Endospores of B subtilis are pyrogenic and activate Mono Mac 6 cells: importance of the CD14 receptor.

The monocytic cell line Mono Mac 6 is sensitive to pyrogens and interleukin-6 secretion is induced after exposure to pyrogens. The aim of this study is to examine the pyrogenic activity and the interleukin-6-inducing capacity of the Gram-positive B. subtilis bacteria, endospores and isolated cell wall components. Furthermore the involvement of CD14 in activation of interleukin-6 release is investigated. All test substances are pyrogenic in the rabbit pyrogen test. The test substance is incubated with monocytic cells (Mono Mac 6) for 24 h and the secreted interleukin-6 is determined in a sandwich immunoassay. B. subtilis bacteria and endospores induce interleukin-6 in a dose-dependent manner. Endospores are less potent than bacteria. Lipoteichoic acid (LTA) isolated from B. subtilis induces interleukin-6 in a dose-dependent manner, whereas muramyl dipeptide (MDP) is unable to induce interleukin-6. Lipopolysaccharides (LPS) dose-dependently induce interleukin-6 release, but the curve differs from that of LTA both in shape and offset. The interleukin-6 secretion induced by LPS, LTA and B. subtilis bacteria can be blocked by 73-85% by an antibody directed against CD14, whereas the antibody only blocks 25% of B. subtilis endospores-induced interleukin-6 release. The results might indicate that B. subtilis endospores use an additional pathway to CD14 to activate mononuclear cells.     

False positive and false negative results in testing for pyrogenic impurities]

Causes of the occurrence of pyrogenic impurities in injection and infusion preparations as well as false-positive and false-negative results possible in the pyrogen test are reported. Possibilities of excluding pyrogenic impurities in the preparations concerned and of avoiding false-positive and false-negative results in the pyrogen test are discussed.     

The effects and properties of sodium nucleinate as a pyrogen working-standard. 9. Pyrogen detection with epinephrine-skin-, dactinomycin- and LAL-tests. The suitability of sodium nucleinate as a pyrogen standard]

Sodium nucleinate (NN) as well as bacterial lipopolysaccharide (LPS) can be detected by epinephrine-skin, dactinomycin and LAL tests. In the quantitative determination of two pyrogen standards for rabbit tests, consisting of NN, a smaller value was found by LAL test for the standard of greatest pyrogenic effect than for that less pyrogenically effective in rabbits. A standard consisting of NN can be used for the pyrogen test in rabbits. But in the future, if necessary a standard consisting of endotoxin will be used, due to its better comparability of results obtained by LAL and rabbit tests.     

TNF alpha measurement in rat and human whole blood as an in vitro method to assay pyrogens and its inhibition by dexamethasone and erythromycin

To ensure the safety of potential drugs, pyrogen tests are traditionally performed in rabbits. New methods have been developed as alternatives to the test to reduce the use of experimental animals. Among these methods there are the Limulus amoebocyte lysate test and the determination of cytokine production by human leukocytes and whole blood. When exposed to a range of concentrations of endotoxins, human and rat whole blood release TNFalpha at amounts that are detectable by a commercially available enzyme-linked immunosorbent assay (ELISA). Our results show that the sensitivity of human and rat blood to endotoxins from Salmonella abortus equi and Escherichia coli is similar. In rat blood, TNFalpha was detected after contact with the pyrogens only in fresh blood, collected on the same day of incubation with the pyrogenic substances. The measurement of TNFalpha production would be a reliable alternative to the rabbit pyrogen test. However, given that the addition of erythromycin and dexamethasone inhibited the production of this cytokine, this method is limited when parenteral formulations contain these two drugs. Similar inhibition has been observed in the rabbit test. Additional experiments will be necessary to demonstrate that the rat whole blood test system is useful and reliable for the pyrogens evaluation.     

Pyrogen testing in vitro using the Limulus test]

The applicability of the Limulus test for the pyrogen test was checked in comparison to the pyrogen test in rabbits. In 6 out of 24 lots of raw materials and drugs pyrogens could be detected by means of the pyrogen test in rabbits. 2 of these 6 lots showed positive reaction in the Limulus test, there were no false positive results. Testing 7 bacterial strains in modified quantity of germs the Limulus test turned out to be more sensitive than the pyrogen test in rabbits. The application of this in vitro test as a complement to the pyrogen test in rabbits for a certain kind of problems is discussed.     

