Citations

Serine protease inhibitors of the Kunitz‐bovine pancreatic trypsin inhibitor (BPTI) family, such as tissue factor pathway inhibitor‐1 Kunitz domain 1 ( KD1 TFPI1) and amyloid precursor protein inhibitor (APPI), are biological regulators of proteolysis and serve as important scaffolds for developing therapeutics. Mesotrypsin is a unique human protease with cancer‐promoting properties which rapidly cleaves and inactivates serine protease inhibitors; because of this, it is a challenging therapeutic target and an excellent model for developing therapeutic inhibitors with improved proteolytic resistance. We hypothesize that stabilization of the Kunitz inhibitor binding loop by disulfide bond anchoring to the scaffold can reduce proteolytic susceptibility and improve binding efficacy. Here we have engineered a new disulfide bond into both KD1 TFPI1 and an APPI variant previously engineered for mesotrypsin affinity. The new disulfide bond anchors the binding loop to the scaffold, resulting in reduced mesotrypsin proteolytic cleavage by 17‐fold and 3‐fold, respectively. To gain insight into the structural basis for improved proteolytic stability, we crystallized mutant KD1 TFPI1 and APPI with mesotrypsin and solved these novel structures. We then performed all‐atom molecular dynamics simulations using these mesotrypsin‐mutant inhibitor complexes compared to models lacking the engineered disulfide bond. Our simulation results support our hypothesis that the cause of decreased proteolysis rates is dampened conformational fluctuations in the binding loop and minimized motion at the enzyme‐inhibitor interface. We also performed simulations using unbound inhibitors to show that stabilization of the binding loop to states similar to the bound configuration correlates with stronger binding affinity. Our disulfide engineering approach is potentially a useful tool in biopharmaceutical development for mesotrypsin, other serine proteases, and possibly for other enzyme targets. Support or Funding Information NIH grant R01CA154387 to E.S.R., European Research Council grant 336041 to N.P., US‐Israel Binational Science Foundation grant BSF2015134 to E.S.R. and N.P. Xray diffraction data were measured at the Berkeley Center for Structural Biology at the Advanced Light Source and at the European Synchrotron Radiation Facility. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Cerebrovascular reactivity to hypercapnia declines with age and low reactivity may be a biomarker of cognitive decline. Central elastic arteries that perfuse the brain stiffen with age, increasing their transmission of pulsatile mechanical forces into the cerebral microvasculature and impairing cerebral microvessel function. Habitual physical activity, however, has been shown to reduce central arterial stiffness and is associated with better cognitive function. Therefore, the purpose of this study was to evaluate cerebrovascular reactivity in young and older healthy adults who habitually exercise. We hypothesized that older adults who habitually exercised would have similar cerebrovascular reactivity when compared with young adults. However, because the underlying mechanisms of cerebral blood flow regulation may differ with age, we also evaluated blood pressure and central hemodynamic measures. We recruited 25 young (YA: age = 27 ± 1 y) and 20 older (OA: age = 59 ± 1 y) habitual exercisers. Middle cerebral artery velocity (MCAv) was recorded using transcranial Doppler ultrasound. In order to assess cerebrovascular reactivity, mean arterial pressure (MAP), end‐tidal carbon dioxide (ETCO 2 ), and MCAv were continuously recorded at rest and during stepwise elevations of inhaled CO 2 . Cerebrovascular conductance index (CVCi) was calculated as MCAv/MAP. Reactivity was calculated as the linear relationship between the change in ETCO 2 and the change in each variable of interest (MCAv, CVCi and MAP). Central arterial stiffness (carotid‐femoral pulse wave velocity, PWV) and central hemodynamics (augmentation index, AIx) were assessed by applanation tonometry. Older adults had higher MAP (YA: 85 ± 2 mmHg; OA: 90 ± 3 mmHg; p<0.05) and AIx (YA: 0 ± 3 %; OA: 20 ± 2 %; p<0.05) at rest compared with young adults. PWV was not statistically different between groups (YA: 4.9 ± 0.3 cm/s; OA: 5.7 ± 0.5 cm/s; p=0.18). MCAv and CVCi reactivity to hypercapnia were similar between young and older adults (MCAv reactivity, YA: 2.0 ± 0.2 cm/s/mmHg; OA: 2.2 ± 0.1 cm/s/mmHg; p=0.46, CVCi reactivity, YA: 0.018 ± 0.002 cm/s/mmHg 2 ; OA: 0.016 ± 0.001 cm/s/mmHg 2 ; p=0.42); however, older adults demonstrated higher MAP reactivity to hypercapnia (YA: 0.3 ± 0.1 a.u.; OA: 0.8 ± 0.1 a.u.; p<0.05). PWV and AIx were not associated with MCAv, CVCi, or MAP reactivity to hypercapnia (p>0.05). Our results suggest that habitual exercise preserves cerebrovascular reactivity; however, MAP reactivity is augmented with aging allowing older adults to sufficiently increase cerebral blood flow during hypercapnia. Support or Funding Information Supported by National Institute of Health grant HL118154 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

