Takeshi Shimazu

Activated platelets enhance microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis.

BACKGROUNDnActivated platelets have been recently reported to produce platelet microparticles and to enhance platelet-leukocyte interaction. The precise role of platelets in systemic inflammatory response syndrome (SIRS) has not been clarified. The objective of this study was to evaluate microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis.nnnMETHODSnTwenty-six patients with severe SIRS (SIRS criteria and serum C-reactive protein > 10 mg/dL) and 12 healthy volunteers were studied. The severe SIRS was caused by trauma in 12 patients and sepsis in 14. Microparticle formation, P-selectin expression on platelets, platelet-monocyte binding, and platelet-polymorphonuclear leukocyte (PMNL) binding were measured by flow cytometry in the presence or absence of ionomycin, N-formyl-methionyl-leucyl-phenylalanine, or anti-CD62p monoclonal antibody. Soluble P-selectin, thrombomodulin, neopterin, and PMNL elastase in blood were also measured.nnnRESULTSnMicroparticle formation, P-selectin expression on platelets, platelet-monocyte binding with or without ionomycin, and platelet-PMNL binding with ionomycin significantly increased in patients with severe SIRS in comparison with values in normal volunteers. The increased platelet-leukocyte binding in severe SIRS patients was markedly inhibited by P-selectin blockade and was not enhanced by N-formyl-methionyl-leucyl-phenylalanine. Soluble P-selectin, thrombomodulin, neopterin, and PMNL elastase in blood also increased in these patients.nnnCONCLUSIONnActivated platelets enhance microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis. Enhanced platelet-leukocyte interaction is dependent on P-selectin expression and may be involved in the systemic inflammatory response after severe inflammatory insult.

Priming, second-hit priming, and apoptosis in leukocytes from trauma patients

BACKGROUNDnPolymorphonuclear leukocytes (PMNL) play important roles in both host defenses and systemic inflammatory responses after insults. The objectives of this study are to examine the serial changes in PMNL priming and apoptosis in severely injured patients and to evaluate the impact of second hits on primed PMNL function and systemic vascular endothelial damage.nnnMETHODSnTwenty-four severely injured patients (mean Injury Severity Score, 31.1 +/- 9.7) were included. Infections were seen as second hits after trauma in seven patients. Oxidative activity, phagocytosis, and apoptosis of PMNL from serial blood samples were measured by flow cytometry. Oxidative activity with no stimulus and with formylmethionyl-leucyl-phenylalanine (FMLP) were analyzed as the priming index and FMLP response, respectively. Interleukin (IL)-6, IL-10, PMNL elastase, and thrombomodulin concentrations in blood were also measured before and after the second hit.nnnRESULTSnThe PMNL priming index was elevated from days 2 to 13, especially days 2 to 5 after injury. FMLP response was enhanced from days 2 to 21 after injury. Apoptosis of PMNL was inhibited for as long as 3 weeks after injury. Infections as second hits after trauma enhanced both the priming index and the FMLP response within 24 hours after diagnosis of infection and increased serum IL-6 concentrations. However, serum thrombomodulin levels were not affected by second hits. All patients with second hits survived.nnnCONCLUSIONnSevere trauma stimulated acute-phase priming in PMNL and inhibited apoptosis. Infections after trauma induced second-hit priming in PMNL, but the unchanged serum levels of thrombomodulin suggest that priming per se may not cause systemic vascular endothelial damage.

The morphology of smoke inhalation injury in sheep.

Pulmonary injury resulting from inhalation of chemical and particulate products of incomplete combustion is one of the principal determinants of mortality following burn injury. In this study, the histopathology of inhalation injury was examined in sheep. Mild, moderate, or severe smoke injury was produced in anesthetized sheep by insufflation with various doses of ambient temperature smoke, generated by burning polyethylene, wood pulp, and nonwoven cellulose pads. A total of 64 sheep were exposed and evaluated at times ranging from 15 minutes to 4 weeks after exposure. Morphologic changes in the lungs were studied using light microscopy and both transmission and scanning electron microscopy. The primary, dose-responsive injury observed was acute cell membrane damage in the trachea and bronchi leading to edema, progressive necrotic tracheobronchitis with pseudomembrane formation, and airway obstruction. These inflammatory and occlusive effects were followed by congestion, alveolar space edema, atelectasis, and bronchopneumonia. Morphologic changes occurring in the alveolar epithelium following high smoke dosage included intracellular edema in type-I cells, changes in the membrane-bound vacuoles of type-II cells, and septal thickening caused by interstitial edema. No capillary endothelial changes were observed.

Enhanced expression of heat shock proteins in activated polymorphonuclear leukocytes in patients with sepsis.

