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Biomedical subjects

W G Junger

Publications and source records attributed to W G Junger.

28 records · Page 2Linked to original sources
PubMed1994

Effects of trauma on immune cell function: impairment of intracellular calcium signaling.

Immunosuppression following injury influences infectious morbidity and mortality. Impaired T-cell activation conceding to inadequate antigen recognition contributes to this immunosuppression. Successful activation and proliferation of T-cells requires precisely specified levels of intracellular calcium thresholds and peak signals. The purpose of this study was to evaluate intracellular calcium signaling following injury. Hospitalized blunt and penetrating trauma patients in a Level 1 Trauma Center following injury and sepsis were tested for immune cell calcium signaling. Peripheral blood mononuclear cells (PBMC) were isolated and calcium signaling tested with Fura-2 AM. PBMC from trauma patients had significantly depressed values of baseline, peak and sustained levels of intracellular calcium prior to and following phytohemagglutinin stimulation when compared to normal controls. This deficit in intracellular calcium signaling is more severe in septic trauma patients (60% reduction). Suppression of calcium signaling appears to be mediated by at least, in part, circulating serum factors. Prostaglandin E2 seems to have a limited contribution to this effect as it is suppressive only when in direct contact with PBMC. Immune cell activation failure can in part be explained by the inadequacy of calcium signaling; restoration of immunocompetence following trauma will have to be addressed by strategies to restore calcium signaling, a vital step necessary for T-cell proliferation following antigen recognition.

Calcium↗
PubMed1994

Hypertonic saline enhances cellular immune function.

Hypertonic saline (HTS) resuscitation improves outcome after trauma. We studied the effect of HTS on immune function. In vitro T-cell proliferation of human and rabbit peripheral blood mononuclear cells (PBMC) was doubled at 25 mM increased extracellular Na+ concentrations. Further increased hypertonicity (more than 40 mM with human cells, and 80 mM with rabbit cells) caused progressive suppression of proliferation. Human and rabbit monocyte functions (tumor necrosis factor production) were augmented by 300% at 30 mM hypertonicity, indicating that HTS-enhanced accessory cell function of monocytes may cause increased T-cell proliferation. Substitution of HTS with KCl also enhanced T-cell proliferation, suggesting an involvement of osmotic effects. HTS (up to 30 mM) increased Ca2+i of nonstimulated human PBMC. HTS injection in rabbits increased cell-mediated immune function (delayed-type hypersensitivity reaction). Our findings suggest that increased plasma osmolality may up-regulate cellular immune function. HTS resuscitation of trauma patients may thus reverse posttraumatic immunosuppression and reduce the risk of sepsis.

Animals↗
PubMed1993

Improved rapid photometric assay for quantitative measurement of PMN migration.

We developed an improved quantitative method to measure in vitro polymorphonuclear leukocyte (PMN) migration using an assembly consisting of a 96-well chamber, polycarbonate filter membrane, and a 96-well microtiter plate. The convenience in setup and counting of migrated cells using this method allows processing of 80 samples and 16 controls in a short assay time of only 2 h. The peroxidase contained in PMNs was used as a marker enzyme to determine the number of migrated cells. Peroxidase released from lysed migrated cells was detected with an enzymatic method utilizing o-dianisidine as substrate. Photometric measurement was performed with a conventional microtiter plate reader at a wavelength of 405 nm. Optical density readings obtained using the enzymatic assay correlated with the number of migrated cells in a linear fashion up to 1 x 10(5) cells/well. The sensitivity of the enzymatic assay was sufficient to determine cell counts as low as 500 PMNs. PMNs lost no measurable amounts of peroxidase during the migration assay when ZAS was used as the chemoattractant. A calibration method was developed to make corrections for variations in the peroxidase content of different cell preparations and changes in the peroxidase content of cells exposed to the chemoattractant. High speed, convenient handling, and the use of standard laboratory equipment result in low cost per assay and make this migration assay ideally suited to research and clinical applications.

Cell Migration Inhibition↗
PubMed1992

Inhibition of human, ovine, and baboon neutrophil elastase with Eglin c and secretory leukocyte proteinase inhibitor.

The association rate constants (kon) of human, ovine, and baboon neutrophil elastase with two recombinant serine proteinase inhibitors (Eglin c, secretory leukocyte proteinase inhibitor) were compared. The association rate constant of sheep leukocyte elastase (SLE) with Eglin c is about 100 times lower (kon = 2.2 x 10(5) M-1s-1) than that of human elastase (kon = 2.4 x 10(7) M-1s-1). Baboon elastase, however, is as effectively blocked with Eglin c (kon = 2.5 x 10(7) M-1s-1) as human elastase. Secretory leukocyte proteinase inhibitor (SLPI) blocks the elastase of all three species with high efficiency; baboon elastase shows the highest association rate constant (kon = 5.6 x 10(7) M-1s-1) followed by human elastase (kon = 4.1 x 10(7) M-1s-1) and finally sheep elastase (kon = 1.2 x 10(7) M-1s-1). These findings demonstrate marked differences in the inhibition kinetic properties of ovine and human elastase. Concerning a future clinical application of proteinase inhibitors, the baboon seems a more suitable model than sheep to evaluate the effects of Eglin c and SLPI, since both inhibitors block baboon and human elastase with comparable efficiency.

