Inducible gene expression of IκB-kinase ε is dependent on nuclear factor-κB in human pulmonary epithelial cells.

While IκB-kinase-ε (IKKε) induces immunomodulatory genes following viral stimuli, its up-regulation by inflammatory cytokines remains under-explored. Since airway epithelial cells respond to airborne insults and potentiate inflammation, IKKε expression was characterized in pulmonary epithelial cell lines (A549, BEAS-2B) and primary human bronchial epithelial cells grown as submersion or differentiated air-liquid interface cultures. IKKε expression was up-regulated by the pro-inflammatory cytokines, interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNFα). Thus, mechanistic interrogations in A549 cells were used to demonstrate the NF-κB dependence of cytokine-induced IKKε. Furthermore, chromatin immunoprecipitation in A549 and BEAS-2B cells revealed robust recruitment of the NF-κB subunit, p65, to one 5' and two intronic regions within the IKKε locus (IKBKE). In addition, IL-1ß and TNFα induced strong RNA polymerase 2 recruitment to the 5' region, the first intron, and the transcription start site. Stable transfection of the p65-binding regions into A549 cells revealed IL-1ß- and TNFα-inducible reporter activity that required NF-κB, but was not repressed by glucocorticoid. While critical NF-κB motifs were identified in the 5' and downstream intronic regions, the first intronic region did not contain functional NF-κB motifs. Thus, IL-1ß- and TNFα-induced IKKε expression involves three NF-κB-binding regions, containing multiple functional NF-κB motifs, and potentially other mechanisms of p65 binding through non-classical NF-κB binding motifs. By enhancing IKKε expression, IL-1ß may prime, or potentiate, responses to alternative stimuli, as modelled by IKKε phosphorylation induced by phorbol 12-myristate 13-acetate. However, since IKKε expression was only partially repressed by glucocorticoid, IKKε-dependent responses could contribute to glucocorticoid-resistant disease.

Comparison of the Genomic Activity of an EP4-Receptor and ß2-Adrenoceptor Agonist in BEAS-2B Human Bronchial Epithelial Cells: In Search of Compartmentalized, cAMP-Dependent Gene Expression.

It has been proposed that inhaled E-prostanoid 4 (EP4)-receptor agonists could represent a new class of bronchodilators for the treatment of asthma that are as effective as ß 2-adrenoceptor agonists. However, the genomic impact of such drugs is unknown despite being potentially deleterious to respiratory health. Herein, we used mRNA-seq to compare the transcriptomic responses produced by 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxo-cyclopentyl]sulphanylpropylsulphanyl] acetic acid (ONO-AE1-329; an EP4-receptor agonist) and vilanterol (a ß 2-adrenoceptor agonist) in BEAS-2B human airway epithelial cells. We also determined if an increase in cAMP mediated by different G protein-coupled receptors (GPCRs) promoted distinct transcriptional signatures by expanding this inquiry to include the adenosine A2B- and I-prostanoid receptor agonists, 2-[[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]-2-pyridinyl]thio]-acetamide (Bay60-6583) and taprostene, respectively. Maximally-effective concentrations of ONO-AE1-329 and vilanterol significantly regulated (q ≤ 0.05; ≥1.5-/≤0.67-fold) 232 and 320 genes, respectively of which 217 were shared. Spearman analysis showed these gene expression changes to be highly rank order correlated, indicating that the functional overlap between the two interventions should be considerable. Unexpectedly, the genomic effects of ONO-AE1-329, vilanterol, Bay 60-6583, and taprostene were also highly rank order correlated. This finding suggests that cAMP generated by any GPCR would initiate the same transcriptional program. Nevertheless, relative to vilanterol, ONO-AE1-329 typically behaved as a partial agonist that varied across transcripts. These data indicate that each ONO-AE1-329-regulated gene differs in sensitivity to cAMP and is defined by a unique receptor occupancy-response relationship. Moreover, if this relatively modest genomic response in BEAS-2B cells is retained in vivo, then inhaled EP4-receptor agonists could represent an alternative, and possibly safer, class of bronchodilators. SIGNIFICANCE STATEMENT: The genomic consequences of ß 2-adrenoceptor agonists in asthma are often overlooked despite being potentially harmful to lung health. We determined that ONO-AE1-329, an EP4-receptor agonist and effective bronchodilator, produced gene expression changes in BEAS-2B cells that were typically modest relative to the ß 2-adrenoceptor agonist vilanterol. Furthermore, ONO-AE1-329 behaved as a partial agonist that varied across transcripts. If this genomic activity is reproduced in vivo, then EP4-receptor agonists could represent an alternative, and possibly safer, class of bronchodilators.

