkinson's diseases. Sickle cell anemia (SCA) is associated with E6V mutation in beta-globin gene that induces hemoglobin (Hb) polymerization under low oxygen or acidic conditions. SCA is characterized by chronic presence of low-level plasma Hb (3-10 µM). Hb is cleared from circulation by macrophage's endocytosis, resulting in degradation of Hb in lysosomes. Acidic lysosome environment may induce Hb polymerization, lysosome dysregulation and autophagy activation.HYPOTHESIS: We hypothesize that endocytosis of HbS induces autophagy in human macrophages.METHODS: The study was approved by Howard University review board (IRB) and all subjects provided written inform consent prior the sample collection. Whole blood samples were obtained from three SCA patients and healthy individuals. Human THP-1 promonocytic cells were differentiated into macrophages with 25 nM PMA for 72 hrs and treated with either purified HbS or HbA (5 µM, Sigma-Aldrich). Total RNA was depleted of cytoplasmic and mitochondrial ribosomal RNA, and strand-specific libraries were constructed using the Illumina ® TruSeq Stranded Total RNA Gold kit. Libraries were sequenced on an Illumina ® NextSeq 500 using 75 bp paired-end sequencing on two v2.5 150 cycle High-Output kits, generating 40-50 million paired-end reads per sample. The sequencing data were mapped to human reference genome using Dragen RNA v.3.8.4 software (Illumina). Comparisons were conducted using Dragen differential expression software v. 3.6.3 software (Illumina). Transmission electron microscopy (TEM) imaging was performed at Talos 200X transmission electron microscope (Thermo Fisher Scientific). CYTO-ID Autophagy detection kit (Enzo Life Science) was used for detection of autophagy in live cells by fluorescence microscopy, and fluorescent microplate assay.RESULTS: In THP-1-derived macrophages treated with HbS compared to HbA, 28362 genes were detected including 322 differentially expressed genes (1.1%, 1.5-fold difference, 187 down and 135 up) at 5% FDR. In THP-1-differentiated macrophages treated with SCD RBC lysate compared to control RBC lysate, 30840 genes were detected including 256 differentially expressed genes (0.83%, 1.5-fold difference, 117 down and 139 up) at 5% FDR.We focused our analysis on 33 autophagy-related genes. Among these 33 genes, 30 genes were up-regulated (1.2-1.7-fold) and only 3 genes were down-regulated in macrophages treated with HbS compared to HbA (Fig.1). These changes were statistically significant for 20 genes (p<0.01). No significant differences were found in expression of autophagy-related genes in macrophages treated either with HbS or SCD RBC lysate.Autophagy activation was validated by CYTO-ID Autophagy detection kit, demonstrating significant autophagy activation in HbS treated macrophages compared to HbA treatment (1.2-fold, p=0.0019). Transmission electron microscopy demonstrated accumulation of autophagosomes in the cells treated with HbS compared to non-treated cells and cells treated with HbA.CONCLUSION: Treatment of macrophages with sickle cell hemoglobin or lysate of red blood cells collected from SCD patients up-regulates expression of autophagy-related genes and increased autophagosome formation.ACKNOWLEDGMENTS: We thank Drs. Castle Raley and Keith Crandall, George Washington University SPH Genomics Core, for sequencing and guidance in data analysis; Dr. Christine Brantner, GW Nanofabrication and Imaging Center, for TEM imaging; and students Kavita Pandya, Gretchen Whitney High School, Cerritos, CA, and Tharindi Jayatilake, Montgomery Blair High School, Silver Spring, MD, for help in analysis of autophagy-related gene expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Increased Activated Plasma Cells in Inflammatory Bowel Disease When Compared to Ischemic Acute Colitis

