Deconvolution of Serum Cortisol Levels by Using Compressed Sensing

The pulsatile release of cortisol from the adrenal glands is controlled by a hierarchical system that involves corticotropin releasing hormone (CRH) from the hypothalamus, adrenocorticotropin hormone (ACTH) from the pituitary, and cortisol from the adrenal glands. Determining the number, timing, and amplitude of the cortisol secretory events and recovering the infusion and clearance rates from serial measurements of serum cortisol levels is a challenging problem. Despite many years of work on this problem, a complete satisfactory solution has been elusive. We formulate this question as a non-convex optimization problem, and solve it using a coordinate descent algorithm that has a principled combination of (i) compressed sensing for recovering the amplitude and timing of the secretory events, and (ii) generalized cross validation for choosing the regularization parameter. Using only the observed serum cortisol levels, we model cortisol secretion from the adrenal glands using a second-order linear differential equation with pulsatile inputs that represent cortisol pulses released in response to pulses of ACTH. Using our algorithm and the assumption that the number of pulses is between 15 to 22 pulses over 24 hours, we successfully deconvolve both simulated datasets and actual 24-hr serum cortisol datasets sampled every 10 minutes from 10 healthy women. Assuming a one-minute resolution for the secretory events, we obtain physiologically plausible timings and amplitudes of each cortisol secretory event with R[superscript 2] above 0.92. Identification of the amplitude and timing of pulsatile hormone release allows (i) quantifying of normal and abnormal secretion patterns towards the goal of understanding pathological neuroendocrine states, and (ii) potentially designing optimal approaches for treating hormonal disorders.National Science Foundation (U.S.). Graduate Research Fellowship ProgramNational Institutes of Health (U.S.) (NIH DP1 OD003646)National Science Foundation (U.S.) (0836720)National Science Foundation (U.S.). Office of Emerging Frontiers in Research and Innovation (EFRI-0735956

Identification of Neural Outgrowth Genes using Genome-Wide RNAi

While genetic screens have identified many genes essential for neurite outgrowth, they have been limited in their ability to identify neural genes that also have earlier critical roles in the gastrula, or neural genes for which maternally contributed RNA compensates for gene mutations in the zygote. To address this, we developed methods to screen the Drosophila genome using RNA-interference (RNAi) on primary neural cells and present the results of the first full-genome RNAi screen in neurons. We used live-cell imaging and quantitative image analysis to characterize the morphological phenotypes of fluorescently labelled primary neurons and glia in response to RNAi-mediated gene knockdown. From the full genome screen, we focused our analysis on 104 evolutionarily conserved genes that when downregulated by RNAi, have morphological defects such as reduced axon extension, excessive branching, loss of fasciculation, and blebbing. To assist in the phenotypic analysis of the large data sets, we generated image analysis algorithms that could assess the statistical significance of the mutant phenotypes. The algorithms were essential for the analysis of the thousands of images generated by the screening process and will become a valuable tool for future genome-wide screens in primary neurons. Our analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. We also found that genes known to be involved in protein and vesicle trafficking showed similar RNAi phenotypes. We confirmed phenotypes of the protein trafficking genes Sec61alpha and Ran GTPase using Drosophila embryo and mouse embryonic cerebral cortical neurons, respectively. Collectively, our results showed that RNAi phenotypes in primary neural culture can parallel in vivo phenotypes, and the screening technique can be used to identify many new genes that have important functions in the nervous system

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Association between lung density changes and radiation pneumonitis after IMRT for lung cancer.

