Clinical relevance of leukocyte-associated endotoxins measured by semi-automatic synthetic luminescent substrate method.

A newly developed semi-automatic synthetic luminescence substrate (SALS) method for measuring endotoxin was compared with the existing turbidimetric kinetic assay (TKA) using leukocyte-rich plasma to verify its usefulness. As a result, the endotoxin levels by this method were higher than that by the existing assay in most specimens, and the time required for measurement was much shorter. In addition, the leukocyte-rich plasma endotoxin level minus the plasma endotoxin levels were named leukocyte-associated endotoxin, and these levels per one leukocyte were compared. As a result, those levels were highly correlated with the endotoxin measurement levels of leukocyte-rich plasma. The correlation coefficient of SALS method was superior to the existing TKA method, the endotoxin level by this method may be close to true endotoxin levels.

Basic Verification of ß-D Glucan in Leukocyte-Rich Plasma for the Diagnosis of Deep Mycosis.

BACKGROUND: Currently, supplementary serological testing for ß-D glucan (BDG) is often selected to diagnose deep mycosis in care covered by the health insurance in Japan. The Wako method used by our center has low sensitivity, and different studies have used different cut-off values due to factors that cause false positives and false negatives. One possible cause of false negatives is the use of platelet-rich plasma (PRP) as the sample material. Because phagocytic white blood cells (WBC) are precipitated by centrifugation and only plasma is measured, it seems unlikely that the actual amount of BDG is being measured when using PRP. Further, a frequent cause of false positives is contamination from blood products and gauze containing BDG. To resolve these issues, the blood cell separator, hydroxyethyl starch, is used to precipitate only the red blood cells to obtain leukocyte-rich plasma (LRP). We hypothesized that it might be possible to improve the diagnostic rate of deep mycosis by measuring the BDG content of plasma containing WBC and fungal components and by comparing the BDG content of PRP and LRP measured simultaneously. MATERIALS AND METHODS: Healthy human blood, albumin-added blood, wrung-out gauze fluid-added blood, and fungal solution-added blood were prepared, and PRP and LRP were prepared using hydroxyethyl starch. The BDG content of each sample was measured using the Wako method and compared. In addition, PRP and LRP of fungal-added blood were Gram-stained and examined under a microscope, and the number of WBCs and phagocytosed fungi was counted visually and compared. RESULTS: Measuring the BDG content of LRP confirmed that there were no false positives with LRP, and in vitro experiments comparing albumin-added false-positive blood to fungal-added blood showed significant differences between PRP and LRP only in the fungal-added blood. CONCLUSION: Calculating the BDG-ratio (LRP/PRP) by measuring both LRP and PRP may eliminate false positives and false negatives of true deep mycosis and improve the diagnostic rate.

Endotoxin contamination of single-use sterile surgical gloves.

Aim: Strict endotoxin limits are enforced for implants and catheters inserted into the body. However, no standard limit has been set for single-use sterile surgical gloves. Materials & methods: Four types of gloves sold in Japan were dipped in saline and that endotoxin levels were measured. Cytokine producing activity of gloves in blood was also measured. Results: Three of the four types of gloves showed endotoxin contamination. We also confirmed an increase in cytokine production in these gloves except one glove in which anionic surfactants was found. Conclusion: The extent to which detected endotoxins enter the body during surgery is controversial, but strict endotoxin limits need to be established.

A dextran-based warming method for preparing leukocyte-rich plasma and its clinical application for endotoxin assay.

We devised a method using dextran for obtaining leukocyte-rich plasma (LRP) to measure endotoxin in blood. In order to find the optimal temperature for obtaining LRP, the measurement results were examined using samples prepared at 37 and 0°C. Sample separation time of LRP was significantly shorter at 37°C than at 0°C. Endotoxin measurement values showed a strong correlation between the two groups but many of the LRPs made at 37°C had measurements above those of the LRPs prepared at 0°C. The diagnostic accuracy for gram-negative bacterial infection was superior for LRP prepared at 37°C, with sensitivity and specificity of 96.8 and 100%, respectively.