Abstract Introduction/Objective Inflammatory bowel disease (IBD) and acute ischemic colitis can both be involved by active colitis. IBD is characterized by crypt architectural distortion, basal lymphoplasmacytosis, and occasional granulomatous changes. However, diagnosis of IBDs is still largely by exclusion of other types of active colitis with similar changes. We previously demonstrated that glucose regulated protein 94 (grp94) is mainly expressed by activated plasma cells. We postulate that increased numbers of grp94-positive plasma cells may support diagnosis of IBDs. Here, we compared IBD and active ischemic colitis for grp94 expression in mucosal plasma cells of colectomy specimens Methods/Case Report Tissue sections from colectomy specimens with active IBD (n = 8) and ischemic colitis (n = 7) were examined for grp94 expression by immunohistochemistry (monoclonal antibody clone 9G10 at dilution of 1:200, Enzo Life Science, Inc Farmindale, NY). The staining intensity and highest number of grp94 in plasma cells per high power field was counted and recorded for each case, and combined scores were calculated as # of plasma cells multiplied by staining intensity (ranging from 0 to 3+). Unpaired student T tests were used to compare these indices between the two groups for statistical significance (p value &amp;lt; 0.05 was considered significantly different) Results (if a Case Study enter NA) Plasma cells in lamina propria identified by grp94 staining showed higher intensity in IBD than ischemic groups. The number of plasma cells and combined scores were also significantly higher in the IBC group than that of ischemic group Conclusion Our data indicates that active plasma cells are much more numerous in IBD than ischemic colitis, supporting the notion that active plasma cells are involved in the development of this disease process. Morphologically, active colitis with increased number of plasma cells appears to be another index favoring the diagnosis of IBD.

Specificity of plasma oxytocin immunoassays: A comparison of commercial assays and sample preparation techniques using oxytocin knockout and wildtype mice

Oxytocin has garnered much interest due to its role in affective states, social behaviors, and diverse physiological functions. However, approaches for measuring endogenous oxytocin concentrations have generated considerable controversy and debate. Common procedures for measuring oxytocin often produce uncorrelated results, and the detected concentrations frequently vary across two orders of magnitude. These findings have led some researchers to argue that immunoassays of plasma oxytocin may be unreliable and nonspecific, particularly when samples are not first processed using an extraction procedure. Here, we assess the specificity of oxytocin immunoassays using plasma samples from wildtype (WT) and oxytocin knockout (KO) mice. Plasma samples from both genotypes were measured using immunoassay and were measured with or without a solid-phase extraction. Using a commercially available kit from Arbor Assays, we demonstrate that both techniques generate a clear contrast between genotypes, with wildtype samples containing high concentrations of oxytocin (unextracted mean = 468 pg/ml; extracted mean = 381 pg/ml), while knockout samples measured below the lower limit of detection. Analytical validations demonstrated good parallelism and spike recovery for both methods. Furthermore, the same wildtype samples measured with both procedures were highly correlated (r = 0.95), although unextracted samples measured at significantly higher concentrations (p = 2.0 ×10−7, Cohen’s d = 2.65). To test the generalizability of these results across immunoassay kits, we performed additional assays with kits from Cayman Chemical and Enzo Life Sciences. The Cayman Chemical kit produced results similar to Arbor Assays with a clean signal differentiating WT and KO plasma, both with and without an extraction step. The Enzo kit also differentiated the genotypes, with correlation between extracted and unextracted samples, but was considerably more susceptible to interference without the extraction, as evidenced by false positive signal in KO plasma samples. The extent to which these results generalize to other species remains unknown and challenging to assess.