Purpose or Objective Definitive chemoradiotherapy (CRT) is potentially curative for locally advanced bronchogenic carcinoma and non-surgical candidates. However, radiation induced pneumonitis (RP) constitutes a major threat to the quality of life. The aim of our work is to investigate the impact of lung density changes as well as other risk factors in predicting RP in patients with lung cancer undergoing CRT using IMRT technique. Material and Methods After IRB approval we queried lung cancer cases treated definitively using CRT with available follow up CT chest datasets. We excluded all patients with prior chest RT, incomplete treatment and cases lacking post-RT images. All patients underwent 4D-CT simulation and the treatment plan was prescribed to cover PTV with 60-66 Gy in 2 Gy fractions using IMRT. Planning CT images with dose distribution were mapped to follow up CT using deformable registration to assess Hounsfield Unit (HU) changes after RT as portrayed in figure (1) excluding any tumor specific changes. Radiation oncologists graded RP by reviewing surveillance encounters using CTCAE v4.03 scale and were blinded to HU changes. Spearman\u27s correlation was plotted for post-RT HU changes against all RP grades. Chi-square and Fisher\u27s exact test were used to study factors associated with RP ≥2. Results After a median follow up of 17 months (2-57) we were able to identify 118 patients who met our inclusion criteria. All had a smoking history with mean pack years (46±29), 51% were males and median age was 70 (45-88). Stage III were the majority (64%) and adenocarcinoma was present in 52.5% followed by squamous and small cell carcinoma (40 & 7.5%). Carboplatin/taxol was the most utilized regimen (54%) followed by platinum/pemetrexed or eteposide (26% & 15%).Grade 1 (G1), a radiological finding; RP was detected in 42 cases (35.6%); grade 2 (G2) & 3 (G3) in 22(18.6%) and 7(5.9%) respectively. The mean corresponding density increases for both lungs - CTV were 10, 26.7, 29.3 & 58.1 HU for G0-3 RP respectively that resulted in a significant Spearman\u27s correlation (r=0.43, p\u3c0.0001) as shown in figure (2). Similar outcomes were depicted for ipsilateral lung - CTV as well as the V20 volumes (p\u3c0.001). Another significant correlation was observed between increasing mean lung dose (MLD) (r=0.26, p=0.01) and V20 (r=0.22, p=0.02) with RP grades, although both were within normal constraints (V20: 21.3±3.9 % and MLD: 13.1±3.9 Gy). All baseline metrics were balanced among RP groups with no significant differences, except that symptomatic RP requiring medical intervention (G2 & 3) had significantly more chronic obstructive lung disease (COPD) (93% vs 67% for G2+ vs G0-1; p=0.006). Conclusion Deformable registration and quantification of HU changes between baseline and follow up CT is strongly predictive for RP and can be integrated in regular follow up protocols after validation in a prospective trial setting. More robust dose constraints need to be tailored for cases with baseline COPD as they are at increased risk for RP. (Figure Presented)

Changing Practice Patterns in the Radiation Treatment Delivery for Locally Advanced Lung Cancer: Results from a Statewide Consortium

Purpose/Objective(s): In 2014, the RTOG 0617 trial defined the standard radiation therapy (RT) dose as 60 Gy with concurrent chemotherapy in locally advanced non-small cell lung cancer (LA-NSCLC) patients. Further analyses of the study showed a decrease in grade 3 pneumonitis with static intensity modulated radiation therapy (sIMRT). Radiation treatment techniques used in routine clinical practice have evolved from 3D conformal radiation therapy (3DCRT) to sIMRT and more recently to rotational IMRT (rIMRT). However, the uptake of lower lung tumor RT dose recommendations and RT treatment techniques used in the “real world” setting is not known. The purpose of this study was to evaluate patterns of practice of both radiation dose and type of radiation technique used in a statewide radiation oncology consortium. Materials/Methods: From 2012 to 2018, 2741 patients with lung cancer treated with curative intent RT at 24 institutions participating in a statewide Radiation Oncology Quality Consortium were enrolled. Patients with small cell lung cancer, previous surgical resection, and patients who did not complete treatment were excluded. Furthermore, for RT dose analyses, outliers of RT doses (\u3c45 Gy and \u3e100 Gy) were excluded. Patients lacking DICOM information regarding treatment plans were excluded for the RT technique analyses. Changes in prescription doses and treatment techniques over time were evaluated. Trends over time in academic (n=4) vs. non-academic centers (n=20) were also analyzed. Academic centers were defined as those with a medical residency training program. Sample t-tests were used to compare means. Results: 1134 patients were analyzed for RT dose analysis, while 1087 patients were evaluated for the RT treatment technique. The mean prescription RT dose delivered to tumor decreased from 63 Gy to 60 Gy (p\u3c0.001) over the 6-year time period. In the earlier years of the data collection (2012-2013), \u3e50% of the cases were treated with 3DCRT and rIMRT was used in ≤5%. However, by 2018, the rate of rIMRT increased to 56.9% (p\u3c0.0001). See Table below. Similar practice patterns were seen in academic and non-academic centers for both RT dose and treatment technique analyses. Conclusion: In this large prospective “real world” study from a statewide consortium of practicing radiation oncologists, prescription mean RT dose used to treat LA-NSCLC has decreased to 60 Gy as per evidence-based guidelines. The primary treatment technique of delivering RT in LA-NSCLC has also changed in this interval. More than half of LA-NSCLC patients are now receiving rIMRT. The clinical implications of potential increases in low dose volumes (e.g., Lung and Heart V5) resulting from rIMRT will have to be evaluated going forward, especially in the changing systemic therapy paradigm that includes immunotherapy. [Figure presented