Synergy between Interleukin-1ß, Interferon-γ, and Glucocorticoids to Induce TLR2 Expression Involves NF-κB, STAT1, and the Glucocorticoid Receptor.

Glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to downregulate inflammatory gene expression and are effective treatments for mild to moderate asthma. However, in severe asthma and virus-induced exacerbations, glucocorticoid therapies are less efficacious, possibly due to reduced repressive ability and/or the increased expression of proinflammatory genes. In human A549 epithelial and primary human bronchial epithelial cells, toll-like receptor (TLR)-2 mRNA and protein were supra-additively induced by interleukin-1ß (IL-1ß) plus dexamethasone (IL-1ß+Dex), interferon-γ (IFN-γ) plus dexamethasone (IFN-γ+Dex), and IL-1ß plus IFN-γ plus dexamethasone (IL-1ß+IFN-γ+Dex). Indeed, ∼34- to 2100-fold increases were apparent at 24 hours for IL-1ß+IFN-γ+Dex, and this was greater than for any single or dual treatment. Using the A549 cell model, TLR2 induction by IL-1ß+IFN-γ+Dex was antagonized by Org34517, a competitive GR antagonist. Further, when combined with IL-1ß, IFN-γ, or IL-1ß+IFN-γ, the enhancements by dexamethasone on TLR2 expression required GR. Likewise, inhibitor of κB kinase 2 inhibitors reduced IL-1ß+IFN-γ+Dex-induced TLR2 expression, and TLR2 expression induced by IL-1ß+Dex, with or without IFN-γ, required the nuclear factor (NF)-κB subunit, p65. Similarly, signal transducer and activator of transcription (STAT)-1 phosphorylation and γ-interferon-activated sequence-dependent transcription were induced by IFN-γ These, along with IL-1ß+IFN-γ+Dex-induced TLR2 expression, were inhibited by Janus kinase (JAK) inhibitors. As IL-1ß+IFN-γ+Dex-induced TLR2 expression also required STAT1, this study reveals cooperation between JAK-STAT1, NF-κB, and GR to upregulate TLR2 expression. Since TLR2 agonism elicits inflammatory responses, we propose that synergies involving TLR2 may occur within the cytokine milieu present in the immunopathology of glucocorticoid-resistant disease, and this could promote glucocorticoid resistance. SIGNIFICANCE STATEMENT: This study highlights that in human pulmonary epithelial cells, glucocorticoids, when combined with the inflammatory cytokines interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ), can synergistically induce the expression of inflammatory genes, such as TLR2. This effect involved positive combinatorial interactions between NF-κB/p65, glucocorticoid receptor, and JAK-STAT1 signaling to synergistically upregulate TLR2 expression. Thus, synergies involving glucocorticoid enhancement of TLR2 expression may occur in the immunopathology of glucocorticoid-resistant inflammatory diseases, including severe asthma.

Nonlinearity in bone-conducted amplitude-modulated cervical vestibular-evoked myogenic potentials: harmonic distortion products.

