Pharmacokinetics of Basiliximab for the Prevention of Graft-versus-Host Disease in Patients Undergoing Hematopoietic Cell Transplantation with Minimal-Intensity Cyclophosphamide and Fludarabine.

STUDY OBJECTIVE: Basiliximab is an immunosuppressive monoclonal antibody used for rejection prevention following solid organ transplantation; the pharmacokinetics (PK) of basiliximab in this setting are known. Basiliximab may also be used for prophylaxis and treatment of graft-versus-host disease (GVHD) in patients undergoing allogeneic hematopoietic cell transplantation (HCT); however, the PK of basiliximab in this setting are not known. Clinical transplant providers expect variation in the volume of distribution and clearance after nonmyeloablative allogeneic transplantation (NMAT) compared with solid organ transplantation. Blood loss, organ site-specific antibody accumulation, and differences in blood product use during the two transplantation approaches may generate differences in basiliximab PK. Therefore, the objective of this study was to describe the PK of basiliximab after its addition to a minimally intense NMAT regimen, in conjunction with cyclosporine, for GVHD prophylaxis in patients with hematologic malignancies. DESIGN: Population PK analysis of a single-center, single-arm, phase II clinical trial. SETTING: Academic cancer research center. PATIENTS: Fourteen adults with hematologic malignancies (acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, myelofibrosis, or severe aplastic anemia) and undergoing NMAT with a fully HLA-matched (10 of 10 antigen matched) related or unrelated donor. MEASUREMENTS AND MAIN RESULTS: Basiliximab was used in conjunction with cyclosporine to deplete activated T cells in vivo as GVHD prophylaxis. We developed a novel competitive enzyme-linked immunosorbent assay (ELISA) method using recombinant interleukin-2 receptor alpha-chain (IL-2Ra) and a commercially available soluble sIL-2R ELISA kit to permit the quantification of serum basiliximab concentrations and characterization of the PK properties of the drug in this patient population. Using a nonlinear mixed effects model with NONMEM software, a one-compartment model with first-order elimination best described the PK, as covariate analysis using stepwise covariate modeling did not improve the base model. CONCLUSION: We suggest a one-compartment population model with first-order elimination to capture the PK profile for basiliximab for this patient population.

Biomarkers of Delirium Duration and Delirium Severity in the ICU.

OBJECTIVES: Both delirium duration and delirium severity are associated with adverse patient outcomes. Serum biomarkers associated with delirium duration and delirium severity in ICU patients have not been reliably identified. We conducted our study to identify peripheral biomarkers representing systemic inflammation, impaired neuroprotection, and astrocyte activation associated with delirium duration, delirium severity, and in-hospital mortality. DESIGN: Observational study. SETTING: Three Indianapolis hospitals. PATIENTS: Three-hundred twenty-one critically ill delirious patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We analyzed the associations between biomarkers collected at delirium onset and delirium-/coma-free days assessed through Richmond Agitation-Sedation Scale/Confusion Assessment Method for the ICU, delirium severity assessed through Confusion Assessment Method for the ICU-7, and in-hospital mortality. After adjusting for age, gender, Acute Physiology and Chronic Health Evaluation II score, Charlson comorbidity score, sepsis diagnosis and study intervention group, interleukin-6, -8, and -10, tumor necrosis factor-α, C-reactive protein, and S-100ß levels in quartile 4 were negatively associated with delirium-/coma-free days by 1 week and 30 days post enrollment. Insulin-like growth factor-1 levels in quartile 4 were not associated with delirium-/coma-free days at both time points. Interleukin-6, -8, and -10, tumor necrosis factor-α, C-reactive protein, and S-100ß levels in quartile 4 were also associated with delirium severity by 1 week. At hospital discharge, interleukin-6, -8, and -10 retained the association but tumor necrosis factor-α, C-reactive protein, and S-100ß lost their associations with delirium severity. Insulin-like growth factor-1 levels in quartile 4 were not associated with delirium severity at both time points. Interleukin-8 and S-100ß levels in quartile 4 were also associated with higher in-hospital mortality. Interleukin-6 and -10, tumor necrosis factor-α, and insulin-like growth factor-1 were not found to be associated with in-hospital mortality. CONCLUSIONS: Biomarkers of systemic inflammation and those for astrocyte and glial activation were associated with longer delirium duration, higher delirium severity, and in-hospital mortality. Utility of these biomarkers early in delirium onset to identify patients at a higher risk of severe and prolonged delirium, and delirium related complications during hospitalization needs to be explored in future studies.

