During 2018, CNUCOP had a total of 22 presentations. Eighteen presentations were from faculty within the Pharmaceutical and Biomedical Sciences Department. There were also four presentations from the Department of Clinical and Administrative Sciences.
Cusick JK, Khansari P, Fitzpatrick L. Use of engaging review application exercises to enhance understanding of critical concepts prior to summative examinations. Presented at the Team Based Learning Collaborative (TBLC) annual meeting, San Diego CA, March 2018.
BACKGROUND: Review application exercises were created in an Immunology class for third-year pharmacy students that utilized Team-Based Learning (TBL) as its pedagogy. The goal was to enhance student understanding of critical concepts prior to summative examinations.
DESCRIPTION: Three distinct exercises were utilized prior to summative examinations. For applications #1 and #2, important topics were divided amongst the teams, to ensure that all topics relevant to an upcoming exam were covered by posters that would be displayed for students to view. Application #1 was conducted over several class periods while applications #2 and #3 were conducted in a single class period. Application Exercise #1: Teams created engaging and informative posters describing assigned subject matter utilizing superheroes or other caricatures. A gallery walk was conducted the day the final posters were due, in which teams of students reviewed key concepts on posters created by fellow students, and graded the posters according to rubrics that emphasized the posters should be engaging, accurate, informative, and most importantly, were effective as a teaching tool. Application Exercise #2: Teams created mnemonics, songs or haikus to highlight assigned material and displayed their creations on a poster. Approximately half of the teams performed their song creation to the rest of the class. Application Exercise #3: A Jeopardy® review game utilizing team clickers and a team leaderboard created a fun and friendly competition in which student teams collectively discussed and answered practice exam questions.
CONCLUSION: The review application exercises were well received by students as indicated by the course evaluations, with many students indicating that the review exercises greatly helped them solidify their knowledge in a way that was fun and engaging. Additionally, the posters served as excellent recruiting tools during the interview process, as the student creations generated positive feedback from visiting applicant candidates not familiar with TBL.
Vinall R and Kreys E. Determination of whether implementation of mini-application exercises can enhance individual student performance in a TBL setting. Presented at the Team Based Learning Collaborative (TBLC) annual meeting, San Diego CA, March 2018.
Introduction: A common concern of TBL is a tendency of some students to depend on team members to answer application exercise questions resulting in inadequate preparation for higher Bloom’s taxonomy questions on exams. The objective of this study was to determine whether implementation of individual applications (iBATs) improves student performance.
Methods: Ninety-eight first year pharmacy students taking the Cell and Molecular Biology & Biochemistry course participated in an unblinded, randomized, controlled, cross-over study. The course consisted of three blocks: block 1 served as a control, iBATs were administered for the two randomized groups in a cross-over manner for only blocks 2 and 3. An independent t-test was used to compare average block exam scores between groups. A paired t-test was used to compare average group z-scores between block 1 exam results and results of the block coinciding with iBAT implementation. A subgroup analysis based on the Bloom’s taxonomy of exam questions was performed. A questionnaire assessed students’ impression of iBAT impact after each block exam.
Results: Exam scores for block 1, 2, or 3 were not significantly different between randomized groups (p-value = 0.35, 0.11, and 0.82, respectively). Subgroup analysis revealed similar results. Implementation of iBATs resulted in an increase of 0.091 z-scores, but was not statistically significant (p= 0.35). Limited to critical thinking questions, an increase of 0.102 z-scores was observed (p= 0.36). Majority of students reported that iBATs increased their ability to identify knowledge gaps (88%), determine their proficiency of critical thinking questions (91%), and increased studying following poor iBAT performance (83%), while few felt that iBATs increased stress (30%), or reduced enjoyment of the course (46%).
Conclusions: Implementation of iBATs did not significantly improve student performance on examinations or specifically on critical thinking exam questions. Generally students felt that iBATs had a positive impact their learning.
Westrick T and Malhotra A. Integrating an Audiology-focused Interprofessional Teaching Activity Into a Pharmacy Curriculum. Presented at American Academy of Audiology meeting, Nashville TN, April 2018.
This classroom teaching poster outlines an interprofessional classroom activity involving audiology and pharmacy graduate students. Audiology students presented on the relationship between loop diuretics and ototoxicity, and participating pharmacy students were surveyed regarding their perceptions of this activity. This poster outlines components of the classroom activity, perceptions of students participating in the activity, and illustrates how this activity can help academic programs close curriculum gaps and fulfill requirements established by academic guidelines and accreditation bodies. Rationale/Purpose: Interprofessional collaboration between students in various healthcare fields has become increasingly prevalent in academic institutions in recent years. In many instances, collaboration between students focuses on analyzing case studies involving multiple disciplines or exploring various roles played by members of the interdisciplinary healthcare team. While these interprofessional activities provide students with an understanding of other allied healthcare professions, there are opportunities for audiology students to interact with students in professions in a teaching capacity.The purpose of this poster is to outline and evaluate the efficacy of an interprofessional classroom teaching activity facilitated by Au. D students in the School of Pharmacy at Pacific University. For the past five years, Au. D. students presented a lecture and coordinated an interactive learning activity as part of a renal-focused course in the Pharm. D. program. The lecture focuses on ototoxicity of loop diuretics, but aspects of cochlear physiology, causes of hearing loss, and an overview of audiology scope of practice are also incorporated. Following the activity, pharmacy students were surveyed and asked to evaluate various components of this classroom activity and to provide feedback regarding the efficacy of this activity in providing a better understanding of the auditory system and ototoxicity of diuretics. A pre and post-activity quiz were also administered to measure learning outcomes. This poster outlines components of the classroom activity, perceptions of students participating in the activity, and illustrates how this activity can help academic programs fulfill curriculum requirements established by oversight guidelines and accreditation bodies. Methods: This poster focuses on the structure of an interprofessional teaching activity and perceptions of participating students. A group of Au. D. students prepared a lecture and an interactive activity focused on loop diuretics and ototoxicity. Components of the lecture and design of the interactive activity component will be outlined. Following the completion of the activity, a voluntary survey was made available to pharmacy students who participated in the activity. The survey was designed to capture details regarding student perceptions and learning outcomes achieved by students participating in this activity. Results and Conclusions: At present time, survey data is still being collected and analyzed. Once data analysis is complete, feedback provided by students will be used not only to serve as a potential measure of the efficacy of this unique interprofessional activity, but will also be used to further develop this and other interprofessional activities facilitated by students at Pacific University and beyond. Data could serve as a means to measure peer-to-peer teaching efficacy, which could in turn be incorporated into not only other areas of audiology curricula, but also interprofessional educational activities outside of audiology. Audiology graduate students teaching pharmacy graduate students about the ototoxicity of loop diuretics is a novel approach in interprofessional education. Not only do the audiology students involved gain experience designing and giving a professional presentation to non-audiology students, but pharmacy students gain a better understanding of the profession of audiology. In turn, both groups of students achieve curriculum requirements set forth by accreditation bodies.
Batra N, Krig S, Sam A, Macias K, Vinall RL et al. Differential expression and localization of the E3 ubiquitin ligase Nrdp1 in African American versus Caucasian American prostate cancer cells. Presented at the American Association for Cancer Research (AACR) Annual Meeting, Chicago IL, April 2018.