Otolith organs of the balance system, the saccule and utricle, encode linear acceleration. Integrity of the saccule is commonly assessed using cervical vestibular-evoked myogenic potentials (cVEMPs) arising from an inhibitory reflex along the vestibulospinal pathway. Conventional approaches to eliciting these responses use brief, transient sounds to elicit onset responses. Here we used long-duration amplitude-modulated (AM) tones to elicit cVEMPs (AMcVEMPs) and analyzed their spectral content for evidence of nonlinear processing consistent with known characteristics of vestibular hair cells. Twelve young adults (ages 21-25) with no hearing or vestibular pathologies participated in this study. AMcVEMPs were elicited by bone-conducted AM tones with a 500-Hz carrier frequency. Eighteen modulation frequencies were used between 7 and 403 Hz. All participants had robust distortion products at harmonics of the modulation frequency. Total harmonic distortion ranged from approximately 10 to 80%. AMcVEMPs contain harmonic distortion products consistent with vestibular hair cell nonlinearities, and this new approach to studying the otolith organs may provide a noninvasive, in vivo method to study nonlinearity of vestibular hair cells in humans.NEW & NOTEWORTHY The otolith balance organs of humans are assessed for basic science and clinical applications by using vestibular-evoked myogenic potentials (VEMPs). Traditionally, VEMPs are elicited with brief, transient sounds to study onset responses. We used long-duration sounds to elicit steady-state VEMPs. This allowed us to measure nonlinear distortion products, consistent with nonlinear processing in vestibular hair cells. This new approach may help to better understand links between otolith organs and balance function.

Comparison of Bone-Conducted Cervical VEMPs Elicited by B71 and B81 Bone Vibrators.

OBJECTIVE: A variety of stimulus delivery methods can elicit vestibular evoked myogenic potentials (VEMPs). The current study compared bone conduction (BC) cervical VEMPs (cVEMPs) across two different clinical bone vibrators. It was hypothesized that the B81 transducer would be more effective for producing larger BC-cVEMP peak to peak amplitudes due to its low-frequency advantages in pure-tone audiometry applications. DESIGN: Twenty young adults under the age of 40 years with no reported history of hearing or balance disorders participated in the study. BC cVEMPs were elicited using two clinical bone transducers: the Radioear B71 bone vibrator and the Radioear B81 bone vibrator. Both transducers were calibrated using the acoustic method of calibration before data collection, and the linear dynamic range of the transducers was determined. Participants were asked to sit and match a fixed electromyography (EMG) target level of 100 µV, while BC cVEMPs were recorded using stimulus frequencies of 250, 500, and 750 Hz. RESULTS: Statistically significant differences in raw amplitude at 250 and 750 Hz between the B71 and B81 were observed; the B71 produced larger peak to peak amplitudes over the B81. At 500 Hz, larger amplitudes were observed with the B71, but results were not statistically significant. The B71 produced significantly lower cVEMP thresholds at all three frequencies. Across both transducers, 500 Hz produced the largest peak to peak amplitude compared with 250 and 750 Hz. Peak to peak amplitude did not increase above 55 dB nHL for 250 and 500 Hz, but amplitude continued to increase at 750 Hz. DISCUSSION: The present study found statistically significant differences in BC-cVEMP amplitude and threshold between the B71 and B81, but results were not what we hypothesized. In general, the B71 elicited larger BC-cVEMP amplitudes and lower thresholds compared with the B81. Additionally, 500 Hz was found to be the best frequency for both BC transducers, contrasting previous studies suggesting lower frequencies yield larger BC-cVEMP amplitudes. It is possible that these average differences could also be clinically significant when looking at individual amplitude differences. Larger peak to peak amplitudes at 500 Hz may be partially due to the underlying physical levels used in the current study, as well as the output spectra of the transducers, and may explain the larger response amplitudes observed at 500 Hz compared with 250 Hz.

Maximum Output and Low-Frequency Limitations of B71 and B81 Clinical Bone Vibrators: Implications for Vestibular Evoked Potentials.

OBJECTIVES: Bone-conducted vestibular evoked myogenic potentials (VEMPs) are tuned to have their maximum amplitude in response to tone bursts at or below 250 Hz. The low-frequency limitations of clinical bone vibrators have not been established for transient, tone burst stimuli at frequencies that are optimal for eliciting VEMPs. DESIGN: Tone bursts with frequencies of 250 to 2000 Hz were delivered to B71 and B81 bone vibrators and their output was examined using an artificial mastoid. The lower-frequency limit of the transducers was evaluated by examining the spectral output of the bone vibrators. Maximum output levels were evaluated by measuring input-output functions across a range of stimulus levels. RESULTS: Both the B71 and B81 could produce transient tone bursts with frequency as low as 400 Hz. However, tone bursts with frequencies of 250 and 315 Hz resulted in output with peak spectral energy at approximately 400 Hz. From 500 to 2000 Hz, maximum output levels within the linear range were between 120 and 128 dB peak force level. The newer B81 bone vibrator had a maximum output approximately 5 dB higher than the B71 at several frequencies. CONCLUSIONS: These findings demonstrate that both transducers can reach levels appropriate to elicit bone-conducted VEMPs, but the low-frequency limitations of these clinical bone vibrators limit tone burst frequency to approximately 400 Hz when attempting to stimulate the otolith organs via tone bursts.

