Serial monitoring of human systemic and xenograft models of leukemia using a novel vascular disrupting agent.

Advances in the treatment of acute leukemia have resulted in significantly improved remission rates, although disease relapse poses a significant risk. By utilizing sensitive, non-invasive imaging guidance, detection of early leukemic infiltration and the extent of residual tumor burden after targeted therapy can be expedited, leading to more efficient treatment planning. We demonstrated marked survival benefit and therapeutic efficacy of a new-generation vascular disrupting agent, combretastatin-A1-diphosphate (OXi4503), using reporter gene-imaging technologies and mice systemically administered luc+ and GFP+ human leukemic cells (LCs). Before treatment, homing of double-transduced cells was serially monitored and whole-body cellular distributions were mapped using bioluminescence imaging (BLI). Imaging findings strongly correlated with quantitative GFP expression levels in solid organs/tissues, suggesting that the measured BLI signal provides a highly sensitive and reliable biomarker of tumor tissue burden in systemic leukemic models. Such optical technologies can thereby serve as robust non-invasive imaging tools for preclinical drug discovery and for rapidly screening promising therapeutic agents to establish potency, treatment efficacy and survival advantage. We further show that GFP+ HL-60 cells reside in close proximity to VE-cadherin- and CD31-expressing endothelial cells, suggesting that the perivascular niche may have a critical role in the maintenance and survival of LCs.

Stromal-derived factor 1-induced megakaryocyte migration and platelet production is dependent on matrix metalloproteinases.

Despite the discovery of thrombopoietin (TPO) and its contribution to megakaryocytopoiesis, the exact mechanisms and sites of platelet production are unknown. It has been shown that mature megakaryocytes (MKs) functionally express the stromal-derived factor 1 (SDF-1) receptor, CXCR4. SDF-1-induced migration of mature MKs through endothelial cell layers results in increased platelet production. Because the migration of polyploid MKs from the bone marrow microenvironment requires remodeling of the perivascular extracellular matrix, it was hypothesized that mature polyploid MKs may express matrix metalloproteinases (MMPs), facilitating their exit into the bone marrow extravascular space. In this report, it is demonstrated that SDF-1 induces the expression and release of gelatinase B (MMP-9) by purified mature polyploid human MKs and an adeno-CXCR4-infected megakaryocytic cell line. Neutralizing antibody to MMP-9, but not MMP-2, blocked SDF-1-induced migration of MKs through reconstituted basement membrane, suggesting that expression of MMP-9 is critical for MK migration. Incubation of mature MKs with a synthetic MMP inhibitor, 5-phenyl-1,10-phenanthrolene, resulted in the inhibition of platelet formation, suggesting that the expression of MMPs is not only critical for megakaryocyte migration but also for subsequent platelet release. Confirming these results, adeno-SDF-1 injection into normal mice resulted in increased platelet counts, a process that could be blocked by a synthetic MMP inhibitor. These results suggest mobilization of MKs involves sequential expression and activation of chemokine receptors such as CXCR4, MMP-9, followed by transendothelial migration. MMP inhibitors may have potential use in the treatment of thrombotic and myeloproliferative disorders. (Blood. 2000;96:4152-4159)

Trace detection of explosives using a membrane-based displacement immunoassay.

A compact membrane-based displacement immunoassay has been designed for rapid detection of explosive compounds 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) at high femtomole levels. The system consists of activated porous membranes, onto which either TNT or RDX antibodies are immobilized, that are inserted into microreactor columns, incorporated into a flow system. The assay is prepared by saturating the immobilized antibody binding sites with labeled antigen. Target analyte is introduced upstream of the microreactor, while the displacement of labeled antigen is monitored downstream using a fluorometer. The concentration of displaced labeled antigen detected is proportional to the concentration of the target analyte introduced into the system. This system provides a reusable and reagentless sensor, suitable for continuous monitoring of explosives, with an operating lifetime of over 50 positive samples. Multiple assays were performed in approximately 5 min at different flow rates, using membranes saturated with varying antibody concentrations. The membrane-based format exhibited a detection limit of approximately 450 fmol for TNT and RDX (100 microl of 1 ng/ml solution) in laboratory samples.