Despite a recent decrease in overall incidence of prostate cancer (CaP), CaP incidence continues to be much higher among men of African origin living in the United States (223.9 men per 100,000) compared to those of European origin (139.9 men per 100,000). Also, the CaP age-specific mortality rates are 2.4 times greater among African-American (AA) compared with European American (EA) men. The causes for these differences are multifactorial, but include genetic effects that contribute to CaP-associated health disparity. We previously showed that the androgen receptor (AR) can suppress levels of the receptor tyrosine kinase ErbB3, a molecule that is known to drive CaP progression, by stimulating the E3 ubiquitin ligase Nrdp1 (also called RNF41 or FLRF). The goals of the current study were to determine whether differential expression and/or localization of Nrdp1
contribute to CaP-associated health disparities, and to further elucidate the mechanisms by which regulation of Nrdp1 levels and localization occurs. Immunohistochemistry (IHC) in prostate tissue determined that nuclear Nrdp1 levels are significantly lower in AA CaP patients (n=19) versus CA CaP patients (n=121) with localized disease (p=0.008). A similar association was observed in CaP cell lines; immunofluorescence (IF) analyses demonstrated MDAPCa2b and E006 (cell lines derived from AA CaP patients) express significantly lower levels of nuclear Nrdp1 compared to LNCaP, CWR22Rv1, C4-2, and C4-2B (cell lines derived from CA CaP patients). Western blot and qPCR determined that Nrdp1 levels are lower in AA versus CA CaP cells (MDAPCa2b express ~2-fold less Nrdp1 protein and mRNA levels compared to LNCaP), and that androgen withdrawal has a bigger impact on Nrdp1 expression levels in AA CaP cells (2-fold decrease in MDAPCa2b compared to ~1.2-fold decrease in LNCaP). Proteasomal regulation of Nrdp1 also occurs; treatment with MG132 resulted in accumulation of Nrdp1 in both AA and CA CaP cell lines. Interestingly, altering androgen and/or AR levels also affected Nrdp1 localization (IF analyses) as well as the ability of Nrdp1 to mediate CaP cell apoptosis (flow cytometry). In summary, we demonstrate that Nrdp1 is differentially expressed in AA versus CA CaP patients as well as in AA versus CA CaP-patient derived cell lines, and that androgen withdrawal has a bigger impact on Nrdp1 expression in AA CaP cell lines. We also demonstrate that androgen/AR is involved in determining Nrdp1 localization, and that Nrdp1 can mediate CaP cell apoptosis in the presence of AR. The combined data, and the knowledge that Nrdp1 regulates ErbB3 levels, suggest that differential expression of Nrdp1 in AA versus CA CaP may contribute to CaP-associated health disparities and may occur as a result of dysregulation of the regulation of Nrdp1 by androgen/AR.
Podium and Poster
True H, Trinh J, Love Q, Badejo A, Rao D, Malhotra A. Raloxifene Compromises Mitochondria, Induces ROS Stress and the Unfolded Protein Response, and Synergizes Gemcitabine's Cytotoxicity in Pancreatic Cancer Cells. Accepted for Presentation at Annual ASPET Meeting, San Diego CA, April 2018.
Abstract: Pancreatic adenocarcinoma (PCa) is a deadly disease associated with high mortality and a very poor 5-year survival rate of only 6-8%. There is a lack of knowledge about how mutations in the human homolog of the Kirsten Rat Sarcoma viral oncogene (KRAS), which occur in 90% of the patient population, lead to its development. This knowledge gap, coupled with substantial chemoresistance, has led to a scarcity in effective therapy to combat PCa. To reduce the bench-to-bedside time for the development of drugs for use in PCa, we repurposed FDA-approved drugs, screening for their cytotoxic potential in KRAS-mutant human PCa cell cultures. Our data show that the FDA-approved raloxifene, a selective estrogen receptor modulator used in the treatment of osteoporosis caused apoptosis of multiple PCa cell lines (MIA PaCa-2, PANC-1, BxPC-3). IC50 for raloxifene was 1.81 + 0.38 μM (MIA PaCa-2), 58.16 + 5.37 μM (PANC-1), and 4.20 + 2.16 μM (BxPC-3). This compared well with the IC50 for gemcitabine, the gold-standard drug used for treating PCa since 1997, values for which were 4.75 + 1.67 μM (MIA PaCa-2), 58.60 + 14.78 μM (PANC-1), and 6.10 + 1.70 μM (BxPC-3) because in each PCa cell line, raloxifene had a lower IC50 than gemcitabine. Further, combination index (CI) analysis using a 1:1 raloxifene-gemcitabine combination showed evidence for synergism with CI values <1 for a broad range of dose concentrations within the three cell lines, and specifically at 50% of cells affected. To investigate the mechanism of cytotoxicity, we examined mitochondrial function and found that in MIA PaCa-2 cells, raloxifene treatment induces reactive oxidative species (ROS) production and decreases mitochondrial membrane potential. Pre-treatment of MIA PaCa-2 cells with the NADPH oxidase inhibitor diphenylene iodonium (DPI) reduced ROS production for each raloxifene concentration used, suggesting that the mitochondria were the source of the ROS. To further investigate the mechanism, we examined the effect of raloxifene treatment on the enzymes involved in the ROS pathway. Our data show that while raloxifene induced the expression of glutathione synthetase, glutathione s-transferase, it had no effect on superoxide dismutase 1 or 2 or glutathione peroxiredoxin. However, catalase expression was decreased in MIA PaCa-2 cells, suggesting that raloxifene induced oxidative stress in these cells, making them susceptible and responsive to further apoptotic stimuli like treatment with gemcitabine. Finally, our data also show that raloxifene induces the unfolded protein response and endoplasmic reticulum stress in PCa cells as evident by an increase in endoplasmic reticulum oxidoreductase 1 (ERO1- α) and cleavage of Activating transcription factor 6 (ATF6), confirming that it helps overcome the chemoresistance of PCa cells.
Lenhard JR, Quach CD, Nguyen TQ, Doan LH, Chau J. Bacterial Brothers in Arms: Cooperation of Staphylococcus aureus and Pseudomonas aeruginosa during Antimicrobial Exposure. Presented at European Congress of Clinical Microbiology and Infectious Diseases, Madrid Spain, April 2018.