BACKGROUNDnHeat shock proteins (HSPs) in cells, as molecular chaperons, have been reported to regulate cell functions. The objective of this study was to investigate the HSP expression in polymorphonuclear leukocytes (PMNLs) from severe septic patients and the relation between the expression of HSPs and PMNL function.nnnMETHODSnIn blood samples from 21 patients with sepsis and serum C-reactive protein levels more than 10 mg/dL, we used flow cytometry to measure expressions of HSP27, HSP60, HSP70, and HSP90; oxidative activity; and levels of apoptosis in PMNLs during sepsis. In in vitro studies, we used cells from 14 healthy volunteers to examine the relation between the expression of HSP70 and PMNL function. Quercetin (30 microM), a suppressor of HSP, and sodium arsenite (100 microM), an inducer of HSP, were used to regulate the expression of HSP70 in PMNLs, and oxidative activity and apoptosis in these cells were measured.nnnRESULTSnIn patients with sepsis, the expressions of HSP27, HSP60, HSP70, and HSP90 and oxidative activity in PMNLs were significantly increased. Apoptosis of these PMNLs was markedly inhibited. In the in vitro studies, administration of sodium arsenite enhanced the expression of HSP70, significantly increased oxidative activity, and inhibited apoptosis. Administration of quercetin before sodium arsenite prevented the expression of HSP70, the increase in oxidative activity, and the inhibition of apoptosis.nnnCONCLUSIONnSepsis causes the enhanced expression of HSPs in activated PMNLs. In PMNLs with enhanced expression of HSP70, oxidative activity is increased and apoptosis is inhibited. The enhanced expression of HSPs may play a role in regulating PMNL function in patients with sepsis.

Half-life of blood carboxyhemoglobin after short-term and long-term exposure to carbon monoxide

BACKGROUNDnIn models of smoke inhalation injury and carbon monoxide poisoning blood carboxy-hemoglobin (COHb) levels decrease faster than predicted by the generally recognized half-life of COHb. We studied the effects of duration of exposure to carbon monoxide (CO) on the subsequent CO elimination.nnnMETHODSnEach of four sheep were insufflated with CO gas mixtures either for a few minutes (short-term exposure) or for several hours (long-term exposure), then ventilated with air for 3 hours. Serial COHb concentrations were analyzed by using a two-compartment, single central outlet mathematical model.nnnRESULTSnShort-term exposures exhibited biphasic decreases of COHb concentration compatible with a two-compartment model; an initial rapid decrease (half-life 5.7 +/- 1.4 minutes) was followed by a slower phase (103 +/- 20.5 minutes). Long-term exposures exhibited almost monophasic decreases, which were nevertheless compatible with the model (half-life, 21.5 +/- 2.1 and 118 +/- 11.2 minutes).nnnCONCLUSIONnThis study demonstrated different patterns of CO elimination curve, which suggests distribution of CO to two compartments having different rates of equilibration.

Delayed Hemispheric Neuronal Loss in Severely Head-Injured Patients

Recent experimental studies have revealed that traumatic brain injury as well as ischemic brain injury can cause chronic progressive neuronal damage. In the present study, we demonstrate previously unreported delayed cerebral atrophy on computerized tomography (CT) scans in severely head-injured patients. Seventeen severely head-injured patients who required mild hypothermia to control intracranial hypertension after the failure of conventional therapies were retrospectively analyzed. All 17 patients survived more than 1 year. Delayed neuronal loss (DNL) was observed in only eight of the 17 patients. Eight patients with DNL required longer durations of mild hypothermia to control intracranial hypertension than nine patients without DNL. Six of these eight patients with DNL achieved functional recovery despite progressive atrophic changes demonstrated on CT scans. On CT scans, DNL was characterized by (1) the sudden appearance at several months postinjury of a low-density area in the hemisphere ipsilateral to the injury; (2) the preservation of essential cortical structure although related white matter structures showed severe atrophic changes; and (3) no spread of the low-density area to the contiguous territory of the other main cerebral artery. It is concluded that focal primary injury to underlying brain, if severe enough, can result in delayed hemispheric atrophy.

Enhanced expression of heat shock proteins in leukocytes from trauma patients.

BACKGROUNDnHeat shock proteins (HSPs) play essential roles as molecular chaperones in cells to assist in the repair of degenerated proteins. The expression of HSPs in polymorphonuclear leukocytes (PMNLs) following insult has not been delineated. The objective of this study was to clarify the serial changes in HSP expression in PMNLs from trauma patients.nnnMETHODSnFifty severely injured patients (mean Injury Severity Score of 31.8 +/- 10.8) and 17 healthy volunteers were included as study subjects. Blood samples were serially obtained at three time points: days 0 to 1, days 2 to 5, and days 6 to 14 after the trauma event. We measured expressions of HSP27, HSP60, HSP70, and HSP90 in permeabilized PMNLs by flow cytometry using a monoclonal antibody generated against each HSP and fluorescein isothiocyanate-conjugated antimouse immunoglobulins as secondary reagents. We also evaluated the expression of HSP70 mRNA in PMNLs by Northern blot hybridization and the expression of HSP70 in PMNLs by fluorescence microscopy.nnnRESULTSnExpressions of HSP27, HSP70, and HSP90 in PMNLs from trauma patients were significantly greater than in PMNLs from healthy volunteers in all three periods (days 0-1, days 2-5, and days 6-14). The expression of HSP60 in PMNLs from trauma patients was significantly greater than normal expression on days 2 to 5 and days 6 to 14. The values for HSP27, HSP60, and HSP70 on days 2 to 5 were significantly higher than those on days 0 to 1. The expression of HSP70 mRNA in PMNLs was significantly enhanced for as long as 2 weeks after trauma compared with that in normal volunteers.nnnCONCLUSIONnSevere trauma causes demonstrated enhanced expression of HSPs in PMNLs during the acute phase. This enhanced expression of HSPs may regulate PMNL functions.

