A case study of applying text analysis to identify possible adverse drug interactions: The case of Adalat (Nifedipine).

Adalat (Nifedipine) is a calcium-channel blocker that is also used as an antihypertensive drug. The drug was approved by the US Food and Drug Administration in 1985 but was discontinued in 1996 on account, among other things, of interactions with other medications. Nonetheless, Adalat is still used in other countries to treat congestive heart failure. We examine all the congestive heart failure electronic health records of the largest medical center in Israel to discover whether, possibly, taking Adalat with other medications is associated with patient death. This study examines a semantic space built by running latent semantic analysis on the entire corpus of congestive heart failure electronic health records of that medical center, encompassing 8 years of data on almost 12,000 patients. Through this semantic space, the most highly correlated medications and medical conditions that co-occurred with Adalat were identified. This was done separately for men and women. The results show that Adalat is correlated with different medications and conditions across genders. The data also suggest that taking Adalat with Captopril (angiotensin-converting enzyme inhibitor) or Rulid (antibiotic) might be dangerous in both genders. The study thus demonstrates the potential of applying latent semantic analysis to identify potentially dangerous drug interactions that may have otherwise gone under the radar.

Quantifying the biomechanics of conception: L-selectin-mediated blastocyst implantation mechanics with engineered "trophospheres".

An estimated 12% of women in the United States suffer from some form of infertility. In vitro fertilization (IVF) is the most common treatment for infertility encompassing over 99% of all assisted reproductive technologies. However, IVF has a low success rate. Live birth rates using IVF can range from 40% in women younger than 35 years to 4% in women older than 42 years. Costs for a successful IVF outcome can be upward of $61,000. The low success rate of IVF has been attributed to the inability of the blastocyst to implant to the uterus. Blastocyst implantation is initiated by L-selectin expressing cells, trophoblasts, binding to L-selectin ligands, primarily sialyl Lewis X (sLeX), on the uterine surface endometrium. Legal and ethical considerations have limited the research on human subjects and tissues, whereas animal models are costly or do not properly mimic human implantation biochemistry. In this work, we describe a cellular model system for quantifying L-selectin adhesion mechanics. L-selectin expression was confirmed in Jeg-3, JAR, and BeWo cell lines, with only Jeg-3 cells exhibiting surface expression. Jeg-3 cells were cultured into three-dimensional spheres, termed "trophospheres," as a mimic to human blastocysts. Detachment assays using a custom-built parallel plate flow chamber show that trophospheres detach from sLeX functionalized slides with 2.75 × 10(-3) dyn of force and 7.5 × 10(-5) dyn-cm of torque. This work marks the first time a three-dimensional cell model has been utilized for quantifying L-selectin binding mechanics related to blastocyst implantation.

Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis.

Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane-binding properties of this protein. Recombinant anthrolysin O (rALO35-512) and two N-terminally truncated versions of ALO (rALO390-512 and rALO403-512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35-512, but not rALO390-512 or rALO403-512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35-512 and rALO403-512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35-512 and rALO403-512, whereas other sterols tested did not support binding. The rALO403-512--membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35-512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides.

Cytokine recognition by human interleukin 5 receptor.

The activation of interleukin 5 (IL-5) receptor is a dynamic process that depends on specific interaction of IL-5 with IL-5 receptor alpha, the formation of oligomeric receptor complexes with receptor beta, and the initiation of cytoplasmic phosphorylation events. These steps culminate in the triggering of a cellular response. Important advances have been made recently in understanding the molecular mechanisms of cytokine recognition, receptor assembly, and signal triggering. Cytokine recognition can be envisioned by relating structure to function in IL-5 and IL-5 receptor alpha. A pair of charge-complementary regions plays an essential role in the specific interaction between IL-5 receptor alpha and IL-5. Moreover, peptide library methodology has led to the discovery of IL-5 receptor alpha antagonists that mimic key elements in IL-5 receptor recognition. Because IL-5 has been implicated in the pathology of eosinophil-related inflammatory diseases, revealing the key recognition elements of IL-5, IL-5 mimetic peptides, and IL-5 receptor alpha could help drive the design of new compounds for therapeutic treatment against allergic inflammatory diseases such as asthma.

Evaluation of glucocorticoid sensitivity in 697 pre-B acute lymphoblastic leukemia cells after overexpression or silencing of MAP kinase phosphatase-1.

