Utilization of I-domain of LFA-1 to Target Drug and Marker Molecules to Leukocytes

The long-term objective of this project is to utilize the I-domain protein for the α-subunit of LFA-1 to target drugs to lymphocytes by binding to ICAM receptors on the cell surface. The short-term goal is to provide proof-of-concept that I-domain conjugated to small molecules can still bind to and uptake by ICAM-1 on the surface of lymphocytes (i.e., Raji cells). To accomplish this goal, the I-domain protein was labeled with FITC at several lysine residues to produce the FITC-I-domain and CD spectroscopy showed that the FITC-I-domain has a secondary structure similar to that of the parent I-domain. The FITC-I-domain was taken up by Raji cells via receptor-mediated endocytosis and its uptake can be blocked by anti-I-domain mAb but not by its isotype control. Antibodies to ICAM-1 enhance the binding of I-domain to ICAM-1, suggesting it binds to ICAM-1 at different sites than the antibodies. The results indicate that fluorophore modification does not alter the binding and uptake properties of the I-domain protein. Thus, I-domain could be useful as a carrier of drug to target ICAM-1-expressing lymphocytes

Understanding medication compliance and persistence from an economics perspective

AbstractObjectivesAn increased understanding of the reasons for noncompliance and lack of persistence with prescribed medication is an important step to improve treatment effectiveness, and thus patient health. Explanations have been attempted from epidemiological, sociological, and psychological perspectives. Economic models (utility maximization, time preferences, health capital, bilateral bargaining, stated preference, and prospect theory) may contribute to the understanding of medication-taking behavior.MethodsEconomic models are applied to medication noncompliance. Traditional consumer choice models under a budget constraint do apply to medication-taking behavior in that increased prices cause decreased utilization. Nevertheless, empiric evidence suggests that budget constraints are not the only factor affecting consumer choice around medicines. Examination of time preference models suggests that the intuitive association between time preference and medication compliance has not been investigated extensively, and has not been proven empirically. The health capital model has theoretical relevance, but has not been applied to compliance. Bilateral bargaining may present an alternative model to concordance of the patient–prescriber relationship, taking account of game-playing by either party. Nevertheless, there is limited empiric evidence to test its usefulness. Stated preference methods have been applied most extensively to medicines use.ResultsEvidence suggests that patients' preferences are consistently affected by side effects, and that preferences change over time, with age and experience. Prospect theory attempts to explain how new information changes risk perceptions and associated behavior but has not been applied empirically to medication use.ConclusionsEconomic models of behavior may contribute to the understanding of medication use, but more empiric work is needed to assess their applicability

Effects of an increase in prescription copayment on utilization of low-sedating antihistamines and nasal steroids

BACKGROUND: Health plans are using 3-tier copayment designs and other methods to control utilization that shifts drug costs to plan members. There is a need to determine the effects of increased member cost sharing on drug utilization and drug costs. OBJECTIVE: To assess the impact of a $10 increase in prescription copayment in a public employer health plan for 2 classes of drugs used for allergic rhinitis. METHODS: Changes in the number of prescriptions dispensed for 2 therapeutic classes low-sedating antihistamines (LSAs) and nasal steroids (NSs) were examined 1 year prior to and 1 year after copayment increase. Relative price effects were measured as arc price elasticity, the ratio of the percent change in prescription utilization over the percent change in price, an indicator of how responsive patients are to the copayment increase. RESULTS: Of 8,643 continuously enrolled health plan beneficiaries, 2,150 patients (24.8$10 increase in copayment per prescription was associated with no statistically significant change in utilization of combined LSA and NS prescriptions, 2.89 per patient in 1998 and 2.94 in 1999 (P = 0.597). Health plan costs for study drugs, unadjusted for inflation, decreased by 16.3% from $86.86 per patient in 1998 to$72.68 in 1999 (P = 0.004). Health plan costs per patient per month (PPPM) for all drugs for the 2,150 allergic rhinitis patients decreased by 13% from $41.33 PPPM in 1998 to$35.93 in 1999 (Pless than0.001), and health plan drug costs for all 8,643 members decreased by 13% from $14.93 per member per month (PMPM) in 1998 to$12.99 in 1999 (Pless than0.001). The actual average copayment increase was $7.23 (a 41$10.98 (71%) for NSs, which was associated with an 11.3% decrease in utilization of NSs and a 10.2% decrease in the number of users of nasals steroids in 1999; the number of nasal steroid prescriptions per patient per year was unchanged at 2.05 in 1999 versus 2.07 in 1998 (P = .842). The combined utilization of LSA and NS prescriptions increased by 8.9% following the increase in copayments for these 2 therapeutically interchangeable drugs for allergic rhinitis. LSA prescriptions were less elastic, with an unadjusted arc elasticity of 0.39, while nasal steroid prescriptions were more responsive to the copayment change, with an unadjusted arc elasticity of .0.22. CONCLUSIONS: An average $10 increase in patient cost sharing per prescription (46.9% copayment increase) was associated with an increase in combined utilization of 2 drug classes used for allergic rhinitis (LSAs and NSs) but no change in the number of prescriptions per patient. Health plan costs decreased significantly for allergic rhinitis drugs, all drugs used by allergic rhinitis patients, and all drugs used by continuously enrolled health plan members. NSs exhibited a greater arc price elasticity compared with low-sedating oral antihistamines