Background: Chronic wound infections are often polymicrobial and may contain pathogens in the small colony variant phenotype (SCVP); however, the pharmacodynamics of commonly used antibacterials are poorly defined in the presence of multiple pathogens. We sought to quantify the in vitro killing of antimicrobials against Pseudomonas aeruginosa co-cultured with either the normal phenotype (NP) or the SCVP of Staphylococcus aureus. Materials/methods: The wild-type P. aeruginosa strain PAO1 was evaluated with either NP MRSA (COL) or its isogenic SCVP (Ia48) in 24-hour static time-killing studies at a 106 CFU/mL inoculum. Using an adapted in vitro wound model with selective plating, concentration arrays consisting of 1/64th, 1/16th, 1/4th, 1x, 4x, and 16x a clinically relevant concentration of gentamicin (6 mg/L), levofloxacin (6 mg/L), vancomycin (20 mg/L), and clindamycin (10 mg/L) were investigated. Pharmacodynamics were quantified using a Hill-type mathematical model that described the areas under the colony-forming unit curves (AUCFUs).Results: In the Hill-type analysis, gentamicin and levofloxacin achieved similar killing against P. aeruginosa in monoculture (Emax gentamicin and levofloxacin: 4.52 and 4.91, R2 > 0.92) and in co-culture with NP MRSA (Emax 4.71 and 4.92, R2 > 0.96) or SCVP MRSA (Emax 4.85 and 4.73, R2 > 0.99); however, 24 mg/L of gentamicin eradicated P. aeruginosa in monoculture by 6 hours, whereas the same concentration was unable to achieve a >3 log10CFU/mL reduction by 24 hours in co-culture experiments. The antimicrobial killing of SCVP MRSA was relatively unaffected by P. aeruginosa. In contrast, clindamycin concentrations ≥2.5 mg/L achieved sustained killing of NP MRSA in monoculture with a maximum 3.5 log10CFU/mL reduction at 24 hours, whereas none of the clindamycin concentrations were able to achieve a 0.5 log10CFU/mL reduction at 24 hours during co-culture with P. aeruginosa. The Hill-type analysis confirmed a reduction in clindamycin killing of NP MRSA in co-culture (Figure 1, Emax 2.00, R2 > 0.90) versus monoculture (Emax 3.37, R2 > 0.99). Conclusions: During the co-culture of S. aureus and P. aeruginosa, the anti-staphylococcal activity of clindamycin is severely attenuated by the presence of P. aeruginosa. These results have implications for antibacterial selection during polymicrobial chronic wound infections.
Smith NM, Lakota EA, Lenhard JR, Bulitta JB, Mergenhagen KA et al. Translating Polymyxin B (PolyB) and Meropenem Synergy against Carbapenem-Resistant Acinetobacter baumannii (CRAB): Moving toward a Rational, Combinatorial Pharmacokinetic Target. Presented at European Congress of Clinical Microbiology and Infectious Diseases, Madrid Spain, April 2018.
Background: CRAB infections represent a major therapeutic problem. Clinicians are tasked with developing treatment strategies based on the use of multiple, individually resistant antibiotics, but without guidance on their optimal use together.
Materials/Methods: A 14 day hollow fiber infection model (HFIM) was used to study dose-escalated PolyB (fCss,avg [fAUCss]= 0.75 mg/L [17.9 mg*h/L], 1.50 [35.7], 2.50 [59.6], 5.00 , and 10.0 ) in combination with meropenem (4g Q8H, 3h PI) versus A. baumannii N16870 (MICmeropenem=16 mg/L, MICPolyB=0.5 mg/L). Pharmacodynamic endpoints were summarized using a mechanism-based mathematical model and the Log Ratio Area (LRA336h). Pharmacokinetic endpoints were summarized as the ratio of dose-normalized exposure between PolyB and meropenem at 24 h. The probability of achieving both pharmacokinetic (optimized joint PolyB and meropenem exposures) and pharmacodynamic (maximized bacterial killing) endpoints was evaluated using 16,000 simulated patients across 16 clinical trial simulations (CTS). Each trial utilized a unique combination of PolyB + meropenem at 50%, 100%, 200%, or 400% of the approved label dosing (ALD).
Results: Against a high bacterial density of CRAB, there was significant bacterial killing at a PolyB fCss,avg of ≥2.5 mg/L [59.6 mg*h/L] + meropenem. A PolyB fCss,avg of 0.75, 1.50, 2.50, 5.00, and 10.0 mg/L + meropenem resulted in a reduction of the LRA336h of 0.26, 0.78, 4.3, 4.3, and 5.3, respectively. PolyB fCss,avg of 0.75 mg/L [17.9] or 1.5mg/L [35.7] + meropenem resulted in rapid killing of >99.9% within 24h but with eventual regrowth, whereas a PolyB fCss,avg of 2.5 mg/L [59.6] + meropenem produced nearly undetectable counts of 102.5 cfu/mL for >10d. CTS showed that ALD of PolyB (1.25mg/kg q12h) achieves a 4.3 log reduction in the LRA336 for 59.5% of subjects (assuming 100% meropenem target attainment), which increased to 98% for PolyB 2.5 mg/kg. Furthermore, 0% of subjects receiving the ALD of meropenem (1g Q8H 30 min infusion) met their pharmacokinetic marker of success (assuming 100% PolyB target attainment).
Conclusions: Treating highly resistant bacterial species often necessitates the un-optimized use of carbapenems or polymyxins. Results from the HFIM and CTS can provide a joint pharmacokinetic target that can guide combinatorial use of meropenem and PolyB.
Talbott G, Le H, Lor P, Jin Z. Reversal of myofibroblast differentiation by phorbol 12-myristate 13-acetate is PKC-independent. Presented at Annual Experimental Biology meeting, San Diego CA, April 2018.
Fibrosis often results in organ dysfunction and failure in diseases, such as: chronic heart failure, hepatic cirrhosis, pulmonary fibrosis, and end-stage renal disease. The differentiation of fibroblasts into myofibroblasts results in the secretion of collagens and other extracellular matrix proteins that limit organ function and underlie the fundamental basis of fibrosis. TGF-β1 induces the differentiation of α-SMA expressing myofibroblasts from fibroblasts. However, the mechanism to induce reversal of myofibroblast differentiation remains elusive. Phorbol 12-myristate 13-acetate (PMA) has been used for the stimulation of lymphocytes and splenocytes. It is also involved in multiple cellular functions by potently activating protein kinase C (PKC). Nevertheless, the effect of PMA on the formation of myofibroblasts is unknown. To investigate whether PMA plays a role in myofibroblast differentiation, NIH 3T3 fibroblasts were cultured in DMEM medium for 48 hours in the presence of DMSO (vehicle control), PMA, or PKC inhibitors (calphostin C, chelerythrine, and staurosporine) alone or plus PMA. The expression of α-smooth muscle actin (SMA, a hallmark of the myofibroblastic phenotype), fibroblast-specific protein 1 (FSP1, a biomarker of fibroblasts), and TGF-β1 in cultured fibroblasts was detected by western blotting and immunofluorescence. Expression of α-MARCK phosphorylation was also detected to examine the activity of PKC activation. Positive staining of α-SMA was observed in some cells in control culture conditions, and these α-SMA positive cells exhibited significant morphology changes with large nuclei and cytoplasm, compared with α-SMA negative fibroblasts. Treatment with PMA 50 ng/mL for 48 hours reduced the expression of α-SMA and TGF-β1, and pretreatment with the three PKC inhibitors mentioned above could not abrogate PMA-induced reduction of α-SMA and TGF-β1. These results indicate that some myofibroblasts are derived from fibroblasts under basic culture conditions. PMA induces reversal of myofibroblast differentiation via a PKC-independent mechanism.
Mohamed I, Thomas S, Searles C. miR-155: a negative modulator of Acute Oscillatory Shear Stress (OSS)-induced Vascular Inflammation and Dysfunction. Accepted for presentation at ATVB/PVD Scientific Meeting, San Francisco CA, May 2018.
