Harold T. Huss

Melittin-induced membrane permeability: a nonosmotic mechanism of cell death.

SummaryDerived from honeybees, melittin is a 26-amino acid, α-helical, membrane-attack protein that efficiently kills mammalian cells. To investigate the contribution of colloid-osmotic effects to the mechanism of cell death, we studied the effect of melittin on lymphocyte membrane permeability and cell volumes. Melittin concentrations of 0.5 to 2.0 μM induced release of membrane permeability markers without total disruption of the cell membrane. At these melittin concentrations, electrical-impedance cytometry demonstrated melittin-induced changes in red blood cell volumes (P<0.01), but no change in lymphocyte cell volumes (P>0.05). Streaming video microscopy, obtaining images of melittintreated lymphocytes at 80-ms intervals, demonstrated a loss of optical density (P<0.001) suggesting a flattening of the cell but no significant increase in cell perimeter (P>0.05). Real-time multiparameter flow cytometry of melittin-treated lymphocytes confirmed simultaneous loss of the cytoplasmic marker, calcein, and uptake of the DNA dye, ethidium homodimer, but demonstrated no increase in forward light scatter. Transmission-electron microscopy of melittin-treated lymphocytes showed normal cell volumes but discontinuities in the cell membrane suggesting direct membrane toxicity. We conclude that melittin causes lymphocyte death by a “leaky patch” mechanism that is independent of colloid-osmotic effects.

Structural adaptations in the murine colon microcirculation associated with hapten-induced inflammation

Background: Blood flowing across the vascular endothelium creates wall shear stress, dependent on velocity of flow and vessel geometry, that tends to disrupt lymphocyte–endothelial cell adhesion. Objective: The microcirculation in a murine model of acute colitis was investigated to identify structural adaptations during acute colitis that may facilitate transmigration. Methods: In 2,4,6-trinitrobenzenesulphonic acid-induced acute colitis, the infiltrating cells and colonic microcirculation was investigated by cellular topographic mapping, corrosion casting and three-dimensional scanning electron microscopy (SEM). Colonic blood velocimetry was performed using intravital microscopy. Results: Clinical and histological parameters suggested a peak inflammatory response at 96 h (p<0.001). The infiltrating cells were spatially related to the mucosal capillary plexus by three-dimensional topographic mapping (p<0.001). In normal mice, corrosion casting and three-dimensional SEM showed a polygonal mucosal plexus supplied by ascending arteries and descending veins. After 2,4,6-trinitrobenzenesulphonic acid stimulation, three-dimensional SEM showed preserved branch angles (p = 0.52) and nominal vessel lengths (p = 0.93), but a significantly dilated mucosal capillary plexus (p<0.001). Intravital microscopy of the mucosal plexus showed a greater than twofold decrease in the velocity of flow (p<0.001). Conclusions: The demonstrable slowing of the velocity of flow despite an increase in volumetric flow suggests that these microvascular adaptations create conditions suitable for leucocyte adhesion and transmigration.

Multiframe particle tracking in intravital imaging: defining Lagrangian coordinates in the microcirculation

The cellular composition of the microcirculation creates blood flow that can be unsteady and nonuniform. To obtain information about nonuniform cellular trajectories, we describe in vivo imaging techniques that provide both detailed tracking of individual particles as well as an approach to simultaneous multicolor particle tracking. Particularly relevant to biologic systems, Lagrangian methods provide information about the fate of individual particles and flow in the system.

Inflammation-responsive focal constrictors in the mouse ear microcirculation

In many capillary exchange beds, blood flow is locally regulated by precapillary sphincter‐like activity. In this study, we used intravascular tracers and scanning electron microscopy to investigate precapillary blood flow regulation in the mouse ear. Gelatin ink injections of the normal mouse ear demonstrated 6.8 ± 2.3 axial vessels with a cutoff of detectable tracer in the early branches: 19 ± 11 focal constrictions were observed along the 1st to 5th order branches of the axial vessels. A perfusion tracer consisting of biotinylated anti‐endothelial lectins (Ricinus Communis Agglutin, Lycopersicon Esculentum and Griffonia Simplicifolia) was circulated for 30 min under physiological conditions. Subsequent enzyme histochemistry demonstrated no significant change in distal perfusion or in the number of focal constrictions (P > 0.05). Furthermore, the focal constrictions were unresponsive to vasodilators such as organic nitrates and prostaglandin E1. By contrast, the presence of oxazolone‐induced inflammation resulted in significant and sustained vasodilatation for more than 96 h (P > 0.001). Scanning electron microscopy demonstrated discrete constricting bands morphologically distinct from known precapillary sphincters. These results suggest that these previously unappreciated inflammation‐responsive precapillary constrictors regulate capillary recruitment in the mouse ear microcirculation.

Hierarchical clustering of monoclonal antibody reactivity patterns in nonhuman species.

Monoclonal antibodies (Mab) are an important resource for defining molecular expression and probing molecular function. The characterization of Mab reactivity patterns, however, can be costly and inefficient in nonhuman experimental systems. To develop a computational approach to the pattern analysis of Mab reactivity, we analyzed a panel of 128 Mab recognizing sheep antigens. Quantitative single parameter flow cytometry histograms were obtained from five cell types isolated from normal animals. The resulting 640 histograms were smoothed using a Gaussian kernel over a range of bandwidths. Histogram features were selected by SiZer—an analytic tool that identifies statistically significant features. The extracted histogram features were compared and grouped using hierarchical clustering. The validity of the clustering was indicated by the accurate pairing of externally verified molecular reactivity. We conclude that our computational algorithm is a potentially useful tool for both Mab classification and molecular taxonomy in nonhuman experimental systems.

Data integration in multi-dimensional data sets: informational asymmetry in the valid correlation of subdivided samples

Background: Flow cytometry is the only currently available high throughput technology that can measure multiple physical and molecular characteristics of individual cells. It is common in flow cytometry to measure a relatively large number of characteristics or features by performing separate experiments on subdivided samples. Correlating data from multiple experiments using certain shared features (e.g. cell size) could provide useful information on the combination pattern of the not shared features. Such correlation, however, are not always reliable. Methods: We developed a method to assess the correlation reliability by estimating the percentage of cells that can be unambiguously correlated between two samples. This method was evaluated using 81 pairs of subdivided samples of microspheres (artificial cells) with known molecular characteristics. Results: Strong correlation (R=0.85) was found between the estimated and actual percentage of unambiguous correlation. Conclusion: The correlation reliability we developed can be used to support data integration of experiments on subdivided samples.