A membrane-based displacement flow immunoassay.

The use of a membrane-based continuous flow displacement immunoassay for detection of nanomolar quantities of explosives is demonstrated, and the kinetics of this system are characterized through experimentation. Antibodies of 2,4,6-trinitrotoluene (TNT) are immobilized onto a porous membrane with surface reactive sites designed to facilitate the covalent binding of the antibody. After saturating the immobilized antibody binding sites with labeled antigen, target analyte is introduced in flow, and the displacement reactions are monitored using a fluorometer. The displaced labeled antigen detected is proportional to the concentration of the analyte introduced to the antibody-labeled antigen complex. Multiple assays were performed at flow rates of 2.0, 1.0, 0.50, and 0.25 mL/min using membranes saturated with varying TNT antibody concentrations. The signal intensity (i.e. the concentration of displaced labeled antigen) was independent of antibody concentration at 1.0 mL/min, but proportional to antibody concentration at 0.25 mL/min. Our data suggests that the lower flow rate created a longer interaction time between the injected analyte and the antibody-labeled antigen complex, resulting in greater displacement of the labeled antigen and higher signal intensities than seen at higher flow rates.

A perspective on myocardial contractility.

Myocardial contractile properties form the cornerstone of the heart's ability to pump blood. Efforts have been made to characterize these properties via classic elasticity theory concepts, which can lead to spurious results, as demonstrated by experiments measuring intramyocardial pressure. Two ways out of these difficulties are identified. One is to start at the cellular level, the other at the chamber level. The latter allows separation of ventricle (source) and arterial (load) effects on measured pressure and flow, distinct from previous definitions of ventricular contractility which tended to lump the two.

Differentiation of intramyocardial fluid pressure from fiber stress.

To characterize the complex force field generated in the ventricular myocardium, intramyocardial pressure (IMP) measurement is used as an indirect means of assessing the distribution of regional wall stress. To resolve the long term confusion associated with this measurement, IMP is divided into its two dominant components: intramyocardial fluid pressure (IFP) and intramyocardial fiber stress (IFS). The intramyocardial response to regional and global contractile function is examined in terms of changes in the magnitude and transmural gradient of IMP recording. The experimental results support the theoretical concept proposed where the hydraulic properties of the myocardium proved to have an influence on cardiac function. To gain a deeper understanding of myocardial function, cellular and subcellular components must be considered.

Assessment of heterogeneity in antibody-antigen displacement reactions.

The intrinsic binding characteristics of monoclonal antibodies are modified upon immobilization onto a solid-phase matrix. Factors such as the distribution in affinity must therefore be taken into consideration in order to predict the kinetics of antibody binding at solid-liquid interfaces. A mathematical analysis is presented herein that allows the assessment of heterogeneity in the affinity of monoclonal antibodies immobilized onto a solid support. This model is based on a modified version of the Sips distribution function adapted to the conditions of a solid-phase displacement assay in flow. An assay for trinitrotoluene (TNT) provides the data to evaluate the extent of heterogeneity introduced by immobilization of antibodies in a flow immunoassay. We determined the index of antibody heterogeneity on two solid supports, controlled-pore glass beads and agarose beads, coated with a monoclonal anti-TNT antibody at varying densities. The data confirm that the threshold for crossover from homogeneous to heterogeneous forms of the reaction isotherm is different in displacement reactions than in association-dissociation reactions. Our analysis shows that the measured displacement isotherm is consistent with a homogeneous or only moderately heterogenous distribution of relative affinities.

An evanescent wave biosensor--Part II: Fluorescent signal acquisition from tapered fiber optic probes.