Introduction: Shear-sensitive micro-RNAs play an integral role in dictating vascular wall pro-inflammatory response and development of atherosclerosis. Previously, our group and others have identified an inverse relationship between micro-RNA-155 (miR-155) expression and inflammation in atheroprone areas of chronic low magnitude oscillatory shear stress (OSS) in vasculature and in-vitro.
Hypothesis: we hypothesized that miR-155 negatively regulates acute OSS-induced vascular inflammation and dysfunction, via modulation of the MAPK-ETS-1 pathway.
Methods: 12-week old C57B/6J wild type (WT) and miR-155 knockout mice (KO) were subjected to abdominal aortic coarctation (AAC), a unique model of acute induction of OSS, for 3-7 days. Downstream acute OSS segments were compared to upstream unidirectional shear stress (USS) segments of thoracic aorta using RT-PCR, western blot and two-way ANOVA followed by Tukey’s multiple comparison analyses.
Results: In WT mice, acute OSS induced vascular inflammation evidenced by upregulation of MCP-1 and VCAM-1 expression in OSS segments compared with USS. This was associated with loss of vascular barrier function as evaluated by extravasation of Evans-blue dye assay along with increased MMP-9 and MMP-3 expression. However, vascular miR-155 levels were also higher in OSS segments compared with USS (n=6-12, P<0.05). Nevertheless, miR-155 KO mice showed enhanced expression and activation of ERK and p-38 MAPKs and downstream ETS-1, VCAM-1 and MMP-9 expression in OSS segments compared with USS versus WT controls (n=3-4, P<0.05). Tail vein injections of miR-155 overexpressing lentivirus particles in WT mice after AAC resulted in further upregulation of miR-155 and abolished OSS-induced upregulation of p-38 and downstream ETS-1, VCAM-1 and MMP-9 expression in OSS segments compared with USS versus scramble controls (n=5-6, P<0.05).
Conclusions: Despite the early upregulation of shear-sensitive miR-155, our data suggest that miR-155 serves as a negative feedback regulator to acute OSS-induced vascular inflammation via inhibition of p-38 and ETS-1. Further studies are in progress to evaluate the effect of exogenous miR-155 on OSS-induced oxidative stress and vascular function, which can serve as basis for developing novel miRNA-based therapeutic modalities.
Fitzpatrick LR, Talbott G et al. al. Ex vivo effects of ROR-gamma T inhibitors on pro-inflammatory cytokine secretion from colonic strips of mice with DSS-induced colitis. Accepted for presentation at Digestive Disease Week Annual Meeting, Washington DC, June 2018.
Abstract: ROR gamma t (RORγt) inhibitors (e.g., GSK 805, VPR 254) have shown efficacy in murine models of colitis. To determine an optimal compound(s) for further in vivo testing, we utilized an ex vivo colonic strip model from mice with DSS-induced colitis. This colitis has been proposed by some investigators to involve IL-17, IL-21 and CCL20, which are under transcriptional control by RORγt. Therefore, we hypothesized that secretion of these cytokines by colonic strips would be attenuated by RORγt inhibitors. Methods: Five RORγt inhibitors were obtained from Visionary Pharmaceuticals (San Diego, CA). Dextran Sulfate Sodium (DSS) was given to male C57BL/6 mice (n = 16) in the drinking water (2% concentration), for a six-day period, to induce colitis. Control mice (n = 8) received water. The ex vivo effects of the RORγt inhibitors (0.05 to 5 μM concentration range) were tested in our 24 hour colonic culture system [Fitzpatrick et al., Inflammopharmacology, 2014]. We utilized ≈ 4 mm colonic strips (n = 4 to 10 per RORγt inhibitor group) from mice with DSS-induced colitis. Some colonic strips were stimulated with IL-1β (10 ng/ml) plus IL-23 (10 ng/ml) to induce enhanced cytokine secretion. The secretion of IL-17, IL-21 and CCL20 were determined in the cell culture media by ELISA. Results: Mice that were administered DSS showed clear evidence of colitis (enhanced disease activity indices and reduced colon lengths). Ex vivo treatment with VPR 254, VPR 425 and GSK 805 were most effective for inhibiting basal IL-17 secretion from colonic strips of mice with colitis. VPR 425 and GSK 805 most effectively attenuated basal (non-stimulated) secretion of IL-21 and CCL20. For dual cytokine stimulated IL-17 secretion, some IL-17 values (pg/ml) were: 112±35 (Vehicle), 87 ± 21 (VPR 254, 5μM), 64 ± 9 (VPR 425, 5 μM) and 45 ± 9 (GSK 805, 5 μM). The calculated IC50 value of VPR 425 for inhibiting stimulated IL-17 secretion was 0.13 μM, while for GSK 805 it is < 0.05 μM. Three compounds (VPR 254, VPR 425 and GSK 805) significantly attenuated (at certain concentrations) dual cytokine stimulated IL-21 secretion from colonic strips of mice with DSS-induced colitis. Overall, the most effective compounds were VPR 425 and GSK 805. Four compounds (VPR 426, VPR 254, VPR 425 and GSK 805) attenuated (to some degree) stimulated CCL20 chemokine secretion from colonic strips of mice with DSS-induced colitis. Chemokine secretion was reduced to the basal level by ex vivo treatment with VPR 425 (0.5 and 5 μM concentrations). Summary: Ex vivo treatment with structurally diverse RORγt inhibitors inhibited basal and stimulated pro-inflammatory cytokine secretion from colonic strips of mice with DSS-induced colitis. Conclusion: These data further contribute to the identification of optimal RORγt inhibitors, which can be used for follow-up in vivo testing in murine models of colitis.
Wang H, Ricklin D, Lambris JD. Complement peptide C4a mitigates LPS-induced endothelium disruption, cytokine production, ERK activation, and [Ca2+] influx from human moncytes ---a new therapeutic intervention for endotoxin shock? Accepted for presentation at International Conference & B2B on Pharma Research and Development, Philadelphia, USA, June 2018.
Activation of the complement cascade is a major effector of the inflammatory response. Cleavage of complement components in the course of activation produces low-molecular-weight peptides, including C3a, C4a, and C5a. Both C3a and C5a have potent anaphylatoxin properties. Our recent study idnetified that C4a mediates effector functions through binding to protease-activated receptors (PAR) 1 and PAR4. Early study demonstrated that animals with complement C4 deficiency were reported to be more susceptable to endotoxic shock, suggesting that C4 protects animals from endotoxic effects. However, the molecular mechanism for C4 protective effect on endotoxin shock in animals is poorly understood. We propose that C4 activation peptide, C4a, possiblly through binding to PAR1/4 on platelets, monocytes, and endothelial cells, inhibits LPS-induced platelet aggregation, IL-1β and TNFα cytokine production from monocytes, and endothelium permeability to achieve C4 protective effects in endotoxin shock animal models. In the present study, we found that pretreatment with C4a to human primary monocytes can significantly inhibit LPS-induced IL-1β and TNFα production. Moreover, LPS-induced ERK phosphorylation and [Ca2+] influx were significantly inhibited by the pretreatment of C4a. Our experiments also revealed that C4a significantly decreased endothelium permeability when human endothelial cells were cultured in the presence of LPS, indicating under endotoxemia condition, C4a prevents endothelium disruption. Our data provide deeper insight into the mechanism of C4’s protective effect on endotoxic shock and would provide a valuable resource for the wider scientific community to generate future therapeutic interventions for the treatment of clinical endotoxemia.