PURPOSE: To determine the effect of modulating MAP kinase phosphatase-1 (MKP-1) expression levels on cell death induced by glucocorticoid (GC) or hydroxyurea (HU) treatment in the human pre-B acute lymphoblastic leukemia cell line 697. METHODS: Stable MKP-1 overexpressing transformants of the 697 pre-B acute lymphoblastic leukemia cell line were created and tested for sensitivity to the GC triamcinolone acetonide (TA) and HU, and compared to a control 697 cell line containing normal MKP-1 expression levels. Small interfering RNAs (siRNAs) were designed to inhibit MKP-1 expression and evaluated for their effect on GC-mediated cell death. RESULTS: MKP-1 overexpression caused a phenotype of partial resistance to HU-induced apoptosis but not to GC-induced apoptosis. Electroporation of siRNAs effectively silenced MKP-1 expression, and increased sensitivity to TA by 9.6+/-1.9%. CONCLUSIONS: Because MKP-1 protects certain tumor cells from chemotherapy-induced apoptosis, its inhibition is being considered as a possible strategy for combination cancer therapy. However, this study suggests that while MKP-1 inhibition may improve the efficacy of DNA damaging agents, it may have only limited utility in combination with glucocorticoids.

Inhibition of glucocorticoid-induced apoptosis by targeting the major splice variants of BIM mRNA with small interfering RNA and short hairpin RNA.

Glucocorticoids (GCs) induce apoptosis in lymphocytes and are effective agents for the treatment of leukemia. The activated glucocorticoid receptor initiates a transcriptional program leading to caspase activation and cell death, but the critical signaling intermediates in GC-induced apoptosis remain largely undefined. We have observed that GC induction of the three major protein products of the Bcl-2 relative Bim (BimEL, BimS, and BimL) correlates with GC sensitivity in a panel of human precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) cell lines. To test the hypothesis that Bim facilitates GC-induced apoptosis, we reduced BIM mRNA levels and Bim protein levels by RNA interference in highly GC-sensitive pre-B ALL cells. Reducing Bim proteins by either electroporation of synthetic small interfering RNA (siRNA) duplexes or lentivirus-mediated stable expression of short hairpin RNA inhibited the activation of caspase-3 and increased cell viability following GC exposure. We also observed that the extent of GC resistance correlated with siRNA silencing potency. siRNA duplexes that reduced only BimEL or BimEL and BimL (but not BimS) exhibited less GC resistance than a potent siRNA that silenced all three major isoforms, implying that induction of all three Bim proteins contributes to cell death. Finally, the modulation of GC-induced apoptosis caused by Bim silencing was independent of Bcl-2 expression levels, negating the hypothesis that the ratio of Bim to Bcl-2 regulates apoptosis. These results offer evidence that the induction of Bim by GC is a required event for the complete apoptotic response in pre-B ALL cells.

TRAIL in the airways.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is an important immunomodulatory factor that may play a role in the structural changes observed in the asthmatic airways. In vitro as well as in vivo studies have evidenced a dual role for TRAIL: it can either function as a pro- or anti-inflammatory cytokine on inflammatory cells, participating in the initiation and resolution of inflammatory and immune responses. TRAIL is expressed in the airways by inflammatory cells infiltrated in the bronchial mucosa, as well as by structural cells of the airway wall including fibroblasts, epithelial, endothelial, and smooth muscle cells. By releasing TRAIL, these different cell types may then participate in the increased levels of TRAIL observed in bronchoalveolar lavage fluid from asthmatic patients. Taken together, this suggests that TRAIL may play a role in inflammation in asthma. However, concerning its role is dual in the modulation of inflammation, further studies are needed to elucidate the precise role of TRAIL in the airways.

Identification of interleukin 8 as an inhibitor of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in the ovarian carcinoma cell line OVCAR3.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to trigger apoptosis in many malignant cells. Whereas cancer cells are responsive to TRAIL-induced cell death when used alone or in combination with other agents, normal cells are known to be relatively less sensitive to the ligand, making it a desirable therapeutic compound to target a variety of cancers. TRAIL induces apoptosis through its interaction with its two proapoptotic death receptors (DRs), DR4 and DR5. In addition, it may also bind the decoy receptors (DcRs), DcR1 and DcR2, which lack an intracellular signaling domain, thus negatively regulating TRAIL-induced apoptosis. Previously, it has been shown that interleukin (IL)-8 is elevated in the ascites of patients with ovarian cancer. Therefore, we examined the role that IL-8 may play in modulating sensitivity to TRAIL-mediated apoptosis. We treated the TRAIL-sensitive cell line OVCAR3 with TRAIL over a period of time with or without pretreatment with IL-8. Here we show the novel findings that IL-8 blocks TRAIL-induced cell death and was able to turn the TRAIL-sensitive cell line into a TRAIL-resistant one. We hypothesized that decreased expression of DRs DR4 and DR5 may contribute to TRAIL resistance. Both reverse transcription-PCR and flow cytometry revealed a decrease in DR4 expression after pretreatment of OVCAR3 cells with IL-8. We have also shown that TRAIL was able to induce caspase-8 cleavage in these cells, whereas pretreatment with IL-8 blocked this caspase cleavage. Through array analysis and confirmation with other techniques, we have determined that IL-8 regulates the expression of a member of the mitogen-activated protein kinase superfamily, p38gamma. These findings provide important insights into the modulation of apoptosis by TRAIL and IL-8 in ovarian cancer. The data suggest a potentially important role of IL-8 in protecting ovarian cancer cells from TRAIL-mediated apoptosis and signify a new potential chemotherapeutic target to augment TRAIL therapy.

