Cytokine induction by pyrogens: comparison of whole blood, mononuclear cells, and TLR-transfectants

Given the shortcomings in the measurement of pyrogenic contamination of pharmaceuticals and/or test substances by means of the rabbit pyrogen test and the Limulus amoebocyte lysate (LAL) test, several in vitro pyrogen tests have been developed based on the measurement of cytokine production by monocytes. In this study we measured cytokine production (IL-6, IL-8, IL-1beta, and TNF) in diluted whole blood (WB), mononuclear cells (MNC), and HEK cells stably transfected with CD14 and Toll-like Receptor-2 (TLR2) or TLR4, after stimulation with both standard pyrogens and contaminated substances. Our study demonstrated that in MNC, IL-6 production was more sensitive to pyrogen stimulation than IL-1beta and TNF production. The sensitivity of WB IL-8 production for pyrogens was comparable with that of MNC IL-6 production, but higher than WB IL-6 production. MNC IL-8 production as readout for pyrogenic stimulation was not useful due to high background IL-8 production. Surprisingly, contaminated culture media potently stimulated WB IL-8 production, but not MNC IL-6 production. Finally, the value of TLR-transfected HEK cells in the detection of pyrogenic contamination as well as the role of IL-10 in interindividual differences in cytokine production, is discussed. To summarize, the results presented herein together with literature data indicate that the measurement of WB IL-8 production may represent an advantageous alternative to the measurement of MNC IL-6 production, for the detection of pyrogenic contamination of pharmaceuticals.     

Isolation and endotoxin activities of lipopolysaccharides from cyanobacterial cultures and complex water blooms and comparison with the effects of heterotrophic bacteria and green alga

Massive cyanobacterial water blooms are serious environmental and health problems worldwide. While some cyanobacterial toxins such as peptide microcystins have been investigated extensively, other toxic components of cyanobacteria (e.g. lipopolysaccharides, LPS) are poorly understood. The present study characterized endotoxin activities of LPS isolated from (i) laboratory cyanobacterial cultures, (ii) cyanobacterial water bloom samples dominated by Microcystis sp., Planktothrix sp., Aphanizomenon sp. and Anabaena sp., (iii) heterotrophic Gram-negative bacteria Escherichia coli, Kluyvera intermedia, Pseudomonas putida and Pseudomonas fluorescens and (iv) green alga Pseudokirchneriella subcapitata. Toxicity results derived with Limulus amebocyte lysate assay (LAL-test) showed that endotoxin activities of LPS from both cyanobacteria and heterotrophic bacteria were comparable and the values were within a similar range (1 x 10(3)-1 x 10(6) Endotoxin Units, EU, per mg of isolated LPS). The highest activities among the cyanobacterial samples were observed in the Aphanizomenon sp. dominated water bloom. The results also suggest generally higher endotoxin activities in complex natural samples than in laboratory cyanobacterial cultures. Further, experiments with the eukaryotic green alga P. subcapitata demonstrated a need for careful purification of the LPS extracts prior to testing with the LAL assay as several contaminants may overestimate endotoxin activities. This study shows relatively high pyrogenicity of LPS from various cyanobacteria. Further research should focus on detailed toxicological and ecotoxicological characterization of LPS in massive cyanobacterial water blooms.     

Purified thromboplastin causes haemostatic abnormalities but not overt DIC in an experimental rabbit model