Basson MC, Wang Q, Chaturvedi LS, Vomhof-DeKrey E. Schlafen 12 Promotes Human Intestinal Epithelial Differentiation via Serpin B12 Modulation of the Deubiquitylation of Transcription Factors Such as CDX2. Accepted for presentation at Digestive Disease Week Annual Meeting, Washington DC, June 2018.
Human enterocytic differentiation is altered during development, fasting, adaptation, and bariatric surgery, but its intracellular control remains unclear. We have previously reported that Schlafen 12 (SLFN12), which decreases in human mucosa after prolonged fasting, regulates Caco-2 sucrase-isomaltase expression in a manner dependent on its binding to Serpin B12 (SERPB12). SLFN12 is chiefly a cytosolic protein, and the rodent Slfn3 still acts even when bound to a nuclear exclusion sequence. We now sought to determine how this cytosolic protein that has no homology to any known signaling protein can influence gene transcription in the nucleus. Both SLFN12 and SERPB12 overexpression increased sucrase-isomaltase promoter activity, mRNA, and protein, while reducing SERPB12 expression prevented the SLFN12 effect. Sucrase-isomaltase induction by both SLFN12 and SERPB12 was attenuated by reducing either of the deubuitylases UCHL5 or USP14, and completely blocked by reducing both. SERPB12 coprecipitated USP14 but not UCHL5 and purified SERPB12 protein stimulated the activity of purified USP14 but not of UCHL5. SLFN12 and SERPB12 increased protein levels of the sucrase-isomaltase-promoter-binding transcription factor Cdx2 without altering Cdx2 mRNA. This was prevented by reducing UCHL5 and USP14. Moreover, reducing Cdx2 with siRNA prevented the induction of sucrase-isomaltase promoter activity by SLFN12. We next validated this SLFN-SERPB12-USP14-Cdx2-sucrase pathway beyond Caco-2 cells. Both SLFN12 and SERPB12 overexpression also stimulated sucrase-isomaltase expression in nonmalignant HIEC6 cells, and siRNA reduction of SERPB12 reduced mucosal ileal sucrase-isomaltase expression in mice, along with expression of two other enterocytic differentiation markers, villin-1 and Glut1. These results suggest that SLFN12 regulates human enterocytic differentiation by a pathway involving SERPB12 and the deubiquitylases, modulating deubiquitylation and cytosolic proteasomal degradation of transcription factors like Cdx2 to regulate gene transcription. This pathway may be targeted to manipulate human enterocytic differentiation in mucosal atrophy, short gut, or obesity.
Title: Student Perceptions of the PCOA- A Multi-institutional Sample. Accepted for presentation at the Annual AACP Meeting, Boston MA, July 2018.
Authors: Daugherty KK, Welch A, Gortney J, Castleberry A, Le U et al.
Background: Questions regarding use of the PCOA to identify at risk students and serve as a high stakes exam have been debated in the literature, but no data exists regarding students’ perceptions of the exam.The purpose of our study was to assess students’ perceptions of the PCOA exam.
Method: A 21-question, electronic survey was developed and beta-tested by faculty representing geographically diverse S/COPs. APPE students from 5 S/COPs completed this survey regarding their PCOA exam study habits, feedback, and results use.
Results: 131 students complete the study with the majority being female. A majority of students indicated that they did not study for the PCOA (65%) or use results of PCOA to prepare for APPE (93%) or NAPLEX (81%) nor did they review results with an advisor (78%). Most students were neutral or disagreed with the statement that doing well on this test was important to them (65%) but most agreed they gave good effort on the test (61%). Students agreed that the following incentives would influence their motivation on the PCOA: if it affected progression (70%), APPE placement (65%), or required remediation (69%).
Conclusion: This study revealed that a majority of students do not study for the PCOA exam and do not discuss their results with faculty advisors in order to improve their area(s) of weakness for APPEs. It is unclear if student perceptions are the result of a lack of understanding of the PCOA’s goals, student time, exam result interpretation and mentoring, or tangible incentives for strong performance.
Chaturvedi LS, Vyas, D. Second generation proteasome inhibitor Carfilzomib (CFZ) Inhibits proliferation and induces apoptosis in human triple-negative breast MDA-MB-231 cancer cells.Accepted for Presentation at ACS-2018 Meeting, Boston MA, August 2018.
Carfilzomib (CFZ) is a selective irreversible second generation proteasome inhibitor is used for the treatment of relapsed and refractory multiple myeloma as anticancer therapy. However, it role and molecular mechanism has not been elucidated in human triple negative breast cancer. Herein, we investigated its antiproliferative and apoptotic effects alone or in combination with Doxorubicin (DOX) on human triple negative (TNBC) MDA-MB-231 breast cancer cells. In these cells, CFZ inhibited proliferation, induced cell cycle alterations and increased apoptosis in a dose and time dependent manner. Its action was compared to Doxorubicin (DOX), a first line chemotherapeutic agent used for TNBC and other cancers. DOX also inhibited proliferation, induced cell cycle alterations and increased apoptosis but was less potent than CFZ. A combination of both CFZ and DOX did not further reduce growth but significantly enhanced apoptosis in comparison to CFZ or DOX alone. The immunoblot analysis of cell cycle protein expression revealed that CFZ increased cyclin-B1 but reduced cyclin-D1 significantly without affecting cyclin-A and cyclin-E1 expression levels. On the other hand, DOX robustly increased cyclin-E1, modestly cyclin-A without affecting cyclin-B1 and cyclin-D1 expression levels. A combination of both CFZ and DOX significantly enhanced expression of cyclin B1 and cyclin-E1 while reducing expression of cyclin-D1. Low doses of either CFZ or DOX alone enhanced the phosphorylation of the DNA double strand break marker gamma-H2AX while higher dose of either drug proved to be less effective on the phosphorylation of gamma-H2AX. Thus our data suggests that irreversible proteasome inhibitor CFZ induces apoptosis, modulates cell cycle proteins, and induces phosphorylation of gamma-H2AX in human triple-negative breast MDA-MB-231 cancer cells. These findings suggest a potential use of CFZ against tumors harboring drug-resistant phenotypes.
Mohamed IN, El-Remessy AB. TXNIP is required for HFD-induced retinal leukostasis, endothelial inflammation and microvascular lesions through autocrine activation of NLRP3 inflammasome. Presented at The XXIII Biennial Meeting of the International Society for Eye Research (ISER). Belfast, Northern Ireland, September 2018.
