Burn Injury Reduces Neutrophil Directional Migration Speed in Microfluidic Devices

Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72–120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.     

Electron microscopy for the diagnosis of tumours.

The role of leukocyte transfusions in the prevention and treatment of infections in adults with granulocytopenia was investigated. Leukocytes were obtained from healthy volunteers by continuous-flow centrifugation. Histocompatibility antigen (HLA)-matched leukocytes were used to assess the prophylactic value of leukocyte transfusions. Seven patients with acute myelogenous leukemia received HLA-matched leukocytes during the period of maximal granulocytopenia associated with initial remission induction therapy; 20 concurrently treated patients who did not receive leukocyte transfusions were the control group. The patients receiving HLA-matched leukocytes had significantly fewer (P = 0.043) infectious episodes (not bacteriologically proven) during the study period, and remission occurred in 5 of the 7, compared with 10 of the 20 controls. In addition, 52 series of two or more ABO-compatible transfusions were given to 50 patients with proven infection or elevated temperature presumed due to infection and a granulocyte count of less than 0.5 X 10(9)/L. Response, indicated by a decrease in temperature, occurred in 23 patients. Leukocyte transfusions thus have an important adjuvant role in the management of patients with severe granulocytopenia.     

Adsorption of cations to phosphatidylinositol 4,5-bisphosphate

We investigated the binding of physiologically and pharmacologically relevant ions to the phosphoinositides by making 31P NMR, electrophoretic mobility, surface potential, and calcium activity measurements. We studied the binding of protons to phosphatidylinositol 4,5-bisphosphate (PIP2) by measuring the effect of pH on the chemical shifts of the 31P NMR signals from the two monoester phosphate groups of PIP2. We studied the binding of potassium, calcium, magnesium, spermine, and gentamicin ions to the phosphoinositides by measuring the effect of these cations on the electrophoretic mobility of multilamellar vesicles formed from mixtures of phosphatidylcholine (PC) and either phosphatidylinositol, phosphatidylinositol 4-phosphate, or PIP2; the adsorption of these cations depends on the surface potential of the membrane and can be described qualitatively by combining the Gouy-Chapman theory with Langmuir adsorption isotherms. Monovalent anionic phospholipids, such as phosphatidylserine and phosphatidylinositol, produce a negative electrostatic potential at the cytoplasmic surface of plasma membranes of erythrocytes, platelets, and other cells. When the electrostatic potential at the surface of a PC/PIP2 bilayer membrane is -30 mV and the aqueous phase contains 0.1 M KCl at pH 7.0, PIP2 binds about one hydrogen and one potassium ion and has a net charge of about -3. Our mobility, surface potential, and electrode measurements suggest that a negligible fraction of the PIP2 molecules in a cell bind calcium ions, but a significant fraction may bind magnesium and spermine ions.     

Effects of body mass and morphology on thermal responses in water

Ten male volunteers were divided into two groups based on body morphology and mass. The large-body mass (LM) group (n = 5) was 16.3 kg heavier and 0.22 cm2 X kg-1 X 10(-2) smaller in surface area-to-mass ratio (AD X wt-1) (P less than 0.05) than the small-body mass (SM) group (n = 5). Both groups were similar in total body fat and skinfold thicknesses (P greater than 0.05). All individuals were immersed for 1 h in stirred water at 26 degrees C during both rest and one intensity of exercise (metabolic rate approximately 550 W). During resting exposures metabolic rate (M) and rectal temperature (Tre) were not different (P greater than 0.05) between the LM and SM groups at min 60. Esophageal temperature (Tes) was higher (P less than 0.05) for the SM group at min 60, although the change in Tes during the 60 min between groups was similar (LM, -0.4 degrees C; SM, -0.2 degrees C). Tissue insulation (I) was lower (P less than 0.05) for SM (0.061 degrees C X m-2 X W-1) compared with the LM group (0.098 degrees C X m-2 X W-1). During exercise M, Tre, Tes, and I were not different (P greater than 0.05) between groups at min 60. These data illustrate that a greater body mass between individuals increases the overall tissue insulation during rest, most likely as a result of a greater volume of muscle tissue to provide insulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypohydration and exercise: effects of heat acclimation, gender, and environment

This study examined the effects of heat acclimation and subject gender on treadmill exercise in comfortable (20 degrees C, 40% rh), hot-dry (49 degrees C, 20% rh), and hot-wet (35 degrees C, 79% rh) environments while subjects were hypo- or euhydrated. Six male and six female subjects, matched for maximal aerobic power and percent body fat, completed two exercise tests in each environment both before and after a 10-day heat acclimation program. One exercise test was completed during euhydration and one during hypohydration (-5.0% from baseline body weight). In general, no significant (P greater than 0.05) differences were noted between men and women at the completion of exercise for rectal temperature (Tre), mean skin temperature (Tsk), or heat rate (HR) during any of the experimental conditions. Hypohydration generally increased Tre and HR values and decreased sweat rate values while not altering Tsk values. In the hypohydration experiments, heat acclimation significantly reduced Tre (0.19 degrees C) and HR (13 beats X min-1) values in the comfortable environment, but only HR values were reduced in hot-dry (21 beats X min-1) and hot-wet (21 beats X min-1) environments. The present findings indicated that men and women respond in a physiologically similar manner to hypohydration during exercise. They also indicated that for hypohydrated subjects heat acclimation decreased thermoregulatory and cardiovascular strain in a comfortable environment, but only cardiovascular strain decreased in hot environments.     