[Effect of Human Serum Albumin on Endotoxin Scattering Photometry].

PURPOSE: Laser scattering photometry (ESP) is a newly developed plasma endotoxin assay method using horseshoe crab amebocyte lysate (AL) that recognizes small particles produced by polymerization of coagulin under the stirring conditions at 1000rpm. We elucidated the effect of human serum album (HSA) in the ESP method. METHODS: AL was dissolved with 630µL of the specimen and a 200-µL aliquot was used for ESP; this conventional protocol was regarded as the ESP630 method. The ESP210 method was also used, i. e. AL was dissolved with 210µL of the specimen and a 200-µL aliquot was used for ESP. RESULTS: Water induced the agglutination, and HSA prolonged the agglutination time depending on its concentration especially in the ESP630 method. The water-induced agglutination was not inhibited by the addition of anti-factor C monoclonal antibody, and amidinophenyl benzoate hydrochloride, used as a clotting enzyme inhibitor, intensively inhibited the water-induced agglutination. Therefore, the water-induced agglutination was suggested to be a false-positive reaction to non-specific activation of the clotting enzyme. The HSA-induced prolongation of the reaction in the national health insurance-covered turbidimetric kinetic assay was not observed. CONCLUSION: HSA or plasma protein seemed to affect the result, especially in the ESP630 method, and a non-specific reaction was found to occur in the ESP methods.

Interleukin-8 induces an elevation in the endotoxin activity assay (EAA) level: does the EAA truly measure the endotoxin level?

The endotoxin activity assay (EAA) is a FDA-approved blood endotoxin assay that is reported as a useful tool for the diagnosis of gram-negative bacterial infection. However, discrepancies between the results of the EAA and those of the limulus amebocyte lysate (LAL) assay have been reported. Thus, we verified these methods. Blood was incubated with anti-endotoxin antibody, the resultant polymorphonuclear activation to produce oxidants was measured and the EAA level calculated. As a reference endotoxin assay, we used an endotoxin-specific LAL assay. Significant increases in plasma LAL assay levels were observed only in patients with sepsis caused by gram-negative bacterial infections, whereas higher EAA levels were observed in almost all the sepsis cases and the SIRS cases, especially those with acute pancreatitis. Graded amounts of LPS (1-10,000 pg/ml) were spiked into normal blood to obtain dose-response curves: a good dose-response curve, from 1 to 1,000 pg/ml, was obtained for the LAL assay. A good dose-response curve was barely obtained for the EAA; the lowest detection limit seemed to be 1,000 pg/ml. Addition of methylprednisolone decreased the EAA levels. Interleukin-8 (IL-8) induced elevation in EAA levels when IL-8 was added to volunteers' blood samples. Overall, the EAA kit could not measure clinically relevant doses of endotoxin. Because IL-8 induced an increase in EAA level, it is suggested that the EAA level reflects the primed state of polymorphonuclear leukocytes.

Evaluation of an endotoxin-specific limulus amebocyte lysate assay using leukocyte-rich plasma for the diagnosis of gram-negative bacterial infection.

We focused our attention on the endotoxin present within and on the surface of white blood cells and attempted to establish a new sample preparation method for endotoxin assays in leukocyte-rich plasma (LRP), taking advantage of the erythrocyte-aggregating property of hydroxyethyl starch. We used an endotoxin-specific turbidimetric kinetic assay, which is the conventional method used to assay endotoxin levels in platelet-rich plasma (PRP). Then, we comparatively assessed the assay results obtained with the endotoxin assay using PRP and LRP. It was found that the sensitivity of endotoxin assay in LRP was 88.5 %, which was superior to 73.1 % of the sensitivity in PRP in the diagnosis of infections caused by gram-negative bacteria. These results suggest that our newly developed LRP endotoxin assay may contribute to an improvement in the rate of sepsis diagnosis.

Experimental application of a synthetic luminescent substrate assay using endotoxin-specific limulus amebocyte lysate to human blood.

