Kikumi Hosotsubo

The influence of surgical stress on T cells : Enhancement of early phase lymphocyte activation

For the control of postoperative infection, it may be important to understand the possible influences of surgical stress on the host immune system.To this end, we examined how the early phase of lymphocyte activation was affected in patients after major surgery (eight patients with esophageal carcinoma and six undergoing cardiac surgery) using a flow cytometric assay based on expression of the early activation antigen, CD69. Freshly isolated T cell in preoperative and postoperative samples did not express CD69. When peripheral blood mononuclear cells were stimulated in vitro, the expression of CD69 was greatly enhanced in both CD4 and CD8 T cells, compared with the preoperative samples. The proportion of de novo CD69-expressing cells in the CD4 subset was approximately 3 times (Postoperative Day 1) and 4 times (Postoperative Days 2, 3, 5, and 7) greater than those preoperatively, whereas the proportion of de novo CD69-expressing cells in the CD8 subset was approximately 1.5 times (Postoperative Days 2 and 5) and 2 times (Postoperative Day 3) greater than those preoperatively. The proportion of CD69+ cells was significantly greater in the CD4+ subset than in the CD8+ subset during the postoperative period. Implications: Our results show that major surgical stress enhances the early phase of lymphocyte activation. The augmentation of activation was greater in CD4 (helper) T cells than in CD8 (cytotoxic) T cells. (Anesth Analg 1998;87:1431-5)

Serial changes in cellular immunity of septic patients with multiple organ-system failure.

Total lymphocyte count, lymphocyte cell-surface markers (OKT3, OKT4, OKT8, and B-1), serum complement factors (C3 and C4), immunoglobulins (IgG, IgA, and IgM), ceruloplasmin (Crl), and transferrin (Trf) were determined weekly for nine septic postoperative patients, all of whom had multiple organ-system failure. The peripheral blood total lymphocyte count, its subpopulation, T-cell subset, and proliferative responses of lymphocyte to phytohemagglutinin (PHA) and concanavalin A (Con A) decreased in all patients. OKT3 and B-l decreased progressively in the four nonsurvivors compared with the five survivors. Although immunoglobulin levels were within the normal range in both groups, they tended to increase in survivors and decrease in nonsurvivors. Serial levels of C3, C4, Crl, and Trf increased in survivors but did not change in nonsurvivors. T-cell function and antibody-producing activity diminished progressively in nonsurvivors. These changes in cellular immunity may represent another manifestation of multiple organ-system failure during sepsis.

Plasma proinflammatory and anti-inflammatory cytokine and catecholamine concentrations as predictors of neurological outcome in acute stroke patients

PurposeProinflammatory and anti-inflammatory cytokines may play a pivotal role in cerebral inflammation, which is implicated in the development of brain injury. Systemic cytokine release is mediated by the sympathetic nervous system and catecholamines. The aim of this study was to investigate which parameters, among plasma levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-α) and the levels of the catecholamines, epinephrine and norepinephrine, contribute to the clinical outcome in acute stroke patients.MethodsThirty-seven acute stroke patients (ischemic, n = 19; hemorrhagic, n = 18) were enrolled. All of them were admitted to our hospital within 8 h after stroke onset. Neurological status was evaluated by a modified National Institute of Health Stroke Scale (mNIHSS) on admission and by a modified Rankin Scale (mRS) at 1 month. An mRS score of 3 or more at 1 month was considered to indicate poor outcome. Serum samples for the cytokine and catecholamine measurements were collected on admission. Plasma levels of IL-1β, IL-6, IL-10, and TNF-α were determined by an enzyme-linked immunosorbent assay (ELISA) method and epinephrine and norepinephrine concentrations were determined by high-performance liquid chromatography with electrochemical detection (HPLC-EC).ResultsIn the ischemic stroke patients, poor outcome was noted in 9 (47%). There were no significant differences in cytokine or catecholamine concentrations between patients with poor and good outcomes, and there was no association between clinical outcome and cytokine and catecholamine concentrations. In the hemorrhagic stroke patients, poor outcome was noted in 10 (56%). IL-6 and IL-10 levels were higher in patients with poor outcome. On logistic regression analysis, higher values of IL-6 were significantly associated with clinical outcome at 1 month (odds ratio [OR], 1.25; 95% confidence interval [CI], 1.02–1.54).ConclusionIn ischemic stroke, plasma cytokines and catecholamines were not predictors of neurological outcome at 1 month. In hemorrhagic stroke, high levels of IL-6 in the early phase indicated a poor neurological outcome.