Our group has shown that high fat diet (HFD) uniquely triggers expression of retinal thioredoxin interacting protein (TXNIP) and that TXNIP is required for activation of NOD-like receptor protein (NLRP3)-inflammasome. Retinas from wild type mice fed with 60%-HFD for 8-18 weeks (WTHFD) showed increased number of adhered leukocytes, barrier dysfunction and degenerated acellular capillaries formation compared to WT mice on normal diet (WT-ND), but not in TXNIP knockout (TKO) mice. Here, we dissect the specific contribution of endothelial- versus leukocyte-TXNIP to HFD-induced leukostasis and NLRP3-inflammasome activation. After 8-weeks of HFD, peripheral blood mononuclear cells (PBMCs) isolated from WT-HFD showed increased adhesion to mouse endothelial cells (EC) by 2-fold, compared with PBMCs from WTND control group. Whereas, PBMCs isolated from TKO-ND or TKO-HFD showed no significant changes compared with WT-ND group. In parallel, isolated PBMCs from obese nondiabetic human subjects also showed increased leukocyte adhesion to cultured human retinal ECs by 1.6-fold. Next, TXNIP overexpression (TXNIP++) in human retinal ECs triggered activation of the NLRP3-inflammasome evidenced by the upregulation of NLRP3 and cleaved-IL-1β expression by 2.5- and 2.4-fold, respectively, and a trend of increased cleaved caspase-1 expression compared with cells transduced with empty vector (EV)-controls. These effects coincided with significant increases in TNF-α, ICAM-1 and PECAM-1 expression by 1.85, 1.5 and 1.65-fold, respectively in the TXNIP++ group compared with EV-controls. Treatment with IL-1β receptor antagonist suppressed the effect of TXNIP overexpression on inflammasome activation and EC inflammation. Finally, after 18-weeks of HFD, retinal vascular branching density was decreased by 0.7-fold in WT-HFD group compared with WT-ND control group. In contrast, TKO mice had comparable vessel branching density to WT-ND controls. Together, our findings highlight the essential role of TXNIP expression on both leukocytes and ECs in activation of NLRP3-inflammasome, leukostasis and inflammation which involves an IL-1β receptor-autocrine fashion. This work establishes the early pre-diabetic impact of HFD-induced obesity on retinal inflammation and microvascular morphological changes independently from frank diabetes. Targeting TXNIP can provide attractive therapeutic modalities against HFD-induced NLRP3-inflammasome activation in both leukocytes and ECs.
ElShamy A, Eng F, Doyle EH et al. Proteomic Analysis By Mass Spectroscopy of Cryoprecipitates from Patients with HCV Associated Cryoglobulinemic Vasculitis Undergoing Direct Acting Antiviral Treatment Compared to Patients with Persistent Cryoglobulinemia after Sustained Viral Response Accepted for Presentation at AASLD Meeting, San Francisco, CA, November 2018.
Background and Aim: Mixed cryoglobulinemia (MC) is strongly associated with HCV infection, but also occurs in autoimmune diseases. HCV cure decreases cryoprecipitates (CPs). We aimed to determine the impact of HCV cure on the composition of CPs and on cryoglobulinemic vasculitis (CryoVas) in patients treated with antiviral drugs.
Methods: CPs were isolated from sera of a) 10 HCV patients enrolled in a prospective trial (NTC0282512) of antiviral treatment (Harvoni/Epclusa) with 24-wk follow-up of CPs and extrahepatic symptoms; b) 5 patients with persistent MC and Cry/Vas 2-15 yr after HCV cure; and c) 4 HCV-negative patients with MC and Sjogrens syndrome or lupus. CPs were analyzed by qRT/PCR for HCV RNA and by Western blotting, mass spectroscopy, and immunofixation. Immunoglobulin (Ig), Ig free light chain (FLC), and components of complement pathways (C’) were quantified in serum. The relationships between concentrations of C4 and concentrations of C1q, C4-binding protein (C4-bp), C1 esterase inhibitor, and C4a were analyzed. CP and C’ components were analyzed by mass spectroscopy.
Results: At baseline, extrahepatic symptoms of the 10 DAA-treated patients included arthropathy (n=8), cutaneous manifestations (n=6) and neuropathy (n=5). All had C4a elevation, an indicator of C4 cleavage. C4a concentration was directly related to CRP concentration and inversely related C4 and C1q concentrations. Total hemolytic complement activity (CH50) was depressed in 7/10, indicating activation of the classical complement pathway. Serum FLC was elevated in all 10 and in other patients with persistent MC. FLC was associated with restriction in CPs. Among HCV+ patients, CPs had 5-20% of the HCV RNA in the sample. The 10 DAA-treated patients cleared HCV by wk 2-3. CryoVas improved in 7/10 and stayed the same in three [Correct?]. CH50 decreased during HCV treatment. CPs of patients with persistent MC after HCV cure were all Type 2 IgM-IgG. Western blots showed that CPs contained IgM, IgG, C1q and C4-bp.
Conclusions: We conducted serial measurements of C’ measurements in serum and performing proteomic analysis of peptides from isolated CPs by mass spectroscopy and obtained new details about CP composition, restriction, and C4 activation. The findings support published data showing that CPs and Cryovas may persist for long periods of time following clearance of HCV, which may indicate a pathogenic similarity to the MC/Cryovas/C4/C1q depletion reported in other autoimmune diseases.
Doyle EH, Rahman A, Aloman C,. Klepper AL, ElShamy A et al. Intrahepatic Plasmacytoid Dendritic Cells Are Polyfunctional and Potent Interferon Alpha Producers during Chronic Hepatitis C Virus Infection. Accepted for Presentation at AASLD Meeting, San Francisco, CA, November 2018 .
Background: Plasmacytoid dendritic cells (pDCs) are “natural” interferon α (IFNα)-producing cells. They play key roles in antiviral defense, autoimmunity, and ischemic injury, but their rarity makes them difficult to study. Previous investigations failed to detect IFNα in hepatitis C virus (HCV)-infected livers, suggesting that HCV may neutralize liver pDCs and shut off intrahepatic IFNα production.
Methods: Because chronic HCV infection remains an important but incompletely understood disease, we used a combination of molecular, biochemical, cytometric, and high-dimensional techniques to analyze pDC fre-quency/function in liver and peripheral blood mononuclear cells of HCV-infected patients and to examine correlations between pDC function and gene expression of matched whole liver and liver mononuclear cells (LMCs).
Results: Data revealed that liver pDCs of HCV-infected patients retain the ability to secrete large quantities of IFNα (>2 million molecules of IFNα/cell/hour) and demonstrated that LMCs produce more IFNα than PBMCs after toll-like receptor (TLR)-7/8 stimulation, p=0.0001. LMCs secreted >14-fold more IFNα than IFNγ. Liver pDC frequency positively correlates with whole liver expression of the “IFNα-response” pathway (R2=0.58, p=0.007) and “monocyte surface” signature (R2=0.54, p=0.01); it had a negative correlation with blood platelet counts (R2=0.43, p=0.008). CyTOF established that only liver pDCs make IFNα after TLR-7/8 stimulation and demonstrated that IFNα+ pDCs are polyfunctional and >90% make IFNα plus 2-4 other cytokines/chemokines.
Conclusions: During chronic HCV infection, liver pDCs are active and retain the ability to make abundant IFNα. Polyfunctional liver pDCs are likely to be key drivers of inflammation and immune activation and they may aggravate thrombocytopenia.
Krig S, Batra N, Steele T, Evans S, Konda A, Sam A, Siddiqui S, Ghosh PM, Vinall RL. Elucidating the role of nuclear Nrdp1 in mediating prostate cancer health disparities. Presented at Society for Basic Urologic Research (SBUR) annual meeting, Rancho Mirage, CA, November 2018.