Influence of surgical approach for decompression on lower eyelid position in thyroid eye disease.

Purpose: Orbital decompression for thyroid eye disease (TED) has been noted to improve lower lid retraction by 0.5-1 mm. We hypothesize that orbital decompression via transconjunctival approach may lead to increased reduction in marginal reflex distance 2 (MRD2) as it involves division of the lower lid retractors. The purpose of this study is to evaluate relative changes in lower lid position for patients undergoing lateral and transconjunctival orbital decompression, respectively.Methods: In this cross-sectional study, all TED patients managed with lateral or transconjunctival orbital decompression for a 3-year period were screened for inclusion. Photographs taken in the primary position preoperatively and three months postoperatively were utilized to evaluate the MRD2 from each patient. Measurements were made utilizing NIH ImageJ software standardized to a corneal diameter. Hertel measurements of proptosis were obtained pre and postoperatively. The primary outcome measure was MRD2 in operative eyes.Results: A total of 131 (86 patients) operative eyes were included in the sample. Mean change MRD2 was not significantly different between the surgical groups (p = 0.07). In multivariate modeling, mean change in MRD2 was significantly associated with change in exophthalmometry, independent of surgical approach.Conclusions: The association between decrease in Hertel measurement and decrease in MRD2 is consistent with the existing literature on the topic. It appears that transconjunctival division of the lower eyelid retractors provides no additional benefit in reducing lower lid retraction relative to change in proptosis.

Relationship between ocular dominance and brow position in patients with blepharoptosis.

PURPOSE: This study aims to determine if ocular dominance plays a role in predicting compensatory eyebrow elevation in cases of ptosis. METHODS: This retrospective observational cohort study screened all individuals presenting to two tertiary oculoplastics practices with complaints of ptosis for entry. Primary position photographs were obtained. Ocular dominance was assessed via a modified Porta test. Ptosis was defined in bilateral cases as marginal reflex distance of <2.5 mm in both eyes and in unilateral cases as either an MRD1 < 2.5 mm or MRD1 of >1 mm lower on one side. Asymmetry in brow height was defined as a difference of >1 mm. Chi square and t-tests were performed. RESULTS: Sixty-eight patients from the both tertiary practices met inclusion criteria (37 male, 31 female). Concordance between the higher brow and the dominant side was 50.0% (n = 22, p > 0.05). Mean brow height on the dominant side (15.5 mm) was not statistically different than brow height on the non-dominant side (15.3 mm, p > 0.05). The concordance between the higher brow and the lower MRD1 eyelid was not significant (45.5%, n = 20, p > 0.05). The difference in mean brow height between the lower and higher MRD1 eyes was not significantly different (-0.11 mm; p > 0.05). This also held true when restricted to unilateral cases (0.28; p > 0.05). CONCLUSIONS: Although asymmetric brow elevation can be noted in patients with ptosis, ocular dominance does not appear to be concordant with this asymmetry. Additionally, brow height does not appear to be concordant with MRD1 in cases of ptosis.

Evaluating the effects of a global pandemic on the operation of an investigational drug service.