Validation of animal models of disseminated intravascular coagulation (DIC) to human DIC is crucial in order to translate findings in research models to treatment modalities for DIC in humans. ISTH classifications of overt and non-overt human DIC have proven to have a high diagnostic accuracy, and we have previously established a rabbit model of non-overt DIC based on the ISTH classification of non-overt DIC. In this rabbit model, we used purified rabbit brain thromboplastin to induce DIC and test applicability of ISTH classifications of overt human DIC.Cardiovascular and haematological parameters from rabbits, either saline-injected or administered a 2.5 mg thromboplastin/kg bolus and a 15 minutes 1.25 mg thromboplastin/kg infusion, were determined at four time points over a 90 minute period. All groups of rabbits were scored at each time point according to the ISTH classifications of overt DIC.Despite the fact that injection of purified thromboplastin resulted in decreased platelet count, increased prothrombin time, activated partial thromboplastin time, level of thrombin-antithrombin complexes and fibrin degradation products, and pulmonary micro-thrombosis, none of the rabbits were diagnosed as having overt DIC according to ISTH classification.We conclude that purified thromboplastin causes haemostatic abnormalities in the rabbit but this experimental model was not diagnosed as overt DIC.     

Recombinant factor C assay for measuring endotoxin in house dust: comparison with LAL, and (1 --> 3)-beta-D-glucans

BACKGROUND: Measurement of exposure to environmental endotoxin is frequently performed using a Limulus amebocyte lysate (LAL) based assay. Recently, a new method has become available with similar sensitivity and potentially greater specificity using recombinant Factor C (rFC) from the horseshoe crab Carcinoscorpius rotundicauda. A preliminary study was carried out to determine the comparability of LAL and rFC in measuring endotoxins in house dust for large scale epidemiologic studies. METHODS: House dust samples were collected from family rooms by vacuuming 1 m2 of the center of the room. Sixty sieved house dust samples were assayed for endotoxin by LAL (Cambrex, KQCL lysate) and rFC (Pyrogene, Cambrex) and for (1 --> 3)-beta-D-glucans by ELISA. The resistant parallel line estimation was used for data analysis of LAL and rFC. A four-parameter logistic fit with inverse prediction was used to calculate (1 --> 3)-beta-D-glucan levels of the samples. RESULTS: The spike recovery was 113.63% (95% CI = 101.69, 125.57%) for LAL and 99.69% (95% CI = 90.14, 109.24%) for rFC assays. The LAL assay gave higher endotoxin estimates compared with rFC. The LAL and rFC estimates were highly correlated (r = 0.86, P < 0.0001). The difference between LAL and rFC endotoxin estimates correlated with the LAL estimates (r = 0.51, P < 0.0001). However, the difference was not correlated with (1 --> 3)-beta-D-glucans. CONCLUSION: LAL and rFC gave comparable results, hence either assay can be used for studies of endotoxin exposure. The current study shows that (1 --> 3)-beta-D-glucan is not a major factor interfering with endotoxin measurements in house dust using a Cambrex KQCL LAL preparation.     

注射用阿奇霉素细菌内毒素检查方法的研究

考察了6批注射用阿奇霉素的细菌内毒素检查法的可行性。按《中国药典》2005年版细菌内毒素检查法。结果可见,浓度为0.2 mg.mL^-1的样品稀释液对标示灵敏度为0.25 EU.mL^-1的鲎试剂无干扰作用。本品稀释为0.2 mg.mL^-1的溶液后,可用灵敏度为0.25 EU.mL-1的鲎试剂进行检测。     

Influenza vaccination and Guillain Barre syndrome

Acute and severe Guillain Barre Syndrome (GBS) cases reported following influenza vaccine to the Vaccine Adverse Events Reporting System (VAERS) database from 1991 through 1999 were examined. Endotoxin concentrations were measured using the Limulus amebocyte lysate assay in influenza vaccines. There were a total of 382 cases of GBS reported to the VAERS database following influenza vaccination (male/female ratio, 1.2). The median onset of GBS following influenza vaccine was 12 days (interquartile range, 7 days to 21 days). There was an increased risk of acute GBS (relative risk, 4.3; 95% confidence interval, 3.0 to 6.4) and severe GBS (relative risk, 8.5; 95% confidence interval, 3.7 to 18.9) in comparison to an adult tetanus-diphtheria (Td) vaccine control group. There were maximums in the incidence of GBS following influenza vaccine that occurred approximately every third year (1993, 1996, and 1998) and statistically significant variation in the incidence of GBS among different influenza manufacturers. Influenza vaccines contained from a 125- to a 1250-fold increase in endotoxin concentrations in comparison to an adult Td vaccine control and endotoxin concentrations varied up to 10-fold among different lots and manufacturers of influenza vaccine. The biologic mechanism for GBS following influenza vaccine may involve the synergistic effects of endotoxin and vaccine-induced autoimmunity. There were minimal potential reporting biases in the data reported to the VAERS database in this study. Patients should make an informed consent decision on whether to take this optional vaccine based upon its safety and efficacy and physicians should vigilantly report GBS following influenza vaccination to the VAERS in the United States so that continued evaluation of the safety of influenza vaccine may be undertaken.     

