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.     

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.     

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.     

A stable long-term hepatocyte culture system for studies of physiologic processes: cytokine stimulation of the acute phase response in rat and human hepatocytes.

Prior studies on the in vitro hepatic acute phase response have involved either hepatoma cell lines or conventional short-term cultures of primary hepatocytes. No data are available on the response of primary hepatocytes in stable long-term culture systems. In this study, the acute phase response of rat and human hepatocytes in a new long-term culture system was examined in response to interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF-alpha). The cultured cells were sandwiched between two layers of collagen in a (double-gel) configuration which has been shown to preserve both hepatocyte function and morphology over prolonged periods of time. The stability of this culture configuration enabled us to investigate, for the first time, the temporal aspects of the response in addition to the effects of the mediators on protein secretion. Exposure of rat hepatocytes to IL-6 after culture for 16 days resulted in a 2-fold reduction of albumin secretion and a 15-fold increase in the secretion rates of fibrinogen and alpha 2-macroglobulin. In all instances, the peak response occurred at 48 h after IL-6 exposure, and all protein secretion rates returned to pretreatment values within 5 days posttreatment. Changes in the mRNA levels of these proteins in response to IL-6 corresponded with those changes seen with the secreted products, indicating pretranslational regulation. Administration of IL-1 beta to rat hepatocyte produced a similar decline of albumin secretion and a 5-fold increase of fibrinogen secretion, whereas alpha 2-macroglobulin secretion remained undisturbed.(ABSTRACT TRUNCATED AT 250 WORDS)     

T-cell therapies for T-cell lymphoma

T-cell lymphomas represent a subpopulation of non-Hodgkin lymphomas with poor outcomes when treated with conventional chemotherapy. A variety of novel agents have been introduced as new treatment strategies either as first-line treatment or in conjunction with chemotherapy. Immunotherapy has been demonstrated to be a promising area for new therapeutics, including monoclonal antibodies and adoptive cellular therapeutics. T-cell therapeutics have been shown to have significant success in the treatment of B-cell malignancies and are rapidly expanding as potential treatment options for other cancers including T-cell lymphomas. Although treating T-cell lymphomas with T-cell therapeutics has unique challenges, multiple targets are currently being studied both preclinically and in clinical trials.     

Thermal performance of quartz capillaries for vitrification

In this paper we report the thermal behavior of a new approach for vitrification. Thermal performance of traditional open pulled straws is compared with a new technique based on the combined use of quartz capillaries with slush nitrogen. This new method of vitrification achieved ultrafast cooling rates of 250,000 °C/min. As a result, a much lower concentration of cryoprotectant was needed to reach vitrification. In fact, a cryoprotectant solution typically used in oocyte slow freezing protocols was shown to remain transparent after cooling to liquid nitrogen temperatures indicating apparent “vitrification”. This approach offers a new and very promising technique for vitrification of cells using low levels of cryoprotectants.