Background: We previously demonstrated that Nrdp1, an E3 ubiquitin ligase, is a transcriptional target of the androgen receptor (AR) and that Nrdp1 levels are reduced by ADT in Caucasian American (CA) CaP cell lines. Nrdp1 regulates levels of multiple targets including ErbB3, a molecule that is associated with ADT resistance. The goal of the current study was to determine whether differential expression and localization of Nrdp1 contributes to CaP health disparities.
Methods: Nrdp1 and AR expression levels and localization in CaP patient biopsies and cell lines were assessed using immunohistochemistry (IHC) and subcellular fractionation/western blot, respectively. Immunoprecipitation was used to identify Nrdp1 binding partners. Manipulation of AR and/or Nrdp1 levels/localization was achieved via knockdown (siRNA), forced overexpression (Nrdp1-FLAG construct), and treatment with enzalutamide or synthetic androgen.
Results: We demonstrate that nuclear Nrdp1 levels are significantly lower in African American (AA) CaP patients (n=19) versus CA CaP patients (n=121) with localized disease (p=0.008), and that AA CaP cell lines also express lower levels of nuclear Nrdp1. Nuclear localization of Nrdp1 has not previously been reported in CaP cells. Knockdown of AR or treatment with enzalutamide reduced levels of nuclear Nrdp1, while treatment with synthetic androgen increased nuclear Nrdp1 levels. Immunoprecipitation experiments verified Nrdp1 can bind to AR. In patients, a strong negative correlation exists between nuclear Nrdp1 and cytoplasmic (inactive) AR (R=-0.64, p<0.001) in AA tumors, but a weak negative correlation between cytoplasmic (inactive) AR and nuclear Nrdp1 in CA (R=-0.37, p<0.001). Forced overexpression of Nrdp1 in CaP cell lines results in increased levels of ubiquitination in both the nucleus and cytoplasm.
Conclusions: Our patient and in vitro data demonstrate that AA cells express significantly lower levels of nuclear Nrdp1 compared to CA CaP cells and that a consequence of this is reduced ubiquitination of nuclear proteins in AA CaP cells. AR is involved in nuclear translocation of Nrdp1, and this can be inhibited by ADT resulting a further reduction in nuclear Nrdp1 levels. Next steps will be to identify the nuclear targets of Nrdp1 and to elucidate their role in CaP and contribution to CaP health disparities.
Vu H Preliminary results of a national public survey on the perception of pharmacists and the barriers to utilizing pharmacy services. Accepted for Presentation at ASHP Meeting, Anaheim, CA, December 2018.
Purpose: The study investigates the public’s perception of pharmacists and the barriers to utilizing clinical pharmacy services. Pharmacist roles and responsibilities have expanded from medications dispensing to complex disease state management. However, patient utilization of clinical pharmacy services remains low. Despite this, the general public’s perception of pharmacists has not yet been adequately studied nationwide. Therefore, there is a pressing need to investigate the barriers to utilizing pharmacy services and the public’s perception of pharmacists to promote the capabilities of pharmacists and to increase patient enrollment in clinical pharmacy services.
Methods:The institutional review board granted exemption status for this study. The initial version of the survey was based on published literature and the input of researchers, pharmacists, and pharmacy students. The survey was test-launched for 3 months in 2017 and then revised following the feedback of 36 participants. The revised survey contained 47 multiple-choice questions and 3 open-ended questions. The first section collected the survey participants’ patient demographic information, frequency of visiting a pharmacy, and most visited types of pharmacies. The second section investigated their knowledge of what services pharmacists provide and recorded what services the public had utilized. The third section examined their perception of, confidence and trust in, and advantages of pharmacists. The forth section investigated the barriers to utilizing pharmacy services. The Likert scale was used with applicable questions. The last section comprised of free text comments to record any additional thoughts about the barriers to utilizing pharmacists and personal views and expectations of pharmacists. Using Survey Monkey Audience Service, the survey was distributed nationwide between April and May 2018 and collected over 2000 responses from people who were at least 18 years old. For preliminary data analysis, the demographic information of the participants was analyzed. Their perceptions of pharmacists, pharmacist roles, and the barriers to utilizing pharmacy clinical services were determined through the Likert scale questions and the free text comments.
Results: A total of 2145 people with an average age of 48. 10 years old answered the survey. Members of all 50 U. S. states took part in the survey. Most of the survey’s participants identified themselves as female (51. 33 percent) and non-Hispanic White (74. 36 percent). Most participants utilized chain pharmacies (48. 63 percent), and most visited a pharmacy once a month (27. 64 percent). Although the majority believed that pharmacists could provide different clinical services besides dispensing medications and providing counsel, very few participants had actually utilized the additional services that pharmacists offer. The participants generally trusted pharmacists to fill their prescriptions correctly, and most of them (about 80. 41 percent) thought that pharmacists were accessible. However, only 22. 29 percent of participants agreed or strongly agreed with the statement that the pharmacists should be able to prescribe medications. The main barriers to utilizing clinical pharmacy services identified through the multiple-choice questions and the free text comments included the lack of advertisement regarding the pharmacy services by pharmacists and other health care professionals, competing services from other healthcare professionals, and a lack of understanding about the rigid training of pharmacists. Conclusion: The preliminary results of the survey identify the differences between the general public’s perception of pharmacists and the important clinical services that pharmacists offer. Pharmacists must take more initiative to educate the general public and even other healthcare professionals about their capabilities and the rigid training that they must undergo. By doing this, pharmacists can better promote the wellness of patients beyond dispensing medications and providing consultation on their proper uses.
Mokrushin ET, Talbott G, Woldemariam T, Fitzpatrick LR. In vitro and ex vivo effects of silymarin fractions on pro-inflammatory cytokine secretion. Accepted for Presentation at ASHP Meeting, Anaheim, CA, December 2018.
Anti-inflammatory drugs are often the first step in the treatment of inflammatory bowel disease (IBD). In an effort to discover novel bioactive natural plant products for the potential future treatment of IBD, we examined the silymarin complex for cytokine inhibition. Silymarin is comprised of four major classes of anti-inflammatory flavonolignans including silibinin, silychristin, silydianin and isosilibinin. Our prior studies determined that certain extracted fractions of silymarin inhibited pro-inflammatory cytokine secretion from macrophage and colonic epithelial cell lines. In this study, we examined in vitro and ex vivo effects of silymarin fractions on pro-inflammatory cytokine secretion.
Colitis was induced in male C57BL/6 mice (n equals 16) by giving 2 percent dextran sulfate sodium (DSS) drinking water for a six-day period. Control mice (n equals 8) were given untreated water. Employing a 24 hour colonic culture system, the ex vivo effects of crude silymarin extract, two different silymarin fractions, as well as commercially derived silybinin and isosilybinin (20 to 200 mcg per ml) were examined. Colonic strips obtained from mice with and without DSS-induced colitis were used; and the secretion of macrophage inflammatory protein 2 (MIP-2) and tumor necrosis factor alpha (TNF-alpha) in cell culture media was determined by ELISA. Further, the effects of silymarin compounds on interleukin 8 (IL-8) and TNF-alpha chemokine secretion induced by colitis supernatant (CS) was characterized with HT-29 colonic epithelial and RAW 264. 7 macrophage cell lines, respectively.