ABSTRACT Purpose: Evidence regarding the utility of systemic steroids in treating patients with cirrhosis and septic shock remains equivocal. This study aimed to evaluate and elucidate the association of steroid use with outcomes and adverse effects in a cohort of patients with cirrhosis and septic shock. Patients and methods: Retrospective cohort study of patients with cirrhosis and septic shock admitted to a tertiary hospital intensive care unit (ICU) from January 2007 to May 2017, using a validated ICU Datamart. Patients who received vasopressors within 6 h of ICU admission were included in the multivariate analysis. The effect of steroids on outcomes was evaluated using multivariable regression, adjusting for confounding variables. Results: Out of 179 admissions of patients with cirrhosis and septic shock, 56 received steroids during the ICU admission. Patients who received steroids received a higher total dose of vasopressors (91.2 mg vs. 39.1 mg, P = 0.04) and had a lower initial lactate level (1.8 mmol/L vs. 2.6 mmol/L, P = 0.007). The multivariate analysis included 117 patients and showed no significant differences in mortality, length of ICU admission, or length of hospital stay. Bleeding events, delirium, and renal-replacement therapy requirements were also not associated with the use of steroids. Conclusion: The use of systemic steroids was more prevalent in cirrhotic patients with higher vasopressor requirements. It was not associated with decreased mortality or increased ICU- and hospital-free days, or to adverse effects.

11007 Background: The proportion of women & underrepresented groups in medicine (URM) in the field of hematology and oncology remains low, particularly in academic leadership positions. Editorial board appointments allow physicians to have a substantial impact on the nature of the published scholarly work and serve as a platform for academic opportunities. We aimed to assess gender and race/ethnicity representation in editorial board positions in hematology and oncology journals. Methods: Editorial leadership board members from 60 journals from oncology, hematology, radiation oncology, and surgical oncology were reviewed, 54 journals were included in the analysis. Gender and race/ethnicity were determined based on publicly available data for editor-in-chief (EiC) and second-in-command (SiC) (including deputy, senior, or associate editors). Descriptive statistics and chi-squared were estimated. Results: A total of 793 editorial board members are included in the analysis. 72.6 % were men and 27.4 % were women. 71.3% of editorial leadership were non-Hispanic white with Asian editorial board members representing the second largest majority at 23.3%. The editorial position was significantly different among men and women (p = 0.038) with women filling only 15.9% (10/63) of the EiC positions. Of these 10 women, the racial breakdown was 90% white and 10% Asian. In the prevalence odds ratio (pOR), women were about half as likely to be in the EiC position compared with men [pOR: 0.47, 95%CI (0.23, 0.95, p = 0.03)]. Women represented 28.4% (207/730) of SiC editorial positions. White editors had the highest representation at 71.0% in the SiC editorial positions, followed by Asian editors at 16.0%. Notable differences were seen in gender proportions between journal specialties (p = 0.001); with surgical oncology and hematology having the lowest female representation at 11.9% and 22.7%, respectively. Conclusions: Women and UIM are markedly underrepresented in leadership roles on Editorial Boards in hematology and oncology journals. Importantly, the representation of minority women physicians in EiC positions is at an inexorable zero which is a sign of unconscious attitudes that may exclude women and minorities from certain positions. It is imperative that we work to move towards a more diverse and inclusive editorial board to ensure critical perspectives are heard and scientific discovery is fostered.[Table: see text]