A synthetic luminescent substrate method, using a mutant-type luciferase whose luminescence intensity is more than ten times as intense as the wild type, was developed recently. We conducted the first basic studies on clinical application of the novel endotoxin measurement method. We assessed and established measurement conditions, including reagent concentrations and reaction time, so that it would be possible to apply the luminescent synthetic substrate method proposed by Noda et al. to measurements in human blood. When we added lipopolysaccharide (LPS) to water, it was possible to measure LPS at a concentration of 0.1 pg/ml, whereas it was possible to measure LPS in tenfold diluted and heated plasma at a concentration of 1 pg/ml. When plasma was further diluted, inhibiting activity decreased considerably. Thus, it will be necessary to completely eliminate the inhibitor present in plasma. However, the shortest time after collecting the specimen in which it was possible to make measurements was 30-40 min, suggesting that if an assay is established, it will be possible to use the method as a novel blood endotoxin assay.

[Study on the leukocyte-rich plasma as the specimen for the measurement of endotoxin].

PURPOSE: In the diagnosis of sepsis, the sensitivity of the endotoxin assay using platelet-rich plasma (PRP) is not as high as expected. In the endotoxin assay using PRP, endotoxin occurring within and on the surface of white blood cells cannot be measured. Thus, we devised a method of preparing leukocyte-rich plasma (LRP) using hydroxyethyl starch (HES) in order to improve the endotoxin assay. METHODS: The endotoxin concentration was measured by an endotoxin-specific Limulus amebocyte lysate assay method: turbidimetric kinetic assay. RESULTS: Only 1% of erythrocytes were recovered in LRP without the hemolysis of erythrocytes. Endotoxin contents in PRP obtained from blood incubated for 0, 30, 60, 120, and 180 min at 37 degrees C in the presence of lipopolysaccharide (50 pg/ml in a final concentration) were 1.18, 1.23, 1.23, 1.39, and 1.25 times higher (mean value from three bloods) than that in PRP, respectively. CONCLUSION: Thus, the superiority of the sensitivity of the LRP-based endotoxin assay over the PRP-based assay was identified. Our newly devised assay method may contribute to improvement of the diagnosis rate of endotoxemia in sepsis.

Suppressive effects of sivelestat on interleukin 8 and TNF-α production from LPS-stimulated granulocytes in whole blood culture.

PURPOSE: The goal of the study was to examine the effects of sivelestat sodium hydrate (sivelestat), a neutrophil elastase inhibitor, on production of cytokines in granulocytes and monocytes, using flow cytometry after cytokine staining in whole blood culture. METHODS: Blood samples were collected from healthy volunteers. Vehicle (control group), lipopolysaccharide (LPS) (LPS group), or LPS + sivelestat (sivelestat group) were added to the whole blood, followed by addition of a protein transport inhibitor in each group. After incubation, staining for cytokines retained in the cells was performed by addition of an anti-interleukin 8 (IL-8) or anti-tumor necrosis factor-α (TNF-α) antibody. The cells were then analyzed using flow cytometry. RESULTS: Granulocytic production of IL-8 induced by 1 ng/ml LPS was significantly (P < 0.05) inhibited by treatment with 1 µg/ml sivelestat, and upregulation of IL-8 by 10 ng/ml LPS was also significantly (P < 0.05) suppressed by 1 and 10 µg/ml sivelestat. Addition of 10 or 100 µg/ml sivelestat significantly (P < 0.05) inhibited the production of TNF-α from granulocytes induced by 10 ng/ml LPS. Sivelestat did not significantly inhibit LPS-induced monocytic production of TNF-α and IL-8. CONCLUSION: Suppression of granulocytic production of IL-8 and TNF-α by sivelestat suggests that this drug may be useful for treatment of morbid conditions involving IL-8 and TNF-α at onset.

Effects of sevoflurane on cytokine balance in patients undergoing coronary artery bypass graft surgery.

