Mathematical framework for activity-based cancer biomarkers

Advances in nanomedicine are providing sophisticated functions to precisely control the behavior of nanoscale drugs and diagnostics. Strategies that coopt protease activity as molecular triggers are increasingly important in nanoparticle design, yet the pharmacokinetics of these systems are challenging to understand without a quantitative framework to reveal nonintuitive associations. We describe a multicompartment mathematical model to predict strategies for ultrasensitive detection of cancer using synthetic biomarkers, a class of activity-based probes that amplify cancer-derived signals into urine as a noninvasive diagnostic. Using a model formulation made of a PEG core conjugated with protease-cleavable peptides, we explore a vast design space and identify guidelines for increasing sensitivity that depend on critical parameters such as enzyme kinetics, dosage, and probe stability. According to this model, synthetic biomarkers that circulate in stealth but then activate at sites of disease have the theoretical capacity to discriminate tumors as small as 5 mm in diameter—a threshold sensitivity that is otherwise challenging for medical imaging and blood biomarkers to achieve. This model may be adapted to describe the behavior of additional activity-based approaches to allow cross-platform comparisons, and to predict allometric scaling across species.The clinical management of cancer is increasingly dependent on the discovery of new biomarkers and the development of ultrasensitive technologies to detect them at a stage when therapeutic interventions may be effective (1, 2). However, despite their growing importance, biomarkers lack predictive power to impact patient outcomes during the earliest stages of disease. The challenges are multifaceted: Biomarkers are shed from tumors at rates that vary by four orders in magnitude (3), are significantly diluted in blood, and circulate for short periods. Recent mathematical studies showed that tumors may remain undetectable with blood biomarkers for an entire decade following tumorigenesis, reaching 1–2.5 cm in diameter (4, 5). To increase sensitivity, major research areas include the development of ultrasensitive in vitro diagnostic platforms (6–11), as well as methods to increase biomarker production by solid tumors (12, 13). These approaches are designed to measure the quantity, or abundance, of a disease biomarker.In contrast to abundance-based methods, activity-based probes are a class of agents that are administered in prodiagnostic form but produce strong diagnostic signals after enzymatic activation (14, 15). These approaches rely on disease-associated enzymes as catalysts to produce a detection signal, of which proteases are particularly potent because the cleavage of peptide bonds is irreversible, and a single protease can cleave many substrates to amplify signals. However, activity-based probes operate within a narrow time window and are activated by off-target tissues. A mathematical model of activity-based probes may aid in examining the critical parameters that determine specificity and sensitivity, and predict the use of activity-based probes in new clinical settings such as predicting disease progression earlier than standards of care (3).To date, pharmacokinetic models have been developed for abundance-based drugs and diagnostics—such as predicting nanoparticle (NP) targeting to tumor vasculature (11), identifying rate-limiting steps in the distribution of drugs within tumors (16–18), providing guidelines to increase NP penetration (19), and modeling NP disassembly at the glomerulus (20). Here, we establish a mathematical framework for synthetic biomarkers, a class of activity-based probes that amplify disease-derived signals into urine for easy analysis (3, 21–24). We use model formulations based on a size-tunable PEG core conjugated with protease substrates in a mouse model of colorectal cancer (CRC). By accounting for key nanomaterial, biochemical, and physiological parameters, our model predicts urine pharmacokinetics and reveals nonintuitive behavior of activity-based biomarkers.     

Lymphoid susceptibility to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin is dependent upon baseline levels of the signaling lipid,phosphatidylinositol‐3,4,5‐triphosphate

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes and other cell types. We have shown that the active subunit, CdtB, exhibits phosphatidylinositol‐3,4,5‐triphosphate (PIP3) phosphatase activity and depletes lymphoid cells of PIP3. Hence we propose that Cdt toxicity results from depletion of this signaling lipid and perturbation of phosphatidylinositol‐3‐kinase (PI‐3K)/PIP3/Akt signaling. We have now focused on the relationship between cell susceptibility to CdtB and differences in the status of baseline PIP3 levels. Our studies demonstrate that the baseline level of PIP3, and likely the dependence of cells on steady‐state activity of the PI‐3K signaling pathway for growth and survival, influence cell susceptibility to the toxic effects of Cdt. Jurkat cells with known defects in both PIP3 degradative enzymes, PTEN and SHIP1, not only contain high baseline levels of PIP3, pAkt, and pGSK3β, but also exhibit high sensitivity to Cdt. In contrast, HUT78 cells, with no known defects in this pathway, contain low levels of PIP3, pAkt, and pGSK3β and likely minimal dependence on the PI‐3K signaling pathway for growth and survival, and exhibit reduced susceptibility to Cdt. These differences in susceptibility to Cdt cannot be explained by differential toxin binding or internalization of the active subunit. Indeed, we now demonstrate that Jurkat and HUT78 cells bind toxin at comparable levels and internalize relatively equal amounts of CdtB. The relevance of these observations to the mode of action of Cdt and its potential role as a virulence factor is discussed.     