HUWE1 Inhibition As a Therapeutic Strategy to Target MYC in MM

BackgroundThe use of proteasome inhibitors to disrupt the ubiquitin proteasome system (UPS) has provided a significant therapeutic advance and highlights the importance of this pathway in Multiple Myeloma (MM). There is increasing interest in developing therapeutic strategies to target enzymes that regulate the UPS, with the aim of gaining more specificity and reducing side-effects. We previously identified elevated expression of the E3 ligase HUWE1 in MM compared to normal cells and demonstrated that inhibition of HUWE1 with shRNA results in growth arrest of MM cell lines (Blood 2016, 128:240). HUWE1 is involved in the regulation of MYC and its expression is deregulated in a number of tumor types. The aim of this study was to elucidate the role and therapeutic potential of inhibiting HUWE1 in MM using small molecule inhibitors.MethodsInhibitors of HUWE1 (BI8622, BI8626) as described in Peter et al. (EMBO Mol Med 2014, 6:1525-1541) were purchased from Syngene. The effect of the inhibitors on MM cell lines (OPM-2, KMS-18, JJN3, MM.1S, MM.1R, ANBL6.WT, ANBL6.BR), patient cells and normal bone marrow (NBM) from healthy donors was assessed using CellTitre-glo and WST1 assays. Cells were analysed for cell cycle status (propidium iodide and EdU staining), apoptosis and β-galactosidase activity. Snapshot Proteomics (AVM Biomed) was performed on HUWE1 knockdown cells to identify novel ubiquitinated substrates of HUWE1. Signalling pathways and substrates of HUWE1 were analysed using UbiQapture-Q (Enzo Life Sciences), Western blotting and RQ-PCR. Calcusyn software was used to calculate a combination index (CI) to assess synergistic anti-MM activity.ResultsUbiQapture assays demonstrated that BI8622 and BI8626 blocked auto-ubiquitination of HUWE1 in a concentration-dependent manner, with an associated decrease in the ubiquitination of MIZ1 (a validated HUWE1 substrate). BI8622 and BI8626 significantly reduced the growth of MM cell lines, including those resistant to dexamethasone (MM.1R) and bortezomib (ANBL6.BR), and patient-derived MM cells (p ≤ 0.01; IC50 range 9-20 µM) without significantly affecting NBM, suggesting a favourable therapeutic index. Furthermore, both compounds inhibit the proliferation of MM cell lines when co-cultured with MM patient-derived bone marrow stromal cells. HUWE1 inhibition induced cell cycle arrest, associated with a reduction of cells undergoing DNA synthesis (p = 0.005), cellular senescence demonstrated by β-galatosidase staining and increased p16 protein expression. HUWE1 inhibition in MM cell lines resulted in a significant decrease in MYC mRNA and protein expression (p < 0.001), accompanied by downregulation of multiple MYC-dependent target genes (CCND2, HSPE1, ODC1, CCNA2, CDC25A; p ≤ 0.02). Proteomic profiling along with ingenuity pathway analysis revealed novel putative substrates of HUWE1 involved in the regulation of MYC. Finally, the combination of HUWE1 inhibitors with proteasome inhibitors (carfilzomib and bortezomib) and dexamethasone results in synergistic anti-MM activity (CI range 0.57 - 1).ConclusionsMYC is frequently dysregulated in MM and represents an attractive therapeutic target. Here we demonstrate that inhibitors of the E3 ligase HUWE1 lead to growth arrest of MM cell lines, associated with decreased expression of MYC and MYC-dependent target genes. While BI8622 and BI8626 have previously been reported to repress MYC activity in models of colorectal cancer, we demonstrate for the first time that in MM this is accompanied with a decrease in MYC expression. Studies are ongoing to elucidate the molecular mechanism of MYC downregulation following HUWE1 inhibition both in vitro and in vivo. In summary, this study demonstrates that HUWE1 is important in the growth and survival of MM cells and presents a novel therapeutic strategy for the inhibition of MYC function in MM. DisclosuresNo relevant conflicts of interest to declare.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314

Relationship between the Length of Sperm Tail Mitochondrial Sheath and Fertility Traits in Boars Used for Artificial Insemination.

The length of sperm tail midpiece, occupied by the mitochondrial sheath (MS), has been correlated with reproductive traits of mice, fish, and birds; however, it is not known whether such a correlation exists in higher order species such as domestic pigs. As the mitochondria provide for sperm motility and generate the fertility-affecting reactive oxygen species (ROS), we hypothesized that MS length correlates with boar semen parameters and artificial insemination (AI) fertility. Sperm samples collected from 57 boars and used for single sire AI were labeled with ProteoStat Aggresome probe (AGG; Enzo Life Sciences) for MS imaging by epifluorescence microscopy and image-based flow cytometry (IBFC). The mean boar MS length was 7.26 ± 0.2 µm, ranging from 6.94 ± 0.18 µm to 7.65 ± 0.31 µm. The absolute longest MS measured was 9.19 µm and the shortest was 5.83 µm. Boars in the high tertile of MS length had significantly higher conception rate (CR; p = 0.05) and sperm parameters. Boars within the high tertile of average number piglets born per litter had significantly shorter MS and more varied MS length than boars in the low tertile (p = 0.04). MS length data correlated with conventional sperm parameters including percent viable and intact acrosomes (p = 0.03), basal:induced oxidation ratio (measure of intracellular ROS levels; p = 0.02) and Comp DNA (chromatin integrity; p = 0.06) along with many flow cytometric AGG parameters in IBFC. Sperm head AGG intensity median absolute deviation had a negative correlation with total born (r = −0.423 p = 0.004). These data reveal a complex relationship between sperm MS length and aggresome abundance to sperm parameters and boar reproductive success in AI service.