OBJECTIVE: The effects of sevoflurane on proinflammatory cytokines related to ischemic-reperfusion injury are not clear. The hypothesis was tested that sevoflurane decreases myocardial ischemic-reperfusion injury by suppressing proinflammatory cytokines. DESIGN: Prospective, randomized study. SETTING: A medical university heart center. PARTICIPANTS: Twenty-three patients undergoing coronary artery bypass surgery allocated randomly into 2 groups. INTERVENTIONS: Anesthesia for 23 patients undergoing coronary artery bypass surgery was maintained using either fentanyl (30 microg/kg) with propofol (2-8 mg/kg/h) in the control group (n = 10) or fentanyl (30 microg/kg) with 0.5% to 1.0% sevoflurane in the sevoflurane group (n = 13). MEASUREMENTS AND MAIN RESULTS: Interleukin (IL)-6, IL-8, IL-10, and IL-1 receptor antagonist (IL-1ra) were measured by enzyme-linked immunosorbent assay. Troponin-T and creatine kinase-MB isoenzyme (CK-MB) were measured by enzyme immunoassay and ultraviolet absorption spectrophotometry, respectively. Serum IL-6 and IL-8 concentrations in both groups increased significantly over baseline from 60 minutes after declamping the aorta (p < 0.001). The increases were greater in the control group than in the sevoflurane group (p < 0.05). Serum IL-10 and IL-1ra concentrations in both groups increased significantly over baseline from 60 minutes after declamping the aorta (p < 0.001). There were no differences between the two groups. Serum troponin-T and CK-MB concentrations increased significantly in both groups from 60 minutes after declamping the aorta (p < 0.001); the increases were greater in the control group (p < 0.05). CONCLUSION: Sevoflurane suppressed the production of IL-6 and IL-8, but not IL-10 and IL-1ra. Changes in the balance between pro- and anti-inflammatory cytokines may be one of the most important mechanisms of myocardial protection caused by sevoflurane.

Nicorandil attenuates NF-kappaB activation, adhesion molecule expression, and cytokine production in patients with coronary artery bypass surgery.

Nicorandil (NCR), a KATP channel opener, has been reported to preserve microvascular integrity in patients with reperfused myocardial infarction. We tested the hypothesis that NCR suppresses myocardial ischemia and reperfusion injury via the attenuation of cytokine production. Forty patients who underwent coronary artery bypass graft surgery were studied. The patients were randomly divided into two groups, i.e., the patients with NCR (4-6 mg/h; N group, n = 20) or without NCR (C group, n = 20). Cardiac surgery was performed under anesthesia using fentanyl and propofol. Blood were sampled at the time of induction of anesthesia, pre-cardiopulmonary bypass, 60 min after aortic occlusion, and 60, 120, and 180 min after declamping the aorta. The activation of NF-kappaB, expression of adhesion molecules, and cytokine production were evaluated in blood samples from the control volunteers by flow cytometric analysis with or without lipopolysaccharide (LPS) stimulation in vitro. Serum IL-6 and IL-8 levels in both groups increased 60 min after declamping the aorta compared with the preoperative value (P < 0.001); the increases of these parameters in N group were lower than those in C group (P < 0.05). Serum creatine kinase with muscle and brain subunits and troponin-T levels increased 60 min after declamping the aorta in two groups (P < 0,001), but the increases of both parameters in N group were lower than those in C group (P < 0.05). NF-kappaB activation, CD11b/CD18 expression, and the production of TNF-alpha, IL-8, and IL-6 in monocytes and granulocytes were inhibited by NCR in vitro. NCR suppressed the increase of inflammatory cytokines such as IL-6 and IL-8 levels, and reduced myocardial reperfusion injury. The inhibition on NF-kappaB activation, adhesion molecule expression, and cytokine production may be one of the important mechanisms of myocardial protection of NCR.

Homologous and heterologous antibody responses to lipopolysaccharide after enterohemorrhagic Escherichia coli infection.

To evaluate antibody responses against lipopolysaccharide (LPS: O157, O26, and O111) in enterohemorrhagic Escherichia coli(EHEC) infection, sera of 24 schoolchildren associated with the Morioka outbreak in 1997 and of 74 sporadic patients suspected of having EHEC infection were examined. Using a positive standard serum, quantitative evaluation of LPS antibodies by an enzyme-linked immunosorbent assay (ELISA) was established. High levels of specific IgM and IgA antibodies against homologous E. coli LPS were present in the acute period and are characteristic of EHEC. This could be used for the serological diagnosis of EHEC infection, except for early infants and the elderly. In addition to the specific homologous response, multiple antibody responses against different serotypes other than those isolated were demonstrated in many cases by qualitative analysis using Western blotting.