Role of apical caspases and glucocorticoid-regulated genes in glucocorticoid-induced apoptosis of pre-B leukemic cells.

Glucocorticoid (GC) sensitivity in hematopoietic cells requires the activation and nuclear translocation of the glucocorticoid receptor (GR) and the subsequent activation of caspases. To gain insight into the caspase cascade responsible for the execution phase of GC-induced apoptosis, 697 pre-B leukemic cells were stably transfected with dominant negative forms of caspase-8, caspase-9, or caspase-10 and the caspase-8 inhibitor CrmA. We observed that inhibition of caspase-9 or caspase-10 activity, but not caspase-8, caused partial resistance of 697 cells to GC-induced apoptosis. Inhibition of multiple caspases through the use of specific peptide inhibitors had an additive effect and caused complete resistance. To identify GR-regulated genes upstream of caspase activation in 697 cells, we performed DNA microarray analysis. 113 genes were identified, which were induced or repressed at least 3-fold by GC. Surprisingly, mitogen-activated protein kinase phosphatase-1 (MKP-1), a GR-induced gene in other cell types, was repressed 3-fold and correlated with an induction of JNK activity. These results suggest the involvement of mitogen activated protein kinases and apical caspase-9 and caspase-10 in the GC-induced apoptosis of pre-B lymphocytes.

Differential expression of TRAIL and TRAIL receptors in allergic asthmatics following segmental antigen challenge: evidence for a role of TRAIL in eosinophil survival.

Asthma is a chronic lung disease exhibiting airway obstruction, hyperresponsiveness, and inflammation, characterized by the infiltration of eosinophils into the airways and the underlying tissue. Prolonged eosinophilic inflammation depends on the balance between the cell's inherent tendency to undergo apoptosis and the local eosinophil-viability enhancing activity. TRAIL, a member of the TNF family, induces apoptosis in most transformed cells; however, its role in health and disease remains unknown. To test the hypothesis that Ag-induced inflammation is associated with TRAIL/TRAIL-R interactions, we used a segmental Ag challenge (SAC) model in ragweed-allergic asthmatics and nonasthmatic patients and analyzed bronchoalveolar lavage (BAL) material for 2 wk. In asthmatic patients, the level of TRAIL in BAL fluid dramatically increased 24 h after SAC, which significantly correlated with BAL eosinophil counts. Immunohistochemical analysis of bronchial biopsies from asthmatic patients demonstrated that TRAIL staining was increased in epithelial, airway smooth muscle, and vascular smooth muscle cells and throughout the interstitial tissue after SAC. This was confirmed by quantitative immunocytochemical image analysis of BAL eosinophils and alveolar macrophages, which demonstrated that expression levels of TRAIL and DcR2 increased, whereas expression levels of the TRAIL-Rs DR4 and DR5 decreased in asthmatic subjects after SAC. We also determined that TRAIL prolongs eosinophil survival ex vivo. These data provide the first in vivo evidence that TRAIL expression is increased in asthmatics following Ag provocation and suggest that modulation of TRAIL and TRAIL-R interactions may play a crucial role in promoting eosinophil survival in asthma.

Inhibition of glucocorticoid-induced apoptosis in 697 pre-B lymphocytes by the mineralocorticoid receptor N-terminal domain.

The glucocorticoid and mineralocorticoid receptors (GR and MR) share considerable structural and functional homology and bind as homodimers to hormone-response elements. We have shown previously that MR and GR can form heterodimers that inhibit transcription from a glucocorticoid (GC)-responsive gene and that this inhibition was mediated by the N-terminal domain (NTD) of MR. In this report, we examined the effect of NTD-MR on GC-induced apoptosis in the GC-sensitive pre-B lymphoma cell line, 697. In GC-treated 697 cells, we demonstrated that stable expression of NTD-MR blocks apoptosis and inhibits proteolytic processing of pro-caspases-3, -8, and -9 and poly(ADP-ribose) polymerase. Importantly, gel shift and immunoprecipitation analyses revealed a direct association between the GR and amino acids 203-603 of NTD-MR. We observed down-regulation of c-Myc and of the anti-apoptotic proteins Bcl-2 and Bfl-1 as well as high levels of the pro-apoptotic proteins Bax and Bid. Conversely, cells stably expressing NTD-MR exhibited increased expression of Bcl-2 and Bfl-1 and diminished levels of Bid and Bax. These data provide a potential mechanism for the observed inhibition of cytochrome c and Smac release from the mitochondria of NTD-MR cells and resultant resistance to GC-induced apoptosis. Thus, NTD-MR may mediate GC effects through heterodimerization with GR and ensuing inhibition of GC-regulated gene transcription.