Differentiation between formation,in plasma,of anaphylatoxin and of endogenous pyrogen

Summary Two anaphylatoxin-forming agents have been investigated with respect to possible pyrogenic effects: the AT forming fraction of cobra venom and agar.The cobra venom fraction produced fever in rabbits. The pyrogenic principle is, however, not identical with the AT forming enzyme. Unlike the latter the pyrogenic principle is stable in acidic solution and destroyed by periodate. It may be a lipopolysaccharide.Rabbit plasma, incubated with agar caused fever in rabbits. Agar also induced pyrogenic activity in saline after it had been incubated in that medium. The active principle proved to be agaropectin, the water-soluble acidic fraction of agar. Agarose was inert. In contrast, anaphylatoxin formation is induced by agarose, not by agaropectin.In rabbit plasma, agaropectin induces the formation of an endogenous pyrogen. This principle can be separated from the agaropectin by DEAE cellulose chromatography. It is further distinguished from the latter by being heat-labile.Besides being activated by different agents the processes of pyrogen and AT formation differ in their requirement for cations. AT formation is blocked by EDTA but pyrogen formation is not. It is concluded that in spite of similarities and common activation by endotoxins the processes of AT and pyrogen formation are different and independent events.     

The antipyretic effect of flurbiprofen.

Flurbiprofen, like its predecessor ibuprofen, possesses antipyretic properties in the endotoxin-fevered rabbit. Comparison of flurbiprofen and ibuprofen at varying dosages, reveals that flurbiprofen is at least 15 times more potent in this species. In goats, flurbiprofen is more potent than in rabbits. Flurbiprofen inhibited the pyrogenic effect of intravenously administered leucocytic pyrogen, but it did not alter the release of leucocytic pyrogen from peritoneal exudate cells in vitro. Moreover, flurbiprofen did not inhibit endotoxin-induced release of endogenous pyrogen in vivo. Incubation of leucocytic pyrogen with flurbiprofen did not alter its pyrogenic poteny. These results suggest that the antipyretic activity of flurbiprofen is due neither to interference with endogenous pyrogen synthesis and release, nor to inactivation of circulating endogenous pyrogen.     

Uridine-induced hyperthermia in the rabbit

Uridine injection in 0.6% saline elevated rabbit temperatures (mean = 0.9 °C) in the USP XX pyrogen test. Hyperthermia was delayed in onset and peaking 3–4 h post injection, but the injection was negative in the limulus amoebocyte lysate (LAL) assay. Uridine from five lots of different sources exceeded USP XX limits in the rabbit pyrogen test and proved negative in the LAL assay. Because the dose of uridine was high, several procedures were used to determine if an impurity was the cause of temperature elevation. Uridine remained pyrogenic in spite of ultrafiltration (10 000 nominal mol. wt), recrystallization and preparative scale HPLC. Sterile filtration and autoclaving also did not affect the response. Hyperthermia, therefore, appears to be an inherent property of uridine. Uridine was also found to release endogenous pyrogen in-vitro from human mononuclear cells. Uridine has been reported to induce fever in man, thus the USP rabbit pyrogen test predicted for the clinical response.     

先锋必(注射用头孢哌酮钠)热原检查法和细菌内毒素检查法的比较性研究

本文阐述了应用热原检查法和细菌内毒素检査法同时对120批先锋必(注射用头孢哌酮钠)样品进行检查,并设立多项对比试验,以确定细菌内毒素检查法取代先锋必热原检查的可行性。结果发现,120批先锋必样品经热原检查和细菌内毒素检查均合格,样品内毒素含量远低于美国药典限值。通过测定加有一定量细菌内毒素的先锋必样品组及对照组的热原反应和鲎试验反应发现,在细菌内毒素浓度为10EU/Mし时,対照组可引发很强的热原反应,而先锋必样品(100MG/ML/KG)组所引发的热原反应明显减弱。说明先锋必在高浓度对热原反应有明显抑制作用。同样,本试验也发现,先锋必在较髙浓度对鲎试验也有明显抑制作用,其最高非抑制浓度为2.5MG/ML。对先锋必内毒素限值测定結果发现,按中国药典兔剂量注射,先锋必内毒素含量达0.1EU/MG时即可引发热原反应,而在0.2EU/MG时热原反应明显。本研究证实,细菌内毒素检査法可以代替热原检查法用于先锋必(注射用头孢哌酮钠)检查,中国药典90版规定的注射用头孢哌酮钠热原检查的兔剂M偏高,对热原反应有抑制作用。     

热原实验计算机管理系统开发与应用

根据《中华人民共和国药典（2020年版）》四部通则1142热原检查法,梳理热原实验全过程的实验流程,并结合实际经验,开发以热原实验过程为主线、家兔状态为辅线的信息管理系统,从而实现了热原实验中家兔全生命周期管理、热原实验全过程及结果可追溯、家兔状态管理的可视化管理。     

Liver function and pyrexia caused by a pyrogen from Escherichia coli, lysergic acid diethylamide and dinitrophenol

Rabbits with liver damaged by carbon tetrachloride do not respond with hyperthermia to a lipopolysaccharide pyrogen from Escherichia coli or dinitrophenol, but lysergic acid diethylamide develops its usual effect. Rabbits with obstructive liver damage react with hyperthermia to all three pyrogenic factors. The results support the concept of a peripheral action of pyrogen. It is suggested that the liver plays a rôle in the process of transforming the bacterial pyrogen into the endogenous pyrogen.     