The correlation between thrombopoietin and platelet count in adult dengue viral infection patients.

AIM: To investigate alteration in humoral regulation during the course of dengue viral infection. METHODS: A prospective analytic study had been conducted involving 40 subjects with dengue viral infection. Subjects were recruited according to consecutive non-probability sampling. Subjects were categorized according to days of illness, platelet counts and serum thrombopoietin (TPO) levels. The plasma TPO levels examinations were done once daily until the platelet counts reached more than 100,000/mm(3). RESULTS: Statistical analysis showed the mean serum TPO levels were increased during thrombocytopenia phase of the disease, and differ significantly from the convalescent phase (mean value 428 pg/ml vs 220.1 pg/ml, p= 0.00). There was also a statistically significant inverse correlation between serum TPO levels and platelet counts (p= 0.00). CONCLUSION: TPO levels were significantly increased in adult patients with dengue infection in which platelets in circulation were markedly reduced, and the TPO levels were inversely related to the platelet counts.

Endotoxin in patients with terminal renal failure undergoing dialysis with re-processing dialyser.

AIM: to determine the level of endotoxin in the blood of patients with renal failure prior to and following hemodialysis using re-processing dialyser to know possibility of pyrogenic reactions in hemodialysis patients. METHODS: this study subjects consisted of 10 patients with terminal renal failure undergoing regular hemodialysis. The collected samples were then sent in frozen condition for endotoxin examination in Japan. The normal level of endotoxin in the blood was < 9.8 pg/ml based on standard E.Coli E.0111 endotoxin quantitatively measured using Limulus Amoebocyte lysate test (the endospecy test). Statistical analysis was performed using paired student test. RESULTS: Ten patients with terminal renal failure who were undergoing hemodialysis were obtained, consisting of 1 female and 9 males. The mean age was 55.5 years (SD 6.74), the mean hemoglobin level 7.26 g/dl (SD 2.19), mean white blood cell (WBC) count 8660/mm(3) (SD 3064.2), and mean albumin level 3.59 g/l (SD 247). The etiologies of renal failure were as follows: glomerulonephritis (GN) 30%, Diabetic nephropathy (DN) 20%, hypertension (HT) 10%, interstitial nephritis (IN) 10%, obstruction/infection (01) 10%, unknown (U) 10%. The mean duration of hemodialysis was 97.9 month (SD 54.86). The mean endotoxin level prior to hemodialysis (ET pre-hemodialysis) was 5.4 pg/dl (SD 8). CONCLUSION: we conclude that terminal renal patients who undergoing re-processing hemodialysis did not have endotoxemia both prior to and following hemodialysis unless if they associated with infection, or other complications.

A silkworm larvae plasma test for detecting peptidoglycan in cerebrospinal fluid is useful for the diagnosis of bacterial meningitis.

The silkworm larvae plasma (SLP) test has been established based on a cascade reaction triggered by either peptidoglycan or (1, 3)-beta-D-glucan to produce melanin. We applied this test to the diagnosis of bacterial meningitis. Cerebrospinal fluid (CSF) obtained from patients with bacterial meningitis due to gram-positive bacteria, gram-negative bacteria, or fungi, showed positive reactions to the test. In contrast, CSF from patients with viral meningitis or noninfectious illnesses gave negative reactions. Therefore, this test seems to be useful for diagnosis of bacterial and fungal meningitis. When this test was used together with two types of limulus tests, an endotoxin-specific test, and a conventional test, meningitis was further characterized as gram-positive, gram-negative or fungal meningitis. The SLP test requires a computerized instrument for quantitative colorimetric measurement. A qualitative alternative of this test also can be accomplished by visually observing the darkening color. Thus, this method can be applied for simple and rapid diagnosis of meningitis.