A Dose-escalation Study of Recombinant Human Interleukin-18 in Combination With Ofatumumab After Autologous Peripheral Blood Stem Cell Transplantation for Lymphoma.

Interleukin-18 (IL-18) is an immunostimulatory cytokine that augments antibody-dependent cellular cytotoxicity mediated by human natural killer cells against antibody-coated lymphoma cells in vitro and that has antitumor activity in animal models. Ofatumumab is a CD20 monoclonal antibody with activity against human B-cell lymphomas. A phase I study of recombinant human (rh) IL-18 given with ofatumumab was undertaken in patients with CD20 lymphoma who had undergone high-dose chemotherapy and autologous peripheral blood stem cell transplantation. Cohorts of 3 patients were given intravenous infusions of ofatumumab 1000 mg weekly for 4 weeks with escalating doses of rhIL-18 as a intravenous infusion weekly for 8 consecutive weeks. Nine male patients with CD20 lymphomas were given ofatumumab in combination with rhIL-18 at doses of 3, 10, and 30 µg/kg. No unexpected or dose-limiting toxicities were observed. The mean reduction from predose levels in the number of peripheral blood natural killer cells after the first rhIL-18 infusion was 91%, 96%, and 97% for the 3, 10, and 30 µg/kg cohorts, respectively. Serum concentrations of interferon-γ and chemokines transiently increased following IL-18 dosing. rhIL-18 can be given in biologically active doses by weekly infusions in combination with ofatumumab after peripheral blood stem cell transplantation to patients with lymphoma. A maximum tolerated dose of rhIL-18 plus ofatumumab was not determined. Further studies of rhIL-18 and CD20 monoclonal antibodies in B-cell malignancies are warranted.

S100 calcium binding protein B as a biomarker of delirium duration in the intensive care unit - an exploratory analysis.

BACKGROUND: Currently, there are no valid and reliable biomarkers to identify delirious patients predisposed to longer delirium duration. We investigated the hypothesis that elevated S100 calcium binding protein B (S100ß) levels will be associated with longer delirium duration in critically ill patients. METHODS: A prospective observational cohort study was performed in the medical, surgical, and progressive intensive care units (ICUs) of a tertiary care, university affiliated, and urban hospital. Sixty-three delirious patients were selected for the analysis, with two samples of S100ß collected on days 1 and 8 of enrollment. The main outcome measure was delirium duration. Using the cutoff of <0.1 ng/mL and ≥0.1 ng/mL as normal and abnormal levels of S100ß, respectively, on day 1 and day 8, four exposure groups were created: Group A, normal S100ß levels on day 1 and day 8; Group B, normal S100ß level on day 1 and abnormal S100ß level on day 8; Group C, abnormal S100ß level on day 1 and normal on day 8; and Group D, abnormal S100ß levels on both day 1 and day 8. RESULTS: Patients with abnormal levels of S100ß showed a trend towards higher delirium duration (P=0.076); Group B (standard deviation) (7.0 [3.2] days), Group C (5.5 [6.3] days), and Group D (5.3 [6.0] days), compared to patients in Group A (3.5 [5.4] days). CONCLUSION: This preliminary investigation identified a potentially novel role for S100ß as a biomarker for delirium duration in critically ill patients. This finding may have important implications for refining future delirium management strategies in ICU patients.

Network analysis of the focal adhesion to invadopodia transition identifies a PI3K-PKCα invasive signaling axis.