PURPOSE: This study is an analysis of the changes to workload and operations of UNC Health's investigational drug service (IDS) brought about by the coronavirus disease 2019 (COVID-19) pandemic. METHODS: Workload statistics were collected and analyzed for trend changes to illustrate operational changes necessitated by the COVID-19 pandemic within the IDS pharmacy at UNC Health. RESULTS: Multiple workload metrics declined at the beginning of the COVID-19 pandemic, followed by an increase in the metrics for many categories as the pandemic continued. Notably, monthly inventory added initially decreased by 37.5%, later leveling off but showing increased variability. Fills dispensed and monitoring visits both decreased by 34.5% from the first quarter (Q1) to Q2 of 2020. Both metrics returned to or slightly exceeded prepandemic levels by the end of the study period in March 2021. Patient enrollment decreased 76% from February to May 2020 before dramatically increasing in Q3 of 2020 and Q1 of 2021 with the initiation of COVID-19 vaccine studies. The average time to study startup increased for trials not related to COVID-19 and decreased for COVID-19-related trials. There has been no major impact on the number of open protocols throughout the course of the pandemic. CONCLUSION: Despite initial decreases in workload following the start of the COVID-19 pandemic, IDS operations returned to and, in some cases, exceeded prepandemic levels.

Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cells.

Roles for the baculoviral inhibitor of apoptosis repeat-containing (BIRC) genes, BIRC2 and BIRC3, may include signaling to the inflammatory transcription factor, nuclear factor-κB (NF-κB) and protection from cell death. However, distinct functions for each BIRC are not well-delineated. Given roles for the epithelium in barrier function and host defence, BIRC2 and BIRC3 expression was characterized in pulmonary epithelial cell lines and primary human bronchial epithelial cells (pHBECs) grown as undifferentiated cells in submersion culture (SC) or as highly differentiated cells at air-liquid interface (ALI). In A549 cells, interleukin-1ß (IL1B) and tumor necrosis factor α (TNF) induced BIRC3 mRNA (~20-50-fold), with maximal protein expression from 6-24 h. Similar effects occurred in BEAS-2B and Calu-3 cells, as well as SC and ALI pHBECs. BIRC2 protein was readily detected in unstimulated cells, but was not markedly modulated by IL1B or TNF. Glucocorticoids (dexamethasone, budesonide) modestly increased BIRC3 mRNA and protein, but showed little effect on BIRC2 expression. In A549 cells, BIRC3 mRNA induced by IL1B was unchanged by glucocorticoids and showed supra-additivity with TNF-plus-glucocorticoid. Supra-additivity was also evident for IL1B-plus-budesonide induced-BIRC3 in SC and ALI pHBECs. Using A549 cells, IL1B- and TNF-induced BIRC3 expression, and to a lesser extent, BIRC2, was prevented by NF-κB inhibition. Glucocorticoid-induced BIRC3 expression was prevented by silencing and antagonism of the glucocorticoid receptor. Whereas TNF, but not IL1B, induced degradation of basal BIRC2 and BIRC3 protein, IL1B- and TNF-induced BIRC3 protein remained stable. Differential regulation by cytokines and glucocorticoids shows BIRC2 protein expression to be consistent with roles in rapid signaling events, whereas cytokine-induced BIRC3 may be more important in later effects. While TNF-induced degradation of both BIRCs may restrict their activity, cytokine-enhanced BIRC3 expression could prime for its function. Finally, shielding from glucocorticoid repression, or further enhancement by glucocorticoid, may indicate a key protective role for BIRC3.

Effects of Tonic Muscle Activation on Amplitude-Modulated Cervical Vestibular Evoked Myogenic Potentials (AMcVEMPs) in Young Females: Preliminary Findings.

Cervical vestibular evoked myogenic potentials (cVEMPs) are usually elicited by transient tonebursts, but when elicited by amplitude-modulated (AM) tones, they can provide new information about cVEMPs. Previous reports of cVEMPs elicited by AM tones, or AMcVEMPs, have not systematically examined the effects of tonic EMG activation on their response properties. Fourteen young, healthy female adults (ages 20-24) with clinically normal audiograms participated in this study. AMcVEMPs were elicited with bone-conducted 500 Hz tones amplitude modulated at a rate of 37 Hz and recorded for five different EMG targets ranging from 0 to 90 µV. Amplitude increased linearly as tonic EMG activation increased. Signal-to-noise ratio (SNR) was minimal at 0 µV, but robust and with equivalent values from 30 to 90 µV; phase coherence and EMG-corrected amplitude had findings similar to SNR across EMG target levels. Interaural asymmetry ratios for SNR and phase coherence were substantially lower than those for raw or corrected amplitude. AMcVEMP amplitude scaled with tonic EMG activation similar to transient cVEMPs. Signal-to-noise ratio, phase coherence, and EMG-corrected amplitude plateaued across a range of EMG values, suggesting that these properties of the response reach their maximum values at relatively low levels of EMG activation and that higher levels of EMG activation are not necessary to record robust AMcVEMPs.