Examination of soluble Fas (sFas) and soluble Fas ligand (sFasL) in patients with burns.

The FasL-Fas system is one of the recognized apoptosis-inducing systems, and has been determined to have important functions in relation to homeostasis and biological defense mechanisms. In this study, we investigated the serum levels of soluble Fas (sFas), soluble FasL (sFasL) and tumor necrosis factor alpha (TNF-alpha) in patients with burns. The sFas levels were found to be significantly higher in the patients who eventually died as compared to those in the patients who survived (3.9+/-1.8ng/ml versus 2.6+/-1.0ng/ml). On the other hand, the sFasL levels were significantly higher in the patients who survived (61.5+/-29.9ng/ml versus 37.2+/-14.4ng/ml) than in those who eventually died. A positive correlation was noted between the TNF-alpha level and the sFas level, and a negative correlation was observed between the TNF-alpha level and the sFasL level. These findings suggest that worsening of the condition of a burns patient may be related to changes in the Fas-FasL system.

Matrix metalloproteinase-1 and cytokines in patients with acute pancreatitis.

INTRODUCTION: Hypercytokinemia is known to occur in severe acute pancreatitis, suggesting that the production, deposition, and degradation of the extracellular matrix (ECM) occur actively as a result of the actions of the complicated cytokine network. AIMS AND METHODOLOGY: To examine the influence of cytokines and growth factors on the ECM in acute pancreatitis from the points of view of the severity of the disease, the complication of multiple organ dysfunction syndrome (MODS), and the prognosis, 25 patients with acute pancreatitis were divided into three groups according to the severity of the condition as assessed by the Ranson score. The serum levels of matrix metalloproteinase (MMP)-1, tissue inhibitor of metalloproteinases (TIMP)-1, the MMP-1.TIMP-1 complex, tumor necrosis factor (TNF)-alpha, and transfer growth factor (TGF)-beta1 were determined by enzyme-linked immunosorbent assay. RESULTS: Comparison of the three groups divided according to the severity of the disease revealed significant differences in the levels of MMP-1 and TNF-alpha among the three groups, with the levels being higher in patients with more severe disease. The TIMP-1/MMP-1 ratio and the TGF-beta1 levels were found to be significantly lower in patients with more severe disease. Comparison between the group with the complication of MODS and the group without the complication showed that the levels of MMP-1, the MMP-1.TIMP-1 complex, and TNF-alpha were significantly higher in the former group than in the latter group. The TIMP-1/MMP-1 ratio and the TGF-beta1 levels were significantly lower in the group with the complication of MODS than in the group without the complication. Comparison between the nonsurvivor group and the survivor group revealed significantly higher levels of MMP-1, TIMP-1, and TNF-alpha in the nonsurvivors than in the survivors. The TIMP-1/MMP-1 ratio and the TGF-beta1 levels were significantly higher in the survivors than in the nonsurvivors. A significant correlation was observed between MMP-1 levels and TNF-alpha levels. On the other hand, a significant negative correlation was noted between MMP-1 levels and TGF-beta1 levels. CONCLUSION: The results of our study suggest that the activity of the ECM catabolic enzyme MMP-1 and cytokines are related to the development of acute pancreatitis.

Surfactant protein A and D (SP-A, AP-D) levels in patients with septic ARDS.

The levels of surfactant protein-A (SP-A) and surfactant protein-D (SP-D) in the serum of patients with septic acute respiratory distress syndrome (ARDS) were determined. Patients with sepsis without ARDS were examined as controls. The mean serum SP-A level in the former group was 37.6 +/- 16.2 ng/ml and in the latter group was 31.8 +/- 9.6 ng/ml. The difference between the two groups was not statistically significant. The maximum serum SP-D level was 83.0 +/- 33.9 ng/ml in the control group of patients without ARDS, and 476.3 +/- 391.2 ng/ml in the patients with ARDS. The level in the latter population was significantly higher than that in the former population. No significant correlation was observed between the SP-A and SP-D levels. These results suggest that the serum SP-D levels may serve as a good diagnostic indicator of ARDS in patients with sepsis.