Difference in the responses after administration of granulocyte colony-stimulating factor in septic patients with relative neutropenia.

OBJECTIVEnThe objective of this study was to classify the clinical responses after administration of granulocyte colony-stimulating factor (G-CSF) in septic patients with relative neutropenia.nnnPATIENTS AND METHODSnWe administered recombinant human G-CSF (2 microg/kg) subcutaneously once a day for 5 days to 30 septic patients with white cell counts below 5,000 cells/mm3. Absolute neutrophil count (ANC), neutrophil differentiation, and serum concentration of G-CSF were determined serially. Bone marrow also was analyzed before and after treatment.nnnRESULTSnNeutrophil responses to G-CSF varied from good (ANC > 10,000/mm3, group G, n = 20) to moderate (ANC < 10,000/mm3, group M, n = 5) to poor (no increase in ANC, group P, n = 5). Before G-CSF administration, the three groups showed no differences in ANC but did show significant differences in serum concentration of G-CSF. G-CSF concentration was 0.16 +/- 0.03 ng/mL in group G, 7.0 +/- 3.0 ng/mL in group M, and 270 +/- 90 ng/mL in group P. Immature neutrophils accounted for 35.0 +/- 3.7% of peripheral leukocytes in group P but only 5.1 +/- 0.6% in group G. Although bone marrow was depressed in all groups before G-CSF treatment, nucleated cell count increased significantly after rhG-CSF treatment in groups G and M. Survival rate after 4 weeks was 90% in group G and 100% in group M; no patient in group P survived.nnnCONCLUSIONnG-CSF administration was effective in septic patients with a low percentage of immature neutrophils and insufficient endogenous G-CSF. It had little effect on patients with a high percentage of immature neutrophils whose G-CSF production was up-regulated and whose bone marrow was severely depressed.

Granulocyte colony-stimulating factor (G-CSF) stiffens leukocytes but attenuates inflammatory response without lung injury in septic patients.

OBJECTIVEnTo determine whether granulocyte colony-stimulating factor (G-CSF) administration changes leukocyte deformability resulting in lung injury in patients with sepsis.nnnMETHODSnTwenty-five consecutive septic patients were divided randomly into two groups. Twelve patients were given recombinant human G-CSF subcutaneously at 2 microg/kg once a day for 5 days (group G). The remaining 13 patients were given sterilized saline as placebo (group N). Leukocyte count; concentrations of C-reactive protein (CRP) and thrombomodulin (TM); respiratory index (RI) and lung injury score (LIS); and APACHE II score and Goris MOF index were determined before and after G-CSF or placebo administration. Leukocyte deformability was observed in a microchannel array etched on a single-crystal silicon tip, which simulates the microvasculature. The number of microchannels obstructed (NOM) by stiffened leukocytes was counted. Transit time (TT), that is, the time taken for 100 microL of whole blood to pass through the microchannel, was determined.nnnRESULTSnG-CSF administration significantly increased leukocyte count and decreased CRP concentration. In group G, both NOM and TT increased significantly 5 days after G-CSF administration; they did not change in group N. However, RI, LIS, and TM did not change, suggesting that no patient developed lung injury.nnnCONCLUSIONnG-CSF causes leukocyte stiffness but attenuates inflammatory response without inducing lung injury in septic patients.

Smoke Inhalation Injury and the Effect of Carbon Monoxide in the Sheep Model

The role of carbon monoxide (CO) in causing the physiologic and anatomic changes characteristic of smoke inhalation injury was evaluated in 34 sheep. The smoke-exposed group received a dose of smoke known to produce mild inhalation injury. The CO group received a pure gas mixture that contained concentrations of oxygen, carbon dioxide, and CO similar to those in the smoke. Cardiopulmonary function was measured immediately after exposure, and 24 and 72 hours after exposure. The CO group showed a transient increase in cardiac output, but the smoke group showed no such response. The CO group maintained normal PaO2 levels during the 72-hour study period; the smoke group gradually developed hypoxemia. The lungs of the CO exposed animals had no discernible histologic changes; lungs of the smoke group showed progressive inflammatory changes. These results indicate that CO per se is not the primary etiologic agent of smoke inhalation injury.