Animals↗
PubMed1992

The enzymatic and release characteristics of sheep neutrophil elastase: a comparison with human neutrophil elastase.

Sheep are often used to study tissue damage following shock after traumatic injury and in the course of other diseases. The processes involved are thought to be caused at least in part by elastase released from polymorphonuclear leukocytes (PMNs). Since little is known about elastase and its role as a mediator of tissue damage in sheep, we studied the biochemical properties and release characteristics to sheep leukocyte elastase (SLE) in comparison of those of human leukocyte elastase (HLE). Both enzymes showed similar molecular masses, amino-acid compositions, N-terminal amino-acid sequences, and abilities to digest elastin substrates. Differences, however, were found in kinetic parameters measured with the elastase-specific substrate N-methoxysuccinyl-(L-alanyl)2-L-prolyl-L- valine-4-nitroanilide (MeoSuc-AAPV-pNa). The Michaelis constant (Km) of ovine elastase was nearly 10 times higher (1.82 mM) than the Km of HLE (0.21 mM). Values of SLE calculated for kcat were 70% and for kcat/Km 8% of corresponding values determined for HLE. In addition, significant differences between sheep and human PMNs were found in in vitro stimulation experiments. In contrast to human PMNs, sheep neutrophils released no active elastase, and only 50 to 70% of the H2O2 produced by human PMNs. This failure to release active elastase could not be explained by a lower elastase content of sheep PMNs, as there were no significant differences found between the elastase contents of sheep and human PMNs. We conclude that elastase liberated by stimulated sheep PMNs is inactivated by a concomitantly released proteinase inhibitor also located within the sheep PMNs.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Alteration in Ca2+ homeostasis by a trauma peptide.

Postinjury tissue inflammation with PMN elastase proteolysis generates immunosuppressive fibronectin peptides (FNDP) impairing chemotaxis, T-cell activation, and proliferation. Excess intracellular Ca2+ ([Ca2+]i) impairs T-cell activation. This study quantifies the changes in [Ca2+]i following exposure to a degradation peptide of fibronectin to determine the mechanism of action of these peptides on calcium homeostasis. Isolated human PBLs were exposed to immunosuppressive concentrations of FNDP after loading with the [Ca2+]i probe FURA-2AM. Resting and sustained [Ca2+]i concentrations were calculated and compared to buffer control. The mechanism of action was determined by pretreatment with: (1) EDTA binding extra cellular Ca2+: [Ca2+]e, (2) the Ca2+ channel blockers verapamil and nifedipine, and (3) inhibition of [Ca2+]i released by dantrolene. Inositol triphosphate (IP3) essential for [Ca2+]i release was measured following T-cell stimulation as well. FNDP caused 200-400% increases in [Ca2+]i concentration relative to buffer control at known suppressive doses. Verapamil and nifedipine partially block [Ca2+]i influx by as much as 50% suggesting the slow Ca2+ (voltage independent) channels are partially responsible for the increased [Ca2+]i seen following FNDP. EDTA completely suppressed [Ca2+]e influx but did not completely inhibit the release of [Ca2+]i although IP3 was 80% suppressed. The increase in [Ca2+]i following FNDP stimulation is due to release of intracellular stores.

Calcium↗
PubMedDate not supplied

Early detection of anastomotic leaks after colorectal surgery by measuring endotoxin in the drainage fluid.

BACKGROUND/AIMS: Early detection of anastomotic leaks after colorectal anastomosis is essential for adequate intervention to prevent peritonitis. We investigated whether the measurement of endotoxin (LPS) concentrations in the drainage has any value for the early detection of anastomotic leaks. MATERIALS AND METHODS: Twenty two patients with colorectal anastomosis were enrolled in this study, 3 developed clinically established signs of anastomotic leaks and 19 recovered without complications. LPS concentrations in the drainage, the total daily excreted LPS amounts, leukocyte and thrombocyte counts, plasma urea and creatinine, and body temperature were measured for up to 8 days after surgery and tested for their value to detect anastomotic leaks. RESULTS: LPS concentrations in the drainage fluid and daily excreted LPS amounts of patients with anastomotic leaks were significantly higher compared to the group without anastomotic leaks. On the third postoperative day, LPS concentrations ranged from 5270 to 6750 pg/ml in patients with anastomotic leaks and from 1 to 1848 pg/ml in patients without complications. Total daily excreted LPS amounts were 270-675 ng/day in patients with anastomotic leak and 0-92 ng/day in patients without anastomotic leaks. Both LPS-related parameters allowed reliable detection of anastomotic leaks on day 3 after surgery (Student's t-Test, p < 0.0005), while leukocyte and thrombocyte counts, plasma urea and creatinine, and body temperatures of both patient groups were not significantly different at any time (p > 0.05). CONCLUSION: We found that the measurement of LPS concentrations in the drainage and the daily excreted LPS amount could be valuable parameters for the early detection of anastomotic leaks as early as on the third post-operative day.

Adult↗