Up-regulation of Fas ligand (FasL) mRNA expression in peripheral blood mononuclear cells (PBMC) after major surgery

FasL, which is expressed mainly on activated lymphocytes, can induce apoptosis (programmed cell death) of cells which express Fas. Fas/FasL interaction is primarily beneficial in maintaining immunological and physiological homeostasis by eliminating unnecessary cells. Dysregulation of the interaction, however, leads to tissue damage. We investigated how Fas/FasL levels changed after major surgery. The major aim of this study was to elucidate the involvement of the Fas/FasL system in postoperative inflammation. The investigation involved 10 patients admitted to the intensive care unit after surgery. Although the percentage of Fas+ cells and the amount of Fas expression tended to increase, there was no significant difference between pre‐ and post‐operative samples. In contrast, the levels of FasL mRNA were dramatically up‐regulated after operation. Post‐operative C‐reactive protein (CRP) levels increased and correlated well with FasL levels (r = 0.91, P < 0.01). Lymphocyte counts decreased after operation and were inversely proportional to FasL levels (r = 0.58, P < 0.05). These results suggest that the enhanced FasL expression is likely to be related to systemic inflammatory responses induced during the perioperative period. FasL up‐regulation may be involved in the aggravation of tissue damage, including lymphocytopenia, in the early post‐operative period.

Improved high-performance liquid chromatographic determination of Amphotericin B in human serum and plasma

An improved reversed-phase high-performance liquid chromatographic procedure is described for the determination of Amphotericin B (AMB) in human serum and plasma. The procedure involves the addition of the internal standard, p-nitroaniline, to the sample (0.1 ml) followed by protein precipitation with acetonitrile. The supernatant is injected directly onto a C8 chromatographic column and eluted with an acetonitrile-aqueous 0.01 M sodium acetate buffer, pH 7.4, mobile phase. A spectrophotometric detector operated at 405 nm is used. Retention times for internal standard and AMB are 5.2 and 6.6 min, respectively. The assay standard curve is linear between 0.05-2.0 micrograms cm-3. Within- and between-run relative standard deviations (RSD) for high and low concentrations of the drug are less than 5.30%. Analytical recovery of added AMB in serum is 98.4-101.4%. Data obtained by microbiological assay correlated well (r = 0.936) with LC results. Some commonly co-administered drugs and high concentrations of bilirubin are shown not to interfere.

Determination of cyclosporin a in whole blood by high-performance liquid chromatography using automated column switching

A fully automated high-performance liquid chromatographic column-switching system is presented for the determination of cyclosporin A in whole blood. After blood proteins were precipitated with acetonitrile, the supernatant was automatically loaded on to a cyanopropyl column for initial separation, and then the fraction containing cyclosporin A was loaded on to a trimethylsilica column for final separation and quantitation. Cyclosporin A was detected by ultraviolet absorption at 205 nm. The minimum detectable concentration of cyclosporin A was 5 ng/ml in 100 microliter of blood. The coefficient of variation of the method was 1.755, 1.748 and 0.655% in whole blood when spiked at the 170, 425 and 850 ng/ml levels, respectively. One assay was completed in 15 min.