In cancer, deregulated signaling can produce an invasive cellular phenotype. We modeled the invasive transition as a theoretical switch between two cytoskeletal structures: focal adhesions and extracellular matrix-degrading invadopodia. We constructed molecular interaction networks of each structure and identified upstream regulatory hubs through computational analyses. We compared these regulatory hubs to the status of signaling components from head and neck carcinomas, which led us to analyze phosphatidylinositol 3-kinase (PI3K) and protein kinase C α (PKCα). Consistent with previous studies, PI3K activity promoted both the formation and the activity of invadopodia. We found that PI3K induction of invadopodia was increased by overexpression of SH2 (Src homology 2) domain-containing inositol 5'-phosphatase 2 (SHIP2), which converts the phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] that is produced by PI3K activity to phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)], which is believed to promote invadopodia formation. Knockdown of PKCα had divergent effects on invadopodia formation, depending on the status of PI3K. Loss of PKCα inhibited invadopodia formation in cells with wild-type PI3K pathway status. Conversely, in cells with constitutively active PI3K (through activating PI3K mutants or lacking the endogenous opposing enzyme PTEN), PKCα knockdown increased invadopodia formation. Mechanistic studies revealed a negative feedback loop from PKCα that dampened PI3K activity and invasive behavior in cells with genetic hyperactivation of the PI3K pathway. These studies demonstrated the potential of network modeling as a discovery tool and identified PI3K and PKCα as interacting regulators of invasive behavior.

A systems biology approach to the global analysis of transcription factors in colorectal cancer.

BACKGROUND: Biological entities do not perform in isolation, and often, it is the nature and degree of interactions among numerous biological entities which ultimately determines any final outcome. Hence, experimental data on any single biological entity can be of limited value when considered only in isolation. To address this, we propose that augmenting individual entity data with the literature will not only better define the entity's own significance but also uncover relationships with novel biological entities.To test this notion, we developed a comprehensive text mining and computational methodology that focused on discovering new targets of one class of molecular entities, transcription factors (TF), within one particular disease, colorectal cancer (CRC). METHODS: We used 39 molecular entities known to be associated with CRC along with six colorectal cancer terms as the bait list, or list of search terms, for mining the biomedical literature to identify CRC-specific genes and proteins. Using the literature-mined data, we constructed a global TF interaction network for CRC. We then developed a multi-level, multi-parametric methodology to identify TFs to CRC. RESULTS: The small bait list, when augmented with literature-mined data, identified a large number of biological entities associated with CRC. The relative importance of these TF and their associated modules was identified using functional and topological features. Additional validation of these highly-ranked TF using the literature strengthened our findings. Some of the novel TF that we identified were: SLUG, RUNX1, IRF1, HIF1A, ATF-2, ABL1, ELK-1 and GATA-1. Some of these TFs are associated with functional modules in known pathways of CRC, including the Beta-catenin/development, immune response, transcription, and DNA damage pathways. CONCLUSIONS: Our methodology of using text mining data and a multi-level, multi-parameter scoring technique was able to identify both known and novel TF that have roles in CRC. Starting with just one TF (SMAD3) in the bait list, the literature mining process identified an additional 116 CRC-associated TFs. Our network-based analysis showed that these TFs all belonged to any of 13 major functional groups that are known to play important roles in CRC. Among these identified TFs, we obtained a novel six-node module consisting of ATF2-P53-JNK1-ELK1-EPHB2-HIF1A, from which the novel JNK1-ELK1 association could potentially be a significant marker for CRC.

Specific tyrosine phosphorylations mediate signal-dependent stimulation of SHIP2 inositol phosphatase activity, while the SH2 domain confers an inhibitory effect to maintain the basal activity.

SH2 domain-containing 5-inositol phosphatase (SHIP2) is implicated in the development of type 2 diabetes and cancer. Tyrosine phosphorylation of SHIP2 is shown to enhance its phosphatase activity. Using IP4 as a substrate, we show here that tyrosines 986, 987, and 1135 are critical for EGF-induced stimulation of SHIP2 activity. SHIP2 with a disrupted SH2 domain (R47G mutation) displays higher constitutive activity than wild-type SHIP2. Deletion of the C-terminus region similarly activates SHIP2. Thus, the SH2 domain of SHIP2, in conjunction with the C-terminus, confers an inhibitory effect to maintain a low basal activity, and signal-induced tyrosine phosphorylations overcome this effect to activate SHIP2.