HL60-IL6法在注射用胶原酶和尿激酶热原检测中的应用研究

目的探讨HL60-IL6单核细胞活化反应法在注射用胶原酶和尿激酶热原检测中的应用。方法参照中国药典2015年版通则1193,确定注射用胶原酶和尿激酶最小有效稀释浓度。通过细胞增殖抑制试验、热原干扰试验和白介素-6(interleukin-6,IL-6)干扰试验探讨对HL60细胞生长,对热原和IL-6测定无影响的供试品浓度,进而确定适用于注射用胶原酶和尿激酶体外热原测定的HL60-IL6方法。用该方法对2批注射用胶原酶、19批注射用尿激酶供试品进行热原测定。结果注射用胶原酶最小有效稀释浓度为0.04U·mL^-1,当浓度≤60U·mL^-1时对HL60细胞生长无影响,浓度≤6 U·mL^-1时对IL-6和热原测定无影响。注射用尿激酶最小有效稀释浓度为100 U·mL^-1,当浓度≤1 000 U·mL^-1时对HL60细胞生长无影响,浓度≤500U·mL^-1时对IL-6和热原测定无影响。因此,适合HL60-IL6单核细胞活化反应法的注射用胶原酶和尿激酶浓度分别为6,500U·mL^-1。用该方法对供试品进行热原测定,结果均未检出热原。结论 HL60-IL6方法适用于注射用胶原酶和尿激酶热原测定。     

Detection of pyrogenicity on medical grade polymer materials using rabbit pyrogen, LAL and ELISA method

The objective of the study is to detect the pyrogenicity of five medical grade gelatinous polymer materials, intended for the manufacturing of capsule for pharmaceutical applications, by an indigenously developed ELISA, LAL and rabbit pyrogen assays. The ELISA methodology includes the incubation of the sample extract with blood from a healthy donor at 37°C. Any pyrogen present in the extract induces the IL-1β which can be determined by ELISA. The rabbit pyrogen and LAL assays were performed as per standards. The result of the ELISA method indicated that all the materials extract induced high level of IL-1β as a marker for pyrogenicity. The rise in temperature of rabbit pyrogen was above 0.5°C in all materials extract. LAL assay induced an endotoxin level above 0.5EU. All the five polymer materials were found pyrogenic in all the assays. The ELISA method is very sensitive because the lowest limit of detection was 10pg/ml endotoxin. Hence it can be concluded that the ELISA method will be an added advantage for the quality control release of a batch of medical products and improving the existing methodologies in the context of reduction and replacement in the use of animal models.     

Ultrasonication of pyrogenic microorganisms improves the detection of pyrogens in the Mono Mac 6 assay.

The monocytic cell line Mono Mac 6 is sensitive to pyrogens. When exposed to pyrogens secretion of interleukin-6 is induced. However, some eukaryotic pyrogenic microorganisms are not detectable. The aim of this study is to introduce a pretreatment of samples to expand the detection range of the assay. The interleukin-6 inducing capacity of a broad spectrum of UV-killed and ultrasonicated microorganisms is examined in Mono Mac 6 cells. The interleukin-6 secretion is determined in a sandwich immunoassay (DELFIA). The Mono Mac 6 assay is able to detect UV-killed Bacillus subtilis, Staphylococcus aureus and Salmonella typhimurium, but neither Candida albicans nor Aspergillus niger. After ultrasonication of the microorganisms it is possible to detect C. albicans and A. niger. The interleukin-6 inducing ability of the examined microorganisms is in no case reduced after ultrasonic treatment. However, ultrasonication of S. aureus results in a 100-fold increase in the interleukin-6 response. Even after ultrasonication Streptococcus faecalis can not be detected. Ultrasonication is an easy and simple method for expanding the detection range in the Mono Mac 6 assay.     

The effects of peptidoglycan, a pyrogenic constituent of gram-positive microorganisms, on the pharmacokinetics of rifampicin

Pharmacokinetics of rifampicin (20 mg/kg orally or i.v.) was determined in calves and rabbits. Seven days later a model pyrogen was administered i.v. to the same animals and 1 hr later the rifampicin administration was repeated. The pharmacokinetic analysis of oral rifampicin was performed using a one-compartment open model with absorption. Intravenously administered rifampicin was analysed by a two-compartment intravascular model. Injection of peptidoglycan in pyrogenic doses led to a significant increase of orally applied rifampicin serum levels in both animal species. The i.v. administration of rifampicin had the same parameters in the control and peptidoglycan experiments. Daily pretreatment of rabbits with small doses of peptidoglycan induced tolerance to the pyrogenic effect. In tolerant animals we did not observe any changes of rifampicin serum levels. Elevated temperature alone was not responsible for observed pharmacokinetic changes leading to the increase of bioavailability of oral rifampicin since another pyrogenic substance (endotoxin) had an opposite effect on pharmacokinetics of previously tested drugs.     