Regional blood flow in respiratory muscles during partial ventilatory assistance in rabbits

We tested the hypothesis that even partial ventilatory assistance would reduce respiratory muscle blood flow to levels similar to those found during control mechanical ventilation (CMV). Three levels of pressure support ventilation (PSV) and 2 CMV settings were compared in 10 rabbits. PSV 0, 6, and 12 cm H2O, under continuous positive airway pressure mode, were applied, and then pressure control ventilation (PCV) values of 6 (36 breaths/min) and 12 cm H2O (18 per breaths/min) were applied to each CMV setting with a muscle relaxant. Using colored microspheres, we measured regional tissue blood flow in respiratory muscles, lower extremities, kidney, and liver. Regional tissue blood flow in the diaphragm during PSV6, PCV6, and PCV12 were less than those during PSV0. During PSV12, blood flow in the crural diaphragm was more than that during PCV12 and similar to that during PSV0. Whereas the transdiaphragmatic pressure of PSV6 was –0.8 ± 1.6 cm H2O, that of PSV12 was –3.1 ± 2.4 cm H2O. Inspiratory asynchrony, arising from an ineffective triggering effort, was observed in PSV12. The ventilatory settings did not affect blood flow of the lower extremities, liver, and kidney. In conclusion, ventilatory settings affected blood flow in the diaphragm. At certain PSV settings, blood flow in the diaphragm was minimal.

Elimination of false-positive limulus amebocyte lysate tests in patients with hyperlipidemia.

Varying concentrations of lipopolysaccharide (LPS) and mannan suspensions were mixed with either saline or plasma from normal volunteers, heated to 100 degrees C for 10 min, and then subjected to the limulus amebocyte lysate test (LAL). A positive LAL in saline required minimum LPS and mannan concentrations of 10(-11) and 10(-5) g/ml, respectively, while the minimum concentrations premixed with plasma were 10(-13) and 10(-9) g/ml, respectively. Thus, use of plasma instead of saline increased assay sensitivity 100-fold for LPS and 10,000-fold for mannan. In the second part of the experiment, normal plasma was separated into lipid and nonlipid phases by Folchs method. LAL analysis of each phase revealed equal sensitivity for LPS and mannan in the nonlipid phase, but no sensitivity in the lipid extract. Subsequently, 200 ml of a fat emulsion (Intralipos) was administered to the normal volunteers, and LAL and lipid analyses were performed. The LAL turned positive in all volunteers. When LAL was positive, triglycerides (TG), chylomicron (Chyl), and very low-density lipoprotein (VLDL) increased significantly compared with when LAL was negative. It is concluded that plasma lipids increase the sensitivity of LAL and directly activate LAL when TG, Chyl, and VLDL concentrations are high. This effect of plasma lipids on LAL can be eliminated by extracting LPS and mannan in the nonlipid phase.

Plasma neutrophil gelatinase-associated lipocalin clearance during venovenous hemodiafiltration

To the Editor Plasma neutrophil gelatinase-associated lipocalin (NGAL) levels are predictors of acute kidney injury (AKI) severity [1, 2]. NGAL may also limit kidney injury [3]. Hemodiafiltration (HDF) may affect plasma NGAL levels because NGAL is probably filtered and absorbed by the filter membrane. We investigated the effect of HDF on plasma NGAL levels in eight AKI patients undergoing intermittent HDF (Osaka University Hospital Ethics Committee No. 9024). HDF by the post-dilution method was performed using an EXCELFLO AEF-10 filter (Asahi Kasei Kuraray Medical Co., Ltd., Tokyo, Japan). The blood flow rate was 80 mL/min. Plasma NGAL levels were measured using the CircuLex NGAL/Lipocalin-2 ELISA Kit (CycLex Co., Ltd. Ina, Japan) before, during, and after HDF. NGAL clearance (ClNGAL) was determined as ClNGAL = (Cpre Cpost)/Cpre 9 80 (mL/min) and the sieving coefficient (SC) as SC = 2 9 Cfil/(Cpre ? Cpost), where Cfil is the concentration in filtered fluid and Cpre and Cpost are the concentrations in preand post-filtered plasma, respectively. The interval between initial HDF and the study was 21.4 ± 24.6 days. Within this period, continuous HDF was applied for 10.1 ± 11.8 days. The total flow rate of the replacement and dialysis fluids was 21.8 ± 2.3 mL/kg/h. Plasma NGAL levels decreased significantly to 83.6 ± 5.6% (percentage of NGAL level at HDF initiation) at the end of HDF (P \ 0.001, repeated measures ANOVA) (Fig. 1). Discontinuation of HDF increased plasma NGAL levels which returned to 90.8 ± 6.3% at 17.5 ± 4.7 h.