SHIP2 phosphoinositol phosphatase positively regulates EGFR-Akt pathway, CXCR4 expression, and cell migration in MDA-MB-231 breast cancer cells.

The phosphoinositol phosphatase SHIP2 is an important regulator of energy metabolism. SHIP2 dephosphorylates phosphatidylinositol 3,4,5 trisphosphates which are critical second messengers in signaling pathways induced by various extracellular stimuli including insulin. SHIP2 also regulates cytoskeleton remodeling, cell adhesion and spreading. In addition, endogenous SHIP2 in HeLa cells regulates receptor endocytosis and ligand-induced EGFR degradation. Further, SHIP2 in MDA-MB-231 breast cancer cells regulates EGFR levels and supports in vitro cell proliferation and in vivo tumor growth and spontaneous metastasis. Here we examine the role of SHIP2 in EGF signaling in breast cancer cells using RNA interference. Our results show that suppression of SHIP2 in MDA-MB-231 breast cancer cells alters EGF and EGFR internalization. Upon SHIP2 silencing, EGF-induced Akt activation was reduced causing decreased nuclear levels of activated Akt. Cytokine receptor CXCR4, a downstream element of EGFR-Akt pathway that plays an important role in metastasis, is down-regulated upon SHIP2 knockdown. Finally, cell adhesion and EGF-induced cell migration were suppressed in SHIP2 silenced cells. These results demonstrate a positive role of SHIP2 in EGF-induced Akt activation, CXCR4 expression, and cell migration in breast cancer cells.

Receptor overexpression or inhibition alters cell surface dynamics of EGF-EGFR interaction: new insights from real-time single molecule analysis.

Binding of Epidermal growth factor (EGF) to epidermal growth factor receptor (EGFR) in two types of cancer cells (HeLa; 5 x 10(4) EGFR/cell) and MDA-MB-468; 2 x 10(6) EGFR/cell) was studied using Total Internal Reflectance Fluorescence (TIRF) microscopy at single molecule precision. Mathematical modeling of the binding kinetics revealed that cells respond differently to the same concentration of EGF depending on the expression level of EGFR. Compared to Hela, MDA-MB-468 cells show; (a) higher number of pre-formed dimers, (b) improved EGF-EGFR interaction at lower ligand concentrations, and (c) shorter time-lapse between first and second EGF binding to the dimer. Treatment with a pharmacological inhibitor of EGFR, AG1478, produced strikingly different binding kinetics where the extent of pre-formed EGFR dimers increased substantially. Thus, single molecule approaches produce novel, quantitative information on signaling mechanisms of significant biological importance. Surface kinetics could also serve as surrogate markers to predict biological outcome of signaling pathways.

High expression of obesity-linked phosphatase SHIP2 in invasive breast cancer correlates with reduced disease-free survival.

SH2-containing 5'-inositol phosphatase (SHIP2) is a known regulator of insulin function. Genetic knockout of SHIP2 in mice causes mild insulin hypersensitivity and prevents high-fat-diet-induced obesity. SHIP2 also regulates actin remodeling and epidermal growth factor receptor (EGFR) turnover and supports breast cancer; and metastatic growth. To determine the clinical significance of SHIP2 expression in breast cancer and its relationship to relevant oncogenic molecules, SHIP2 expression was determined immunohistochemically in 285 primary breast cancers; 140 ductal carcinomas in situ (DCIS) and 145 invasive carcinomas. Forty-five percent of the specimens showed high SHIP2 levels in cancer cells while only 15% of adjacent normal cells expressed high SHIP2 levels (p < 0.0001). In cancer cells, the risk of SHIP2 overexpression is elevated (a) in women aged < or =50 years (relative risk, RR = 4.13; 95% confidence interval, CI, 2.5-6.9) compared to women aged >50 years (RR = 2.37; 95% CI 1.6-3.5; p = 0.0003), and (b) in invasive carcinomas (RR = 3.52; 95% CI 2.3-5.5) compared with DCIS (RR = 2.22; 95% CI 1.5-3.5; p = 0.0009). Patients with higher SHIP2 levels in invasive carcinomas had significantly reduced disease-free (p = 0.0025) and overall survival periods (p = 0.0228). In invasive carcinomas, SHIP2 correlated with estrogen receptor absence (p = 0.003) and EGFR presence (p = 0.0147). In conclusion, SHIP2 is an important biomarker for breast cancer.