Endoplasmic reticulum chaperone Gp96 controls actomyosin dynamics and protects against pore-forming toxins

This research was originally published in the Journal of Biological Chemistry. Suman Ghosh, Heather E. Shinogle, Nadezhda A. Galeva, Rick T. Dobrowsky, and Brian S. J. Blagg. Endoplasmic Reticulum-resident Heat Shock Protein 90 (HSP90) Isoform Glucose-regulated Protein 94 (GRP94) Regulates Cell Polarity and Cancer Cell Migration by Affecting Intracellular Transport. Journal of Biological Chemistry. 2016; 291, 8309-8323.Heat shock protein 90 (HSP90) is a molecular chaperone that is up-regulated in cancer and is required for the folding of numerous signaling proteins. Consequently, HSP90 represents an ideal target for the development of new anti-cancer agents. The human HSP90 isoform, glucose-regulated protein 94 (GRP94), resides in the endoplasmic reticulum and regulates secretory pathways, integrins, and Toll-like receptors, which contribute to regulating immunity and metastasis. However, the cellular function of GRP94 remains underinvestigated. We report that GRP94 knockdown cells are defective in intracellular transport and, consequently, negatively impact the trafficking of F-actin toward the cellular cortex, integrin α2 and integrin αL toward the cell membrane and filopodia, and secretory vesicles containing the HSP90α-AHA1-survivin complex toward the leading edge. As a result, GRP94 knockdown cells form a multipolar spindle instead of bipolar morphology and consequently manifest a defect in cell migration and adhesion

Antigen-specific blocking of immunological synapse formation using bifunctional peptide, Utilization of I-domain of LFA-1 to Target Drug and Marker Molecules to Leukocytes

This is the publisher's version.The long-term objective of this project is to utilize the I-domain protein for the alpha-subunit of LFA-1 to target drugs to lymphocytes by binding to ICAM receptors on the cell surface. The short-term goal is to provide proof-of-concept that I-domain conjugated to small molecules can still bind to and uptake by ICAM-1 on the surface of lymphocytes (i.e., Raji cells). To accomplish this goal, the I-domain protein was labeled with FITC at several lysine residues to produce the FITC-I-domain and CD spectroscopy showed that the FITC-I-domain has a secondary structure similar to that of the parent I-domain. The FITC-I-domain was taken up by Raji cells via receptor-mediated endocytosis and its uptake can be blocked by anti-I-domain mAb but not by its isotype control. Antibodies to ICAM-1 enhance the binding of I-domain to ICAM-1, suggesting it binds to ICAM-1 at different sites than the antibodies. The results in-dicate that fluorophore modification does not alter the binding and uptake properties of the I-domain protein. Thus, I-domain could be useful as a carrier of drug to target ICAM-1-expressing lymphocytes

Hsp90 C‑Terminal Inhibitors Exhibit Antimigratory Activity by Disrupting the Hsp90α/Aha1 Complex in PC3-MM2 Cells

Human Hsp90 isoforms are molecular chaperones that are often up-regulated in malignances and represent a primary target for Hsp90 inhibitors undergoing clinical evaluation. Hsp90α is a stress-inducible isoform of Hsp90 that plays a significant role in apoptosis and metastasis. Though Hsp90α is secreted into the extracellular space under metastatic conditions, its role in cancer biology is poorly understood. We report that Hsp90α associates with the Aha1 co-chaperone and found this complex to localize in secretory vesicles and at the leading edge of migrating cells. Knockdown of Hsp90α resulted in a defect in cell migration. The functional role of Hsp90α/Aha1 was studied by treating the cells with various novobiocin-based Hsp90 C-terminal inhibitors. These inhibitors disrupted the Hsp90α/Aha1 complex, caused a cytoplasmic redistribution of Hsp90α and Aha1, and decreased cell migration. Structure–function studies determined that disruption of Hsp90α/Aha1 association and inhibition of cell migration correlated with the presence of a benzamide side chain, since an acetamide substituted analog was less effective. Our results show that disruption of Hsp90α/Aha1 interactions with novobiocin-based Hsp90 C-terminal inhibitors may limit the metastatic potential of tumors