Association of Plasma Heat Shock Protein 70 with Disease Severity, Smoking and Lung Function of Patients with Chronic Obstructive Pulmonary Disease.

Extracellular heat shock protein 70 (eHsp70) might modulate immune responses in chronic obstructive pulmonary disease (COPD). The aim of the study was to explore eHsp70 concentration in stable COPD, its association with disease severity and smoking status as well as its diagnostic performance in COPD assessment. Plasma samples were collected from 137 COPD patients and 95 healthy individuals, and concentration of eHsp70 was assessed by commercially available enzyme-linked immunosorbent assay (ELISA) kit (Enzo Life Science, Farmingdale, NY, USA). COPD patients were subdivided regarding airflow obstruction severity and symptoms severity according to the Global Initiative for COPD (GOLD) guidelines. eHsp70 concentration increased in COPD patients when compared to controls and increased with the severity of airflow limitation as well as symptoms burden and exacerbation history. eHsp70 concentration did not differ among COPD patients based on smoking status, yet it increased in healthy smokers compared to healthy nonsmokers. In addition, eHsp70 negatively correlated with lung function parameters forced expiratory volume in one second (FEV1) and FEV1/ forced vital capacity (FVC), and positively with COPD multicomponent indices BODCAT (BMI, airflow obstruction, dyspnea, CAT score), BODEx (BMI, airflow obstruction, dyspnea, previous exacerbations), CODEx (Charlson’s comorbidity index, airflow obstruction, dyspnea, previous exacerbations) and DOSE (dyspnea, airflow obstruction, smoking status, previous exacerbations) With great predictive value (OR = 7.63) obtained from univariate logistic regression, eHsp70 correctly classified 76% of cases. eHsp70 is associated with COPD prediction and disease severity and might have the potential for becoming an additional biomarker in COPD assessment.

Urinary Angiotensinogen is Essential for Renal Potassium Handling

Apart from the well‐known systemic renin‐angiotensin system (RAS), the urinary RAS acts as a local autocrine/paracrine system along the nephron. The regulation of renal K+ handling by systemic vs urinary RAS remains unclear. Liver‐specific (Alb‐Cre) angiotensinogen knockout (LKO) and liver + proximal tubule (Alb‐Cre + KAP‐Cre) angiotensinogen knockout (DKO) were utilized in this study. WT, LKO, and DKO mice were kept on either a regular diet (RD) or a high K diet (HK; 5% K+) for 10 days. Mice were acclimated to metabolic cages twice before collecting urine for 24 hours. Mice were then euthanized to collect blood and kidneys. Plasma and urinary [Na+] and [K+] were measured by flame photometry. Plasma and urinary angiotensin II (Ang II) concentrations were measured by ELISA (Enzo Life Sciences). Plasma and urinary aldosterone (Aldo) concentrations were measured by ELISA (DRG Diagnostics). Urinary creatinine (Cr) concentrations were measured by colorimetric assays (Cayman Chemical). Plasma [Ang II] was significantly lower in LKO, compared to WT, and the value in DKO was not significantly different from zero. Urinary [Ang II] was significantly lower in DKO compared to WT and LKO. Plasma [K+] was significantly higher in DKO but not LKO compared to WT on HK. Urinary K:Cr ratio was significantly lower in DKO compared to WT on HK. However, plasma and urinary [Aldo] were not different between DKO and WT on HK. Overall, our results showed that urinary RAS but not systemic RAS plays an essential role in renal K handling.Support or Funding InformationThis project was funded by F30 DK108456.