9548 Background: A randomized, double-blind, placebo-controlled phase IIb trial of the tumor lysate, particle loaded, dendritic cell (TLPLDC) vaccine was conducted to prevent recurrence in patients (pts) with resected stage III/IV melanoma. Two methods for dendritic cell (DC) harvest were used for vaccine production, including no pretreatment or pretreatment with granulocyte-colony stimulating factor (G-CSF) in an attempt to reduce blood draw volumes. This analysis investigates differences in clinical outcomes and RNA gene expression between these DC harvest methods for TLPLDC vaccine creation. Methods: The TLPLDC vaccine is created by loading autologous tumor lysate into yeast cell wall particles (YCWPs) and exposing them to phagocytosis by DCs. By investigator/pt choice, pts had 120mL of blood drawn for DC harvest, or pts received 300μg of G-CSF for pre-DC mobilization and a 50-70 mL blood draw 24-48 hours later. Total vaccine production time was 72 hrs. Pts were randomized 2:1 to receive TLPLDC or placebo (DCs exposed to empty YCWPs). 1-1.5 x10^6 cells/dose were injected intradermally at 0, 1, 2, 6, 12, and 18 months. Differences in disease free survival (DFS) and overall survival (OS) were evaluated by Kaplan Meier analysis between pts who did not receive pretreatment (TLPLDC), pts who did receive pretreatment with G-CSF (TLPLDC+G), and pts receiving placebo. RNA-seq analysis was performed on the total RNA of pts’ prepared TLPLDC vaccines to assess gene expression. Relative RNA expression (RRE) was compared between TLPLDC and TLPLDC+G. Results: As previously reported, 144 pts were randomized: 103 received TLPLDC (46 TLPLDC, 57 TLPLDC+G) and 41 received placebo. There were no significant clinicopathologic or treatment differences between the three treatment arms. Survival was significantly improved in TLPLDC compared to TLPLDC+G or placebo, including 36-month OS (92.9% vs 62.8% vs 72.3% respectively, p = 0.022) and DFS (51.8% vs 23.4% vs 27.1%, p = 0.027). When compared to TLPLDC+G (n = 3) vaccine, RNA-seq from TLPLDC vaccine (n = 3) showed upregulation of genes associated with DC maturation, including HLA-DMB (RRE: 3.60), IFIT1 (3.38), CD27 (3.26), IFI44L (3.24), MX1 (2.96), HLA-DQA1 (2.67), HLA-DRA (2.40), CD49D (2.34) and CD74 (2.09), while downregulated genes were associated with DC suppression or immaturity including SERPINA1 (RRE:7.8), TLR2 (6.65), CCR1 (5.11), IL10 (4.19), CD93 (3.84) and CD14 (3.25). Conclusions: Pts receiving TLPLDC vaccine had significantly improved OS and DFS, while outcomes remained similar between those who received TLPLDC+G vs placebo. Pts who did not receive G-CSF had higher expression of genes linked to DC maturation and antigen processing and presentation, likely explaining the improvement in clinical efficacy. A phase III trial to further assess the TLPLDC vaccine to prevent recurrence is planned. Clinical trial information: NCT02301611.

5040 Background: PARPi have shown promise in mCRPC pts with mutations in DNA repair, but ATM- and BRCA2-altered pts may respond differently to PARPi. We hypothesized that differences may also exist for taxane therapy, aiding in treatment sequencing decisions. Methods: mCRPC pts (N = 137) with deleterious ATM or BRCA2 mutations who received taxanes, PARPi, or both were identified from 8 US academic centers. Demographic, treatment, and survival data were collected. Kaplan-Meier analyses were performed for time-to-treatment-discontinuation (TTD), as well as overall survival (OS), from time of first taxane or PARPi therapy. Cox hazard ratio (HR) regression analyses were performed, adjusting for Gleason sum (≤7 vs. 8-10). For OS, receipt of subsequent therapies following first taxane or PARPi was also included as a covariate. Results: 50 ATM- and 87 BRCA2-mutated pts were identified. 40/50 (80%) of ATM-mutated pts received taxane only or taxane prior to PARPi, while 10/50 (20%) received PARPi only or PARPi prior to taxane. ATM-mutated pts showed a trend towards longer TTD when taxane was given first vs PARPi given first (P = 0.08, adjusted HR for taxane treatment 0.50 [95% CI: 0.24–1.08]). Considering all pts who received taxane first, ATM-mutated pts had longer TTD than BRCA2-mutated pts who received taxane first ( P= 0.04, adjusted HR for ATM 0.61 [CI: 0.37–0.99]). Among ATM-mutated pts, OS was longer in those receiving taxane first ( P= 0.06, adjusted HR for taxane treatment 0.33 [CI: 0.10–1.05]). Among BRCA2-mutated pts, 43/87 (49%) received taxane first and 44/87 (51%) received PARPi first. BRCA2-mutated pts had longer TTD when PARPi was given first vs taxane given first ( P< 0.0001, adjusted HR for PARPi treatment 0.32 [CI: 0.19–0.56]). Considering all pts who received PARPi first, BRCA2-mutated pts also had longer TTD than ATM-mutated pts who received PARPi first ( P= 0.0031, adjusted HR for BRCA2 0.29 [CI: 0.12–0.66]). There was no significant OS difference in BRCA2-mutated pts regarding which treatment was given first ( P= 0.63, adjusted HR for PARPi treatment 1.18 [CI: 0.59–2.35]). Conclusions: Our data in ATM- and BRCA2-mutated mCRPC pts suggests a trend towards improved clinical outcomes when taxanes are used prior to PARPi in ATM-mutated pts, while the reverse sequence appears to be better for BRCA2-mutated pts.[Table: see text]