A biosensor was developed using antibodies, fluorescence and the evanescent wave to detect antigen binding at the surface of an optical fiber. Cladding was removed from the core along the distal end of a step-index optical fiber, and recognition antibodies were immobilized on the declad core to form the probe sensing region. Immersing the declad probe in aqueous solution creates a V-number mismatch between the immersed probe and the clad fiber. Probes created with reduced sensing region radius exhibited improved response by decreasing the V-number mismatch. Tapering the radius of this region has further improved probe response. Ray tracing analysis of the tapered probe demonstrated that the evanescent wave penetration depth increases along the length of the taper. Experiments correlating position of refraction along the taper with launch angle at the proximal end were realized in the ray tracing model. An evanescent wave immunoassay was performed with a series of the tapered fiber probes, each tapered from the fiber core radius (100 microns) to different end radii. An end radius of 29 microns was found to produce maximal signal from the tapered probe. Factors leading to the determination of the optimized probe are discussed.

Effect of antibody density on the displacement kinetics of a flow immunoassay.

This study investigates the effect of antibody density on the kinetics of a solid-phase displacement immunoassay. Conducted in flow under nonequilibrium conditions, the assay utilizes a monoclonal antibody to the cocaine metabolite benzoylecgonine, which has been immobilized onto Sepharose beads and saturated with fluorophore labeled antigen. Displacement of antibody-bound labeled antigen by non-labeled antigen occurs when sample is introduced in the buffer flow. Comparison of matrices coated with two different antibody densities revealed that the displacement efficiency is a function of the density of antibody-bound labeled antigen. A higher density of antibody provides a higher amount of displaced labeled antigen, but the displacement efficiency of the assay is decreased. The effect of antibody density on the immunoassay kinetics was analyzed using a mathematical formulation developed to characterize antibody-antigen interactions at solid-liquid interfaces. Higher antibody density proved to be associated with a lower apparent dissociation rate constant. The implications of these results on the design of immunoassays in flow are discussed.

Pressure generation in a contracting myocyte.

The central hypothesis of this investigation is that a shortening myocyte generates a time-varying transmural pressure, or intracellular pressure. A mathematical model was formulated for a single myocyte, consisting of a fluid-filled cylindrical shell with axially arranged contractile filaments, to quantitate the fiber-fluid interaction. In this model, the intracellular pressure mediates the interaction between myofilament force, cell shortening, and the mechanical properties of the sarcolemma. Shortening of myofibrils, which are embedded in the fluid-filled myocytes, deforms the myocyte, thereby altering its transmural fluid pressure. This increase in transmural pressure counteracts fiber shortening, hence constituting an internal load to shortening. The shortening of the myocyte is accompanied by thickening, due to the incompressible nature of its contents. Consequently, the overall contractile performance of the cell is integrally linked to the generation of intracellular pressure. The model manifests a positive transmural pressure during shortening, but not without shortening. The pressure in the myocyte, therefore, is not a direct function of the force generated, but rather of shortening. Intracellular pressure was measured through a fluid-filled glass micropipette (5 mu ID) employing a servo-nulling pressure transducer in a standard micropuncture technique. Measured intracellular pressure in a contracting isolated skeletal myocyte of the giant barnacle is observed to be dynamically related to shortening, but not to tension without shortening. The relation between the force of contraction, cell shortening, and intracellular pressure was assessed during both isotonic and isometric contractions. The results support the prediction that isometric, or nondeforming, contractions will not develop intracellular pressure and identify a reason for relengthening of the myocytes during relaxation.

Optical immunosensors.

The development of biochemical detection systems that use antibodies as the recognition element, also known as immunosensors, has generated considerable interest from science and industry alike. This is due to the practically inexhaustible number of applications for which such a technology could conceivable lend itself, such as medicine, process control, and environmental monitoring. The potentially high sensitivity of such systems makes them ideal for situations where the utmost speed and accuracy are demanded. Moreover, there is theoretically no limit to the chemical and biological moieties for which an immunosensor can be manufactured. This critical review emphasizes both the biological and transduction aspects of optical immunosensors. The basic concepts of antibody-antigen interactions in solution are reviewed, and factors affecting the kinetics at the solid-liquid interface are characterized. In addition, the aspects of reagent immobilization, inherent to immunosensor design, are discussed. Finally, a comprehensive overview describing the mechanisms, advantages, and shortcomings of several immunosensing formats such as fiber-optic, planar waveguide, surface plasmon resonance, and continuous-flow immunosensor is presented. The future of these sensors, we well as the integration of fluorescence lifetime sensing and fluorescent polarization technologies with immunosensing technologies, is contemplated, posing the question: What place will immunosensors secure in tomorrow's market?