Phosphoinositol phosphatase SHIP2 promotes cancer development and metastasis coupled with alterations in EGF receptor turnover.

Phosphoinositol phosphatases are important regulators of signaling pathways relevant to both diabetes and cancer. A 3'-phosphoinositol phosphatase, phosphatase homologous to tensin (PTEN), is both a tumor suppressor and a negative regulator of insulin action. A 5'-phosphoinositol phosphatase, SH2-containing 5'-inositol phosphatase (SHIP2), regulates insulin signaling and its genetic knockout prevents high-fat diet-induced obesity in mice. SHIP2 also regulates cytoskeleton remodeling and receptor endocytosis. This and the fact that both PTEN and SHIP2 act on the same substrate suggest a potential role for SHIP2 in cancer. Here we report that, in direct contrast to PTEN, SHIP2 protein expression is elevated in a number of breast cancer cell lines. RNA interference-mediated silencing of SHIP2 in MDA-231 cells suppresses epidermal growth factor receptor (EGFR) levels by means of enhanced receptor degradation. Furthermore, endogenous SHIP2 in MDA-231 breast cancer cells supports in vitro cell proliferation, increases cellular sensitivity to drugs targeting the EGFR and supports cancer development and metastasis in nude mice. In addition, significantly high proportions (44%; P = 0.0001) of clinical specimens of breast cancer tissues in comparison with non-cancerous breast tissues contain elevated expression of SHIP2 protein. Taken together, our results demonstrate that SHIP2 is a clinically relevant novel anticancer target that links perturbed metabolism to cancer development.

SH2-containing 5'-inositol phosphatase, SHIP2, regulates cytoskeleton organization and ligand-dependent down-regulation of the epidermal growth factor receptor.

Phosphoinositide lipid second messengers are integral components of signaling pathways mediated by insulin, growth factors, and integrins. SHIP2 dephosphorylates phosphatidylinositol 3,4,5-trisphosphate generated by the activated phosphatidylinositol 3'-kinase. SHIP2 down-regulates insulin signaling and is present at higher levels in diabetes and obesity. SHIP2 associates with p130Cas and filamin, regulators of cell adhesion/migration and cytoskeleton, influencing cell adhesion/spreading. Type I collagen specifically induces Src-mediated tyrosine phosphorylation of SHIP2. To better understand SHIP2 function, we employed RNA interference (RNAi) approach to silence the expression of the endogenous SHIP2 in HeLa cells. Suppression of SHIP2 levels caused severe F-actin deformities characterized by weak cortical actin and peripheral actin spikes. SHIP2 RNAi cells displayed cell-spreading defects involving a notable absence of focal contact structures and the formation of multiple slender membrane protrusions capped by actin spikes. Furthermore, decreased SHIP2 levels altered distribution of early endocytic antigen 1 (EEA1)-positive endocytic vesicles and of vesicles containing internalized epidermal growth factor (EGF) and transferrin. EGF treatment of SHIP2 RNAi cells led to the following: enhanced EGF receptor (EGFR) degradation; increased EGFR ubiquitination; and increased association of EGFR with c-Cbl ubiquitin ligase. Taken together, these experiments demonstrate that SHIP2 functions in the maintenance and dynamic remodeling of actin structures as well as in endocytosis, having a major impact on ligand-induced EGFR internalization and degradation. Accordingly, we suggest that, in HeLa cells, SHIP2 plays a distinct role in signaling pathways mediated by integrins and growth factor receptors.