Cytotoxicity and the Effect of Temperature on Physical Properties and Chemical Composition of a New Calcium Silicate–based Root Canal Sealer

IntroductionThe suitability of EndoSequence BC Sealer (BC Sealer; Brasseler USA, Savannah, GA) for warm vertical compaction has been questioned. The aim was to evaluate the cytotoxicity and the effect of heating on the physicochemical properties of a new calcium-based root canal sealer (EndoSequence BC Sealer HiFlow [HiFlow]) in comparison with EndoSequence BC Sealer. MethodsHuman periodontal ligament fibroblasts were incubated for 1, 2, or 3 days with material extracts of different concentrations, and cell viability was evaluated by Cell Counting Kit-8 (Enzo Life Sciences Inc, Burlington, Ontario, Canada). The setting time, flow, film thickness, microhardness, radiopacity, and radiopacity of the 2 sealers were measured according to ISO 6786/2012. The continuous changes in viscosity were tested by a stress-controlled rheometer at shear rates ranging from 0.01–10 s−1 and different temperatures, and chemical composition was assessed by Fourier-transform infrared spectroscopy. ResultsCell viability was significantly decreased on day 3 for the 1:4 diluted extract from both materials. The setting time, microhardness, and solubility of HiFlow were similar to BC Sealer at 37°C and 100°C. HiFlow had significantly higher flow and radiopacity than BC Sealer at room temperature (P < .05), and when heated, HiFlow retained its higher flow and lower film thickness (P < .05). Both sealers showed decreasing viscosity with increasing shear rate, and at a shear rate of 0.01 and 0.1 s−1, HiFlow exhibited lower viscosity than BC Sealer at all temperatures measured. The chemical composition of the 2 sealers was not changed by heating. ConclusionsHiFlow showed better performance on flow/viscosity and film thickness, especially under high temperatures, which are generated by the commonly used warm vertical compaction technique.

KLK6 as a Predictor of Ovarian Malignancy

Objective: To measure the levels of KLK6 and CA125 tumor marker and compare them as a predictor of epithelial type ovarian malignancy. KLK6 and CA125 were taken from blood serum of 60 ovarian tumor patients who met the inclusion criteria in consecutive sampling. CA125 quantitative examination and KLK6 titer were performed by ELISA. Surgical tissue from Dr. Soetomo Hospital, Surabaya was examined histopathologically at the Anatomical Pathology Laboratory of the Faculty of Medicine Airlangga University, Surabaya. Methods: This study is a diagnostic test using a cross-sectional design with a total sample of 60 ovarian tumor patients from obstetrics and gynecology outpatient clinic, Soetomo Hospital, Obstetrics and Gynecology Department, Faculty of Medicine, Airlangga University, Surabaya. CA125 was examined by ELISA, while KLK6 was examined using an ELISA reagent kit from Enzo Life Sciences Inc., USA. Results: This study examined a total sample of 60 patients, 30 patients (50%) with benign ovarian tumors, 26 patients (43.33%) with stage I ovarian cancer and 4 patients (6.66%) with stage II ovarian cancer. The best cut off for KLK6 is 3.14ng/mL and the standard CA125 cut-off is 35U/mL. The sensitivity and specifi city of Ca125 are 70.00; 33.33, the sensitivity and specifi city of KLK6 are 50.00; 100.00, PPV and NPV of Ca125 are 51.22; 52.63, PPV and NPV of KLK6 are 100.00; 66.67. Conclusion: KLK6 showed high specifi city for the diagnosis of ovarian cancer. This can improve the diagnostic accuracy of Ca125 which has high sensitivity but low specifi city. A combined panel of Ca125 and KLK6 showed high diagnostic effi ciency for early-stage ovarian cancer.