Phacoemulsifier occlusion break surge volume reduction.

PURPOSE: To compare the volumetric occlusion break surge responses of phacoemulsification units from 1 company over 3 generations under varying vacuum limits and target intraocular pressure (IOP) settings. SETTING: Alcon Research, Ltd., Lake Forest, California, USA. DESIGN: Experimental study. METHODS: Three generations of phacoemulsification units (Infiniti Vision System, Centurion Vision System, and Centurion Vision System with Active Sentry upgrades) were tested. Volumetric surge responses were measured after occlusion breaks at vacuum limits of 200 mm Hg, 300 mm Hg, 400 mm Hg, 500 mm Hg, and 600 mm Hg and target IOPs of 30 mm Hg, 55 mm Hg, and 80 mm Hg. An acrylic test chamber with a piston attached to 3 springs modeled the human eye in this study. The springs were calibrated to mimic volumetric changes in the eye over a wide range of IOPs. RESULTS: Occlusion break surge volumes varied from 17.4 µL to 153 µL, corresponding to 7% and 61%, respectively, of the aqueous volume in the average phakic eye and to 4% and 33% of the aqueous volume in the average aphakic eye. CONCLUSION: Occlusion break surge volumes decreased with increasing target IOP, decreasing vacuum limit, and each generational increment in the phacoemulsification system.

Aqueous volume loss associated with occlusion break surge in phacoemulsifiers from 4 different manufacturers.

PURPOSE: To evaluate aqueous volume losses associated with occlusion breaks at varying vacuum limits in phacoemulsification systems from 4 different manufacturers. SETTING: Alcon Research Ltd., Lake Forest, California, USA. DESIGN: Experimental study. METHODS: The anterior chamber was modeled using the spring eye model. Systems tested included the Centurion, Whitestar Signature, Stellaris PC, and EVA. Occlusion breaks were actuated at vacuum limits of 200, 300, 400, 500, and 600 mm Hg and a target intraocular pressure of 55 mm Hg. RESULTS: The model eye piston reached its measurement limit just below 600 mm Hg on the EVA and just above 400 mm Hg on the Stellaris PC. Higher vacuum limits could not be tested on these 2 units. Surge volumes varied from 17 to 77 µL on the Centurion, 30 to 103 µL on the Whitestar Signature, 67 µL to 163 µL on the Stellaris PC, and 47 to 165 µL on the EVA. Assuming an average phakic eye aqueous volume of 250 µL, these µL values correspond to percent aqueous volume losses of 7% to 31% on the Centurion, 12% to 41% on the Whitestar Signature, 27% to 65% on the Stellaris PC, and 19% to 66% on the EVA. Surge responses increased on all machines with each increment in vacuum limit. The Centurion had the lowest surge volumes across all vacuum limits. CONCLUSIONS: Occlusion break surge volumes vary considerably across phacoemulsification platforms. Severe chamber shallowing might occur if an occlusion break occurs under high vacuum on some systems.

Anesthesia and use of a sling system to facilitate transvaginal laparoscopy in a black rhinoceros (Diceros bicornis minor).

Transvaginal laparoscopy to allow assessment of ovarian pathology and to attempt retrieval of oocytes was facilitated in a captive, female black rhinoceros (Diceros bicornis minor) through the use of a sling on two separate occasions. Following induction of anesthesia with an opioid-based combination, the rhinoceros was intubated and maintained on isoflurane in oxygen. The use of the sling and volume controlled inhalation anesthesia allowed for maintenance of appropriate anatomic positioning, analgesia, and insufflation of the abdominal cavity for laparoscopy during both procedures.