18种氨基酸原料的细菌内毒素干扰试验的研究

采用中国药典１９９５年版收载的内毒素检查方法和参考有关文献进行试验。结果表明用鲎试剂灵敏度（λ）０．５ＥＵ／ｍｌ相对应的有效浓度进行试验,有蛋氨酸等１１种氨基酸无干扰作用。     

Bacteria and endotoxin in meconium-stained amniotic fluid at term: could intra-amniotic infection cause meconium passage?

Abstract

Background: Meconium-stained amniotic fluid (MSAF) is a common occurrence among women in spontaneous labor at term, and has been associated with adverse outcomes in both mother and neonate. MSAF is a risk factor for microbial invasion of the amniotic cavity (MIAC) and preterm birth among women with preterm labor and intact membranes. We now report the frequency of MIAC and the presence of bacterial endotoxin in the amniotic fluid of patients with MSAF at term.

Materials and methods: We conducted a cross-sectional study including women in presumed preterm labor because of uncertain dates who underwent amniocentesis, and were later determined to be at term (n?=?108). Patients were allocated into two groups: (1) MSAF (n?=?66) and (2) clear amniotic fluid (n?=?42). The presence of bacteria was determined by microbiologic techniques, and endotoxin was detected using the Limulus amebocyte lysate (LAL) gel clot assay. Statistical analyses were performed to test for normality and bivariate comparisons.

Results: Bacteria were more frequently present in patients with MSAF compared to those with clear amniotic fluid [19.6% (13/66) versus 4.7% (2/42); p?<?0.05]. The microorganisms were Gram-negative rods (n?=?7), Ureaplasma urealyticum (n?=?4), Gram-positive rods (n?=?2) and Mycoplasma hominis (n?=?1). The LAL gel clot assay was positive in 46.9% (31/66) of patients with MSAF, and in 4.7% (2/42) of those with clear amniotic fluid (p?<?0.001). After heat treatment, the frequency of a positive LAL gel clot assay remained higher in the MSAF group [18.1% (12/66) versus 2.3% (1/42), p?<?0.05]. Median amniotic fluid IL-6 concentration (ng/mL) was higher [1.3 (0.7–1.9) versus 0.6 (0.3–1.2), p?=?0.04], and median amniotic fluid glucose concentration (mg/dL) was lower [6 (0–8.9) versus 9 (7.4–12.6), p?<?0.001] in the MSAF group, than in those with clear amniotic fluid.

Conclusion: MSAF at term was associated with an increased incidence of MIAC. The index of suspicion for an infection-related process in postpartum women and their neonates should be increased in the presence of MSAF.     

显色基质法在盐酸左氧氟沙星注射液热原检测中的应用

目的 建立快速的盐酸左氧氟沙星注射液细菌内毒素定量检查法,替代凝胶法.方法 使用显色基质鲎试剂对不同批号的盐酸左氧氟沙星注射液分别进行干扰试验,考察确立盐酸左氧氟沙星注射液内毒素定量检查法.结果 表明标准曲线的线性相关系数r≥0.98,盐酸左氧氟沙星注射液在1：32稀释级别无干扰作用.结论 显色基质法与凝胶法比较,有灵敏、快速、能定量、重复性好的特点,可用于盐酸左氧氟沙星内毒素含量的测定.