Dry and moist heat sterilisation cannot inactivate pyrogenicity of Gram positive microorganisms.

In the monocytic cell line Mono Mac 6 pyrogens induce interleukin-6 secretion dose dependently. The aim of this study is to examine the interleukin-6 inducing capacity of Gram positive Staphylococcus aureus and Bacillus subtilis endospores after moist and dry heat sterilisation. Moist heat sterilisation of B. subtilis endospores for 15 min at 121 degrees C and 134 degrees C can only reduce the interleukin-6 inducing capacity to 57% and 63%, respectively, compared to untreated. Moist heat sterilisation of S. aureus for 60 min at 121 degrees C and 134 degrees C does not reduce the interleukin-6 inducing capacity of S. aureus. On the contrary moist heat sterilisation at 134 degrees C for 10, 20 and 40 min significantly increases the interleukin-6 inducing capacity compared to untreated S. aureus. This is confirmed in the rabbit pyrogen test. Dry heat sterilisation of B. subtilis endospores at 220 degrees C for 45 min reduces the interleukin-6 inducing capacity to 2% compared to untreated endospores. Dry heat treatment of S. aureus at 220 degrees C for 30 min only reduces the activity to 55%. However, after 250 degrees C for 30 min or 220 degrees C for 6h there is no detectable activity of S. aureus. In conclusion, neither the interleukin-6 inducing activity nor the pyrogenicity of S. aureus and endospores of B. subtilis can be inactivated by the heat sterilisation procedures described by the European Pharmacopoeia.     

Effect of moist heat sterilisation on the pyrogenicity of cell wall components from Staphylococcus aureus

As opposed to endotoxins very little is known about the heat resistance of Gram positive pyrogens. The aim of this study is to examine the pyrogenic activity of the cell wall components lipoteichoic acid and peptidoglycan from Staphylococcus aureus after moist heat sterilisation. The pyrogenic activity is determined as the ability of the substances to induce interleukin-6 secretion in Mono Mac 6 cells.The standard terminal moist heat sterilisation procedures (121 °C for 15 min and 134 °C for 3 min) are not able to inactivate the pyrogenic activity of S. aureus lipoteichoic acid and peptidoglycan. However after longer treatment times the pyrogenic activity of lipoteichoic acid is removed at both 121 °C and 134 °C. In contrast the activity of peptidoglycan is not removed after 160 min at neither 121 °C nor 134 °C where only a 2-log reduction is obtained. In conclusion the terminal moist heat sterilisation procedures described by the European Pharmacopoeia are not able to inactivate the interleukin-6 inducing activity of S. aureus lipoteichoic acid and peptidoglycan.     

塞克硝唑注射液中细菌内毒素的定量检测研究

目的建立塞克硝唑注射液中细菌内毒素定量分析的方法.方法采用动态比浊定量法考察塞克硝唑注射液对鲎试剂的干扰作用,并与热原检查法进行对比试验分析.结果塞克硝唑注射液的2倍稀释液用动态浊度法定量检测时已无干扰,内毒素回收率在50%～200%范围内,样品检查结果与家兔法一致.结论可用动态浊度法对塞克硝唑注射液中的细菌内毒素进行质量控制.     

A highly sensitive pyrogen test for antibiotics I: detection of trace amounts of endotoxin in injectable sodium ampicillin preparations

The rabbit pyrogen test (specified in the pharmacopeia) and the Limulus amebocyte lysate (LAL) test are influenced by high concentrations of certain antibiotics. Therefore, it has not been possible to detect trace amounts of endotoxin which may contaminate these antibiotics. To detect trace amounts of endotoxin in injectable sodium ampicillin, an ultrafiltration technique was utilized which removed the antibiotic and left a solution which contained predominantly the endotoxin. After ultrafiltration, a trace amount of pyrogen (which otherwise could not be detected) was found using both the rabbit pyrogen and the LAL tests. The endotoxin was also determined quantitatively using a chromogenic endotoxin reagent which is made by combining the Limulus amebocyte lysate and a synthetic substrate with a suitable chromophore.     

检测热原的新方案

综述了利用外致热原激活体内血单核细胞（ＭＮＣ）释放细胞因子如白介素－１（ＩＬ－１）、肿瘤坏死因子（ＴＮＦ－α）等即所谓的内致热原的原理，将待检样品与ＭＮＣ共同培养，检测其上清液中的ＩＬ－１或ＴＮＦ－α含量，这一体外检测致热原的新方法。