Engineering organ perfusion protocols: NMR analysis of hepatocyte isolation from perfused rat liver

The development and use of an extracorporeal liver support device depends upon the isolation of a large number of viable, functioning hepatocytes from whole or partial livers. Current practice, however, produces nonoptimal yields, given that a large percentage of hepatocytes initially present are not successfully isolated. The normal hepatocyte isolation protocol consists of sequential perfusion with calcium chelating and collagenase buffers, and then separation of viable hepatocytes from non-viable and nonparenchymal cells, usually on the basis of cell density. In order to improve understanding regarding the metabolic and perfusion state of the liver during this perfusion protocol, ATP, pH, and tissue perfusion were evaluated using nuclear magnetic resonance (NMR). Perfusion with calcium chelating buffer was found to have minimal effect on the metabolic and perfusion parameters, whereas subsequent perfusion with collagenase buffer produced large declines in ATP, pH, and homogeneity of perfusion within 3 min. Perfusion with calcium-chelating buffer alone, or perfusion with calcium chelating buffer followed by a short period of ischemia to mimic the perfusion disruption of collagenase, did not produce the same decline in metabolic parameters. This NMR data suggested that enhancing the early perfusion and penetration of collagenase or prolonging the nontoxic calcium-chelation step may improve the yield and/or functionality of isolated cells. Therefore, several altered perfusion protocols were evaluated in terms of yield of viable parenchymal hepatocytes and hepatocyte albumin production. Although increasing the perfusion flow rate and initial perfusion with inactive (cold) collagenase did not produce significant improvements when compared with the control protocol (control cell yield 226 +/- 42 x 10(6) viable hepatocytes for 10- to 14-week-old female Lewis rat), prolonging and enhancing the calcium-chelating perfusion step or increasing the collagenase concentration did yield a significantly great number of viable parenchymal hepatocytes (393 +/- 44 and 328 +/- 39 x 10(6) viable hepatocytes, respectively) with no change in albumin production per seeded viable cell. (c) 1994 John Wiley & Sons, Inc.     

Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential.

A thermodynamic model was used to evaluate and optimize a rapid three-step nonequilibrium freezing protocol for one-cell mouse embryos in the absence of cryoprotectants (CPAs) that avoided lethal intracellular ice formation (IIF). Biophysical parameters of one-cell mouse embryos were determined at subzero temperatures using cryomicroscopic investigations (i.e., the water permeability of the plasma membrane, its temperature dependence, and the parameters for heterogeneous IIF). The parameters were then incorporated into the thermodynamic model, which predicted the likelihood of IIF. Model predictions showed that IIF could be prevented at a cooling rate of 120 degrees C/min when a 5-min holding period was inserted at -10 degrees C to assure cellular dehydration. This predicted freezing protocol, which avoided IIF in the absence of CPAs, was two orders of magnitude faster than conventional embryo cryopreservation cooling rates of between 0.5 and 1 degree C/min. At slow cooling rates, embryos predominantly follow the equilibrium phase diagram and do not undergo IIF, but mechanisms other than IIF (e.g., high electrolyte concentrations, mechanical effects, and others) cause cellular damage. We tested the predictions of our thermodynamic model using a programmable freezer and confirmed the theoretical predictions. The membrane integrity of one-cell mouse embryos, as assessed by fluorescein diacetate retention, was approximately 80% after freezing down to -45 degrees C by the rapid nonequilibrium protocol derived from our model. The fact that embryos could be rapidly frozen in the absence of CPAs without damage to the plasma membrane as assessed by fluorescein diacetate retention is a new and exciting finding. Further refinements of this protocol is necessary to retain the developmental competence of the embryos.     

Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide.

A three-part, coupled model of cell dehydration, nucleation, and crystal growth was used to study intracellular ice formation (IIF) in cultured hepatocytes frozen in the presence of dimethyl sulfoxide (DMSO). Heterogeneous nucleation temperatures were predicted as a function of DMSO concentration and were in good agreement with experimental data. Simulated freezing protocols correctly predicted and explained experimentally observed effects of cooling rate, warming rate, and storage temperature on hepatocyte function. For cells cooled to -40 degrees C, no IIF occurred for cooling rates less than 10 degrees C/min. IIF did occur at faster cooling rates, and the predicted volume of intracellular ice increased with increasing cooling rate. Cells cooled at 5 degrees C/min to -80 degrees C were shown to undergo nucleation at -46.8 degrees C, with the consequence that storage temperatures above this value resulted in high viability independent of warming rate, whereas colder storage temperatures resulted in cell injury for slow warming rates. Cell damage correlated positively with predicted intracellular ice volume, and an upper limit for the critical ice content was estimated to be 3.7% of the isotonic water content. The power of the model was limited by difficulties in estimating the cytosol viscosity and membrane permeability as functions of DMSO concentration at low temperatures.     

Using Molecular Dynamics to Predict Factors Affecting Binding Strength and Magnetic Relaxivity of MRI Contrast Agents

We demonstrate the use of molecular dynamics and molecular mechanics methods to calculate properties and behavior of metal-chelate complexes that can be used as MRI contrast agents. Static and dynamic properties of several known agents were calculated and compared with experiment. We calculated the static properties such as the q-values (number of inner shell waters) and binding distances of chelate atoms to the metal ion for a set of chelates with known X-ray structure. The dynamic flexibility of the chelate arms was also calculated. These computations were extended to a series of exploratory chelate structures in order to estimate their potential as MRI contrast agents. We have also calculated for the first time the NMR relaxivity of an MRI contrast agent using a long (5 nsec) molecular dynamics simulation. Our predictions are promising enough that the method should prove useful for evaluating novel candidate compounds before they are synthesized. One novel static property, the projected area of chelate atoms onto a virtual surface centered on the metal ion (gnomonic projection), was found to give an effective measure of how well the chelate atoms use the free space around the metal ion.