Peripheral vascular effects on auscultatory blood pressure measurement.

Experiments were conducted to examine the accuracy of the conventional auscultatory method of blood pressure measurement. The influence of the physiologic state of the vascular system in the forearm distal to the site of Korotkoff sound recording and its impact on the precision of the measured blood pressure is discussed. The peripheral resistance in the arm distal to the cuff was changed noninvasively by heating and cooling effects and by induction of reactive hyperemia. All interventions were preceded by an investigation of their effect on central blood pressure to distinguish local effects from changes in central blood pressure. These interventions were sufficiently moderate to make their effect on central blood pressure, recorded in the other arm, statistically insignificant (i.e., changes in systolic [p < 0.3] and diastolic [p < 0.02]). Nevertheless, such alterations were found to modify the amplitude of the Korotkoff sound, which can manifest itself as an apparent change in arterial blood pressure that is readily discerned by the human ear. The increase in diastolic pressure for the cooling experiments was statistically significant (p < 0.001). Moreover, both measured systolic (p < 0.004) and diastolic (p < 0.001) pressure decreases during the reactive hyperemia experiments were statistically significant. The findings demonstrate that alteration in vascular state generates perplexing changes in blood pressure, hence confirming experimental observations by earlier investigators as well as predictions by our model studies.

Kinetics of antibody binding at solid-liquid interfaces in flow.

We have developed the theoretical framework for a displacement immunoassay conducted in flow under nonequilibrium conditions. Using a repetitive displacement technique, we determined the displacement rate and apparent dissociation rate constant at different flow rates. Our data suggest that the kinetics are best described by a first-order function. The displacement efficiency, the displacement rate, and therefore the apparent dissociation rate constant were calculated and demonstrated to be flow rate dependent. The theoretical framework developed in this study was successful in predicting the behavior of antigen displacement in flow.

Intramyocardial pressure: interaction of myocardial fluid pressure and fiber stress.

Previous measurements of intramyocardial pressure (IMP) have yielded systolic pressures that range from values lower than to far exceeding systolic left ventricular pressure (LVP). This study identifies a possible mechanism underlying these divergent observations by building on established morphology of the ventricular wall. It is hypothesized here that the generation of fiber stress as a manifestation of myocardial contraction increases fluid pressure in the myocytes and the interstitial spaces. This increase in fluid pressure in turn generates the pressure in the ventricular cavity. Thus there are two quantities of interest: intramyocardial fluid pressure (IFP) and intramyocardial fiber stress (IFS). To test the hypothesis, we conducted experiments on conditioned dogs, utilizing a side-mounted catheter-tip strain gauge transducer to sense IMP as the sum of IFP and some component of IFS. In addition, a recessed end-tip fiber-optic transducer with its sensing element shielded from local myocardial fibers was employed to sense IFP. Both IFP and IMP were measured at various depths in the left ventricular free wall. The effects of inotropic interventions by administration of epinephrine and propranolol, mechanical interventions via clamping of the aorta and ligation of the left anterior descending coronary artery, and neural interventions by stimulation of the ansa subclavian of the stellate ganglion and right vagus were recorded. A transmural gradient in the wall for both IMP and IFP was observed. Systolic values of IFP recorded in the endocardium match those of LVP, with peak IMP exceeding both. The results support the hypothesis and offer an interpretation of the long-standing controversy regarding the magnitude of IMP with respect to LVP.

Baroreceptor responses derived from a fundamental concept.

A model is presented that relates the change in baroreceptor firing rate to a step change in blood pressure. This relationship is nonlinear since the alteration in rate of firing depends on the current rate of firing. It is shown that this simple relationship embodies all currently established baroreceptor response modes. The model needs refinement to allow for effects arising from the properties of the tissue matrix in which the receptors are embedded. Further analysis is precluded at present owing to paucity of quantitative experimental data.