Potent reduction of plasma lipoprotein (a) with an antisense oligonucleotide in human subjects does not affect ex vivo fibrinolysis

It is postulated that lipoprotein (a) [Lp(a)] inhibits fibrinolysis, but this hypothesis has not been tested in humans due to the lack of specific Lp(a) lowering agents. Patients with elevated Lp(a) were randomized to antisense oligonucleotide [IONIS-APO(a)Rx] directed to apo(a) (n = 7) or placebo (n = 10). Ex vivo plasma lysis times and antigen concentrations of plasminogen, factor XI, plasminogen activator inhibitor 1, thrombin activatable fibrinolysis inhibitor, and fibrinogen at baseline, day 85/92/99 (peak drug effect), and day 190 (3 months off drug) were measured. The mean ± SD baseline Lp(a) levels were 477.3 ± 55.9 nmol/l in IONIS-APO(a)Rx and 362.1 ± 89.9 nmol/l in placebo. The mean± SD percentage change in Lp(a) for IONIS-APO(a)Rx was -69.3 ± 12.2% versus -5.4 ± 6.9% placebo (P < 0.0010) at day 85/92/99 and -15.6 ± 8.9% versus 3.2 ± 12.2% (P = 0.003) at day 190. Clot lysis times and coagulation/fibrinolysis-related biomarkers showed no significant differences between IONIS-APO(a)Rx and placebo at all time points. Clot lysis times were not affected by exogenously added Lp(a) at concentrations up to 200 nmol/l to plasma with very low (12.5 nmol/l) Lp(a) levels, whereas recombinant apo(a) had a potent antifibrinolytic effect. In conclusion, potent reductions of Lp(a) in patients with highly elevated Lp(a) levels do not affect ex vivo measures of fibrinolysis; the relevance of any putative antifibrinolytic effects of Lp(a) in vivo needs further study.

Repression of Death Receptor–Mediated Apoptosis of Hepatocytes by Hepatitis B Virus e Antigen

Hepatitis B virus (HBV) e antigen (HBeAg) is associated with viral persistence and pathogenesis. Resistance of HBV-infected hepatocytes to apoptosis is seen as one of the primary promotors for HBV chronicity and malignancy. Fas receptor/ligand (Fas/FasL) and the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) system plays a key role in hepatic death during HBV infection. We found that HBeAg mediates resistance of hepatocytes to FasL or TRAIL-induced apoptosis. Introduction of HBeAg into human hepatocytes rendered resistance to FasL or TRAIL cytotoxicity in a p53-dependent manner. HBeAg further inhibited the expression of p53, total Fas, membrane-bound Fas, TNF receptor superfamily member 10a, and TNF receptor superfamily member 10b at both mRNA and protein levels. Incontrast, HBeAg enhanced the expression of soluble forms of Fas through facilitation of Fas alternative mRNA splicing. In a mouse model, expression of HBeAg in mice injected with recombinant adenovirus-associated virus 8 inhibited agonistic anti-Fas antibody-induced hepatic apoptosis. Xenograft tumorigenicity assay also found that HBeAg-induced carcinogenesis was resistant to the proapoptotic effect of TRAIL and chemotherapeutic drugs. These results indicate that HBeAg may prevent hepatocytes from FasL and TRAIL-induced apoptosis by regulating the expression of the proapoptotic and antiapoptotic forms of death receptors, which may contribute to the survival and persistence of infected hepatocytes during HBV infection.