The beta subunit of the interleukin-2 receptor mediates interleukin-2 induction of anti-CD3 redirected cytotoxic capability in large granular lymphocytes

The interleukin 2 (IL 2) receptor was studied in three cases of large granular lymphocyte (LGL) lymphocytosis. All cases were nonreactive with anti-Tac monoclonal antibody (MoAb; recognizing the p55 alpha subunit of the IL 2 receptor). Sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoretic analysis (PAGE) of cells to which radio-labeled rIL 2 had been chemically crosslinked revealed uniform expression of the p70/75 beta subunit of the IL 2 receptor in the absence of the alpha subunit. Stimulation of this receptor with 2 nmol/L rIL 2 for five days led to acquisition of anti-CD3 redirected cytotoxicity. This was accompanied by a fivefold to tenfold elevation in the activity of intracellular N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl esterase, an LGL granule marker enzyme. These effects of IL 2 did not require induction of the Tac peptide.     

A Biomechanical Comparison of Three Different Posterior Fixation Constructs Used for C6–C7 Cervical Spine Immobilization: A Finite Element Study

The intralaminar screw construct has been recently introduced in C6–C7 fixation. The aim of the study is to compare the stability afforded by three different C7 posterior fixation techniques using a three-dimensional finite element model of a C6–C7 cervical spine motion segment. Finite element models representing three different cervical anchor types (C7 intralaminar screw, C7 lateral mass screw, and C7 pedicle screw) were developed. Range of motion (ROM) and maximum von Mises stresses in the vertebra for the three screw techniques were compared under pure moments in flexion, extension, lateral bending, and axial rotation. ROM for pedicle screw construct was less than the lateral mass screw construct and intralaminar screw construct in the three principal directions. The maximum von Misses stress was observed in the C7 vertebra around the pedicle in all the three screw constructs. Maximum von Mises stress in pedicle screw construct was less than the lateral mass screw construct and intralaminar screw construct in all loading modes. This study demonstrated that the pedicle screw fixation is the strongest instrumentation method for C6–C7 fixation. Pedicle screw fixation resulted in least stresses around the C7 pedicle-vertebral body complex. However, if pedicle fixation is not favorable, the laminar screw can be a better option compared to the lateral mass screw because the stress around the pedicle-vertebral body complex and ROM predicted for laminar screw construct was smaller than those of lateral mass screw construct.     

Case report. Pelvic rib/digit.

In summary, a case of a pelvic rib/digit is presented. Although there is no surgical proof that the finding does indeed represent a sacral rib, its radiographic findings are characteristic enough to warrant this diagnosis. In some respects, the lesion is similar to other cases that have appeared in the literature, but it differs in that it has features of both a pelvic rib and pelvic digit, suggesting a spectrum of development of these anomalous bony structures.     

Advances in imaging of new targets for pharmacological intervention in stroke: real-time tracking of T-cells in the ischaemic brain

Background and purpose:

T-cells may play a role in the evolution of ischaemic damage and repair, but the ability to image these cells in the living brain after a stroke has been limited. We aim to extend the technique of real-time in situ brain imaging of T-cells, previously shown in models of immunological diseases, to models of experimental stroke.

Experimental approach:

Male C57BL6 mice (6–8 weeks) (n= 3) received a total of 2–5 × 10⁶ carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled lymphocytes from donor C57BL6 mice via i.v. injection by adoptive transfer. Twenty-four hours later, recipient mice underwent permanent left distal middle cerebral artery occlusion (MCAO) by electrocoagulation or by sham surgery under isoflurane anaesthesia. Female hCD2-green fluorescent protein (GFP) transgenic mice that exhibit GFP-labelled T-cells underwent MCAO. At 24 or 48 h post-MCAO, a sagittal brain slice (1500 µm thick) containing cortical branches of the occluded middle cerebral artery (MCA) was dissected and used for multiphoton laser scanning microscopy (MPLSM).

Key results:

Our results provide direct observations for the first time of dynamic T-cell behaviour in living brain tissue in real time and herein proved the feasibility of MPLSM for ex vivo live imaging of immune response after experimental stroke.

Conclusions and Implications:

It is hoped that these advances in the imaging of immune cells will provide information that can be harnessed to a therapeutic advantage.This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x