Prominent inhibition of MIP-2 and TNF-alpha secretion from colonic strips of mice with and without DSS-induced colitis was observed with various silymarin treatments (crude extract, fractions 2 and 5, silibinin, isosilibinin). Further, dose dependent inhibition of dual cytokine (interleukin 23 and interleukin 1 beta) stimulated secretion of interleukin 17 from colonic strips of mice with and without DSS induced colitis was found with silymarin treatments. Finally, significant attenuation of TNF-alpha from CS stimulated RAW 264. 7 cells was found for crude silymarin extract and isosilibinin treatments. Inhibition of IL-8 secretion from CS stimulated HT-29 colonic epithelial cell line was observed for isosilibinin.
Our results imply that a promising therapeutic approach for anti-colitis drugs could be with silymarin derived compounds. Specifically, the overall data profile suggests that fraction 2 (containing predominantly isosilibinin), or isosilibinin itself, would be optimal for follow-up in vivo testing in animal models of IBD. Potentially, this research path could lead to further identification of specific silymarin derived flavonolignans for potential therapeutic use in patients with IBD.
Asadirad M, Talbott G, Satow B, Woldemariam T, Malhotra A. Preclinical Investigation of Middle Eastern Diet as Adjuvant for Pancreatic Cancer Therapy. Accepted for Presentation at ASHP Meeting, Anaheim, CA, December 2018.
Purpose: Pancreatic adenocarcinoma (PCa) is an aggressive disease with a high mortality and only a 6-8% survival rate over a 5-year period following diagnosis. Chemotherapy with gemcitabine or the FOLFIRINOX remains ineffective due to chemoresistance. Our previous work showed that mitochondrial renewal pathways prevent the accumulation of damaged mitochondria in response to therapy in the PCa cells. We hypothesized that this inhibits apoptosis and drug response. Here we explored the use of Middle Eastern dietary components (MEDC) in mitigating mitochondrial renewal. Our goal is elucidate the mechanism by which MEDC overcome chemoresistance in PCa.
Methods: We prepared crude aqueous and acetone extracts from six spices and plants used in Middle Eastern and Indian cuisines, including artichoke (Cynara scolymus), curry leaf (Murraya koenigii), dill (Anethum graveolens), noni (Morinda citrifolia), olive (Olea europaea), rose (Rosa damascena), and sumac (Rhus coriaria). Briefly, macerated 5 g plant samples were extracted with either water or acetone for 24 hours, freeze dried or the solvent evaporated, and the percent yield of the dried residue was determined. To identify possible novel peptides, a Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) method was developed using a water:methanol, gradient 100:0 to 0:100 % over 20 minutes for separation of the compounds. The RP-HPLC LaChrom Elite® Hitachi System is equipped with a photodiode-array detector (L-2455). To determine bioactivity, we treated human PCa cells lines, MIA PaCa-2 and PANC-1 with exponentially increasing doses of these crude aqueous extracts at 300 ng/mL, 3 μg/mL, 30 μg/mL, and 300 μg/mL for 48 hours followed by an MTT-based cell viability assay. We selected MIA PaCa-2 cells because they harbor the established gene mutation in the Kirsten Rat Sarcoma viral oncogene (KRAS), found in 90% patients while PANC-1 cells were included to compare and contrast the effects of MEDC.
Results: Sumac and artichoke aqueous extracts induced cytotoxicity. To identify specific compounds, we used a fractional approach based on our preparative HPLC, preparing and re-testing three fractions from the crude extracts of sumac. In the course of our bioassay-guided screening of compounds, 100 mg of the residue was chromatographed onto a silica gel column and gradient eluted using dichloromethane/methanol (gradient, 0, 100%, v/v). All the fractions and eluates were monitored by thin layer chromatography (TLC) using precoated silica gel 60 F254, 0.25 mm aluminum backed plates (Merck). As our fractions showed cytotoxicity in both the PCa cell lines, LC-MS based analysis of these fractions is currently underway to identify the specific bioactive compounds. To address whether the observed cytotoxicity of the crude aqueous extracts of sumac and artichoke was limited to PCa cells, we compared cytotoxicity in NIH-3T3 cells as a non-cancer control. Sumac and artichoke aqueous extracts allowed greater cell viability at comparative doses in NIH-3T3 cells when compared to MIA PaCa-2 and PANC-1. Additionally, we also tested the effect of a 1:1 combination of the aqueous extracts of sumac and artichoke on the viability of the two PCa cell lines and found evidence for change due to combination.
Conclusions: Our results suggest that sumac and artichoke are cytotoxic to the selected PCa cell lines. We are currently investigating the effect of these agents on caspase-3 and 7 activation in PCa cells. Our future work will explore possible mechanisms by testing the effect of these extracts on the production of reactive oxidative species in the mitochondria. Our work will address the gap in knowledge of mt-renewal process in PCa, especially in response to dietary components.
Nguyen V, Vu H. Interpreting the national hospital ambulatory medical care survey to determine the trend of opioid and non-opioid prescriptions in the emergency department from 2013 to 2015. Accepted for Presentation at ASHP Meeting, Anaheim, CA, December 2018.
Prescription opioid overdoses are a leading cause of death and an epidemic in the United States. Interpreting the trends of opioid versus non-opioid pain management in the emergency departments (ED) may help identify potential factors leading to overprescribing of opioids. We hypothesized that opioid prescribing rates have increased over time and are greater than non-opioid prescriptions. Further, we aim to identify and analyze the opioid prescribing factors to improve the management of patient care and promote awareness to public health systems.
Research investigators will review the national hospital ambulatory medical care (NHAMCS) ED data from 2013-2015 and determine the trends between patients managed with non-opioid and opioid treatments. Furthermore, the investigator will use a statistical analysis software, statistical analysis system (SAS), to filter and determine patient-specific trends. Once the trends are determined between the non-opioid and opioid treatment groups, the investigator will interpret and extrapolate the rationale for the potential prescribing factors such as the following: average age, age category, gender, ethnicity, type of medical insurance, region, ED waiting time, ED length of visit, metropolitan statistical area status, and pain scale.
Opioids were prescribed for 36.51 percent (P value is less than 0.0001) of all ED discharges. Non-opioids were prescribed for 63.49 percent (P value is less than 0.0001) of all ED discharges. From 2013 to 2014 and 2014 to 2015, opioid prescriptions in the ED reduced by 1.04 percent and 6.24 percent, respectively. From 2013 to 2014 and 2014 to 2015, non-opioid prescriptions in the ED reduced by 7.67 percent and 12.73 percent, respectively. The average age for prescribed opioids and non-opioids in the ED was 44 and 45, respectively. The top 3 age category receiving opioids and non-opioids were 18 to 30, 31 to 40, and 41 to 50. Patients-specific factors associated with a non-Hispanic white population, female gender, and private insurance represented a greater association with opioid treatments (P value is less than 0.05) than non-opioid treatments.
We found no temporal trend towards increased prescribing opioids and non-opioids from 2013 to 2015. Our results suggest that opioid over-prescribing involves on a number of factors and not solely dependent in the ED.