Endotoxin preparations

Parr et al. [61] showed that lipid A has the same antitumoral effect as whole endotoxin preparations on murine L5178Y lymphoma. The effects of LPS and synthetic lipid A treatments were compared by Shimizu et al. [158-161] on Meth A fibrosarcoma in BALB/c mouse. The antitumoral activity of different lipids A has also been investigated. Ribi et al. [162] used an extract from S. typhimurium containing lipid A, which when injected directly into hepatocarcinoma line 10 tumors in guinea pigs shows an antitumoral effect. This activity is attributed to a monophosphoryl diglucosamine derivative of lipid A [163], Synthetic lipid A analogs also proved to be active in this system [164], as well as... 

We showed the existence of a toxic and a nontoxic lipid A fraction in the acid hydrolyzed endotoxin preparation (23). These two fractions were separated and the composition was determined on the purified components so that we could relate specific structural features of lipid A to both toxicity and tumor regression activity (23). 

Ludwig, J. D., Avis, K. E. Recovery of endotoxin preparations from the surface of glass capilliary tubes. J Parenter Drug Assoc 43 276 (1989). 

Wahl, L.M., Rosenstreich, D.L., Glode, L.M., Sandberg, A.L., Mergenhagen, S.E. Defective prostaglandin synthesis by C3H/HeJ mouse macrophages stimulated with endotoxin preparation. Infect Immun 23 (1979) 8-13. 

Naturally occurring bacterial endotoxins contain the lipid, carbohydrate, and protein makeup of the outer cell membrane of GNB (Fig. 1). However, most of the commercial endotoxin preparations have been purified by various extraction procedures and are generally free of nucleic acids, proteins, phospholipids, and other bacterial cell components. The primary chemical configuration that remains after purification is apolysaccharide structure that is covalently bound to a lipid component called Lipid A. Based on its chemical nature, which is common to various bacterial families, this substance is referred to as lipopolysaccharide (LPS). Although the terms endotoxin and LPS are often used interchangeably, most reference endotoxin standards are purified preparations that are more correctly described as LPS. 

In 1969, Greisman and Hornick compared three purified endotoxin preparations on a dose-per-weight basis in rabbits and healthy adult males. Their results showed that rabbits and humans require approximately the same amount of endotoxin on a weight basis to induce threshold pyrogenic responses. 

Pyrogenicity or endotoxin content. The pyro-genicity of a specified dose of product when administered to rabbits can be assayed by a standard pharmacopoeial method but the trend is to replace this with an in vitro assay for endotoxin (Chapter 19). The capacity of the product to induce gelation of Limulus polypbemus amoebocyte lysate is determined against a reference endotoxin preparation and the result is expressed as IU of endotoxin. 

Reconstitution of T-ceU deficiencies with thymic hormones has not been successhil even though the various hormone preparations induce prothymocyte differentiation and functions of mature T-ceUs. They do not regulate the maturation of thymocytes in the thymus. In contrast, IL-2, endotoxin, thymic epithehal cell products, but not interleukin 1, were found to promote functional maturation of immature thymocytes. Two classes of dmgs show thymomimetic actions (Table 2). Levamisole [14769-73-4], sodium salt of diethyl dithiocarbamate (imuthiol) and certain... 

Standardization and Testing". The final vaccine is tested for safety, potency, and residual chemicals. Safety includes testing for endotoxin and stetihty. Potency is evaluated by quantitative determination of the amount of hemagglutinin in the vaccine. Antibody to this glycoprotein is associated with protection. The single radial immunodiffusion (SKID) technique is used to standardi2e the mass of this protein in comparison to a reference preparation. 

For enzymes intended for parenteral use, the manufacturer must assure that the enzyme preparation is essentially pure and free of endotoxins. Electrophoretic and immunologic tests provide the requisite evidence of purity and homogeneity. Most importandy, the manufacturer must remove toxic impurities, eg, bacterial hpopolysacchati.de (endotoxins) which might cause severe toxic reactions such as anaphylactic shock, fever, and vascular coUapse. 

Preparations of the bacterium Bacillus thuringiensis (BT) are applied as sprays to control insect pests on agricultural crops. The bacterium produces endotoxins that are highly toxic to insects. 

The second B. thuringiensis toxin, the /3-exotoxin has a much broader spectrum encompassing the Lepidoptera, Coleoptera and Diptera. It is an adenine nucleotide, probably an ATP analogue which acts by competitively inhibiting enzymes which catalyse the hydrolysis of ATP and pyrophosphate. This compound, however, is toxic when administered to mammals so that commercial preparations of the B. thuringiensis 5-endotoxin are obtained from strains which do not produce the j8-exotoxin. 

Despite rigorous implementation of GMP, most biopharmaceutical preparations will be contaminated with low levels of Gram-negative bacteria at some stage of manufacture. These bacteria shed endotoxin into the product stream, which is not removed during subsequent bacterial filtration steps. This is one of many reasons why GMP dictates that the level of bioburden in the product stream should be minimized at all stages of manufacture. 

Another issue of relevance is that certain biopharmaceuticals (e.g. cytokines such as 1L-1 and TNF Chapter 9) themselves induce a natural pyrogenic response. This rules out use of the rabbit-based assay for detection of exogenous pyrogens in such products. Such difficulties have led to the increased use of an in vitro assay the Limulus ameobocyte lysate (LAL) test. This is based upon endotoxin-stimulated coagulation of amoebocyte lysate obtained from horseshoe crabs. This test is now the most widely used assay for the detection of endotoxins in biopharmaceutical and other pharmaceutical preparations. 

Its major disadvantage is its selectivity it only detects endotoxin-based pyrogens. In practice, however, endotoxin represents the pyrogen that is by far the most likely to be present in pharmaceutical products. The LAL method is used extensively within the industry. It is used not only to detect endotoxin in finished parenteral preparations, but also in WFI and in biological fluids, such as serum or cerebrospinal fluid. 

We have prepared pure dog Hp by our method for human Hp type 1-1. Rabbit-antidog Hp serum was easily obtained. It was found to cross-react with human Hp. The Hp-level in dog plasma increases after injection of endotoxin or turpentine (G7), and, as in humans, it increases after surgical trauma (A9) and disappears after Hb injection (VI). In rats, guinea pigs, and rabbits the plasma Hp level rises markedly after induction of different types of inflammatory states (II, M5, R3), but the absolute values never reach the high level seen in pathological conditions in man. 

Roth RI, Levin J. Measurement of endotoxin levels in hemoglobin preparations. Meth Enzymol 1994 231 75. 

After weighing the dust filters, the amount of endotoxin was determined by shaking them In 10 ml of pyrogen free water and preparing serial dilutions. Llmulus lysate (Cape Cod Associates Inc.) was added to the dilutions according to the manufacturer s recommendations. The last dilution giving a stable clot was read as the Escherichia coll endotoxin equivalent concentration. Dilutions were