Salivary Stress Biomarkers During the Lake Placid Ironman® Ultraendurance Event

Biomarkers such as salivary IgA (sIgA) have been established as valid, reliable, and non-invasive stress markers. sIgA concentrations have been reported to decrease following periods of high physiological stress, such as that experienced during ultra-endurance events. Heat shock protein 70 (HSP70), a molecular chaperone, has been assessed primarily as a plasma/serum stress biomarker. The role of HSP70 in saliva, how it responds to extremely stressful exercise events and its correlation to changes in sIgA remain unclear. PURPOSE: To test the hypothesis that salivary HSP70 can be used as a salivary stress biomarker correlated to sIgA during an ultraendurance event. METHODS: Thirty-three subjects competing in the Lake Placid Ironman triathlon participated (all data, mean±SD: 38±8 yrs, 178.4±8.9 cm, 76.3±10.4 kg, 10.8±3.8% body fat, finish time 708±90 min). Environmental symptoms questionnaires (ESQ) were administered before (PRE) and 1 hour after the race (POST). Hydration status was assessed via urine specific gravity (USG). Saliva samples were collected PRE, POST, and 1 day post-race (AMPOST) and analyzed for IgA (Salimetrics) and HSP70 (Enzo Life Sciences) by ELISA according to manufacturer instructions. Significant differences among time points were analyzed by repeated measures ANOVA and LSD post hoc tests. RESULTS: Subjects experienced significant stress with completion of the race (708±90 min finish time, ESQ 1hPOST (20±8) vs. PRE (5±3, p<0.05), POST RPE 18±2). sIgA was decreased POST (41.71 ±20.46 μg·ml-1) vs. PRE (48.88 ±17.20 μg·ml-1, p=0.05) and vs. AMPOST (37.76 ±27.02 μg·ml-1, p=0.006). Salivary HSP70 was increased POST (3.91±3.29 ng·ml-1) vs. PRE (1.68±1.93 ng·ml-1 p=0.0002). CONCLUSIONS: Salivary HSP70 was detectable using a commercially available ultrasensitive HSP70 ELISA. Extracellular HSP70 in the oral cavity may be a non-invasive marker of stress during an ultraendurance event and is correlated with more common salivary stress marker sIgA.

SAT-106 Plasma Interleukine-6 (IL-6) Concentration Is a Determinant of Free-Living Weight Change in Healthy Humans

Background: IL-6 is a multifunctional cytokine secreted by leukocytes and endothelial cells in multiple organs. Mice with IL-6 deficiency show mature-onset obesity due to decreased energy expenditure (EE) but no change in food intake. In humans, IL-6 concentration increases with obesity; however, the causal relationship with future weight gain is unclear. We investigated if fasting plasma IL-6 concentration predicts weight change in lean and overweight individuals. Methods: While residing in our clinical research unit, forty-nine healthy, weight-stable volunteers (37.5±10.8 y, 26.7±4.0 kg/m2BMI, 29.7±9.1% body fat; mean±SD, 39 men) with normal glucose regulation had 24-h EE measurements in a whole-room indirect calorimeter during energy balance and consuming a standard diet (50% carbohydrate, 20% protein). After at least 3 days on weight-maintaining diet and an overnight fast, plasma was collected for measurement of IL-6 concentrations by ELISA (Enzo Life Sciences, Farmingdale, NY; intra-assay CV=7.9%, inter-assay CV=10.5%, range=1.52-50 pg/mL) on 6 different days with values averaged to increase precision. Dual-energy X-ray absorptiometry (DXA) was used to measure body composition. Volunteers returned for follow up assessment of body weight and composition by DXA after 6 months (n=38) and 1 year (n=32). Results: Fasting IL-6 concentrations (geometric mean, 95% CI: 11.5, 10.0-13.2 pg/mL) did not differ by gender or race (p>0.05) nor were associated with age, BMI, % body fat, fat mass (FM), or fat-free mass (FFM). After adjustment for body composition and other known EE determinants, fasting IL-6 concentration was not associated with 24-h EE (p=0.33), sleeping EE (p=0.85), or respiratory quotient (p=0.76). However, higher IL-6 concentration was associated with weight gain at 6 months (r=0.51, p=0.001) and at 1 year (r=0.45, p=0.009), reflecting increases in FM (r=0.42, p=0.01 and r=0.50, p=0.04, respectively), but not FFM (p=0.09 and p=0.40). In a linear model, fasting IL-6 concentration at baseline was an independent predictor of weight gain at 6 months and 1 year [β=13.2 (95% CI: 5.7-20.8) and 15.1 kg (1.6-28.7) per 10-fold increase, p=0.001 and 0.03; respectively] after accounting for baseline weight, age, and sex. Conclusion: Although not influencing EE, higher plasma concentration of IL-6 predicts future weight and FM gain in humans, suggesting a potentially novel role of IL-6 in food intake and overeating.

Characteristics and Potential Biomarkers of Adult Sickle Cell Patients with Chronic Pain

A paradigm shift is occurring in our understanding of pain in patients with sickle cell disease (SCD). Vaso-occlusive episodes (VOEs) are crises of acute nociceptive pain, and have long been recognized as a hallmark of SCD (Brandow et al., 2017). While patients with SCD are traditionally considered to be at “steady state” and pain free between VOEs, recent studies have shown that a significant number of adults with SCD (~30%) experience daily chronic pain (>50% of the time in the past 6 months) (Smith et al., 2008). Although the precise mechanisms underlying this evolution from acute episodic to chronic pain are not well known, some contributing factors include age, chronic inflammation, organ damage, and opioid induced hyperalgesia (Stoicea et al., 2015; Rees et al., 2010). A recent study in a mouse model of SCD showed that mast cell activation was an important contributor to neurogenic inflammation and chronic pain (Vincent et al., 2013). We previously reported that SCD patients with chronic pain were older and had higher levels of mast cell activation markers, plasma tryptase and substance P, compared to those without chronic pain (Kuei et al., 2015). Recently, nerve growth factor (NGF) has been implicated in pathogenesis of some chronic pain syndromes (osteoarthritis), and clinical trials with anti-NGF monoclonal antibodies have been shown to result in superior pain control compared to placebo, opioids and NSAIDs. Here we report the results of our extended study of the evolution of chronic pain in SCD. A total of 72 subjects (age 15-66) were enrolled: 10 in the 15-19 age group, 19 in 20-29, 21 in 30-39, and 22 in ≥ 40. Patients transfused within the past 3 months and those who had an ED visit or hospitalization within the past 2 weeks were excluded. Information on the frequency of VOEs, presence or absence of chronic pain, HU therapy, opioid use (as mg morphine equivalents within the past 6 months), other medications and routine laboratory data (CBC, retics, chemistry panel, HbF) were collected. 4 mL of EDTA blood was collected at steady state and the plasma was separated by centrifugation and kept at -80 C. Plasma tryptase, substance P, and NGF levels were assayed by ELISA using kits from Biomatik inc (catalog# EKU07922), Enzo Life Sciences (catalog# ADI-900-018), and R&D Systems (catalog# DY256), respectively. Pressure pain threshold (PPTh) was measured using a hand-held digital algometer (AlgoMed, Medoc, Israel) four times at each of the following anatomic muscle groups on the left side of the body and then averaged for analysis: masseter, trapezius, and ulna in this consecutive order. Cutaneous mechanical pain was assessed using a Von Frey monofilament on the back of the subject's left hand. A baseline of one stimulus and then two separate series of 10 repeated stimuli each were conducted. The subject was asked to rate the pain on a scale of 0 to 10 (MFB, MF1 and MF2). Overall, 34 patients had chronic pain and 38 did not; there was an age dependent increase in the frequency of chronic pain, VOE frequency, opioid use and Von Frey MF values. Similarly, QST showed significantly lower pressure pain thresholds in subjects with chronic pain at ulna and trapezius (p=0.026 and 0.024 respectively). As expected, opioid use (daily morphine equivalents) was significantly higher in the chronic pain patients (52.8 mg vs 6.94 mg, p=0.009), suggesting a correlation between opioid use and hyperalgesia. Tryptase and substance P levels were higher in chronic pain patients, though the difference did not reach statistical significance. NGF levels were significantly higher in the chronic pain group (1126 pg/ml vs 473 pg/ml, p=0.051). Our results confirm previous observations that there is an age dependent increase in the proportion of patients with chronic pain (Table 1, Fig. 1-3). The trend towards higher levels of tryptase and substance P is in support of mast cell activation and neurogenic inflammation as a contributing factor to chronic pain (Vincent et al., 2013). To our knowledge, this is the first study of NGF as a possible contributing factor to chronic pain in SCD. If confirmed in larger multi-center studies, these observations could provide a rationale for novel interventions for chronic pain in SCD, via inhibition of mast cell activation/c-kit (tyrosine kinase inhibitors) or via repurposing of existing anti-NGF monoclonal antibodies as an alternative to opioids, whose inefficacy in chronic pain is well documented. DisclosuresKutlar:Novartis: Consultancy, Honoraria, Other: Personal fees, Research Funding; Bluebird Bio: Other: DSMB Member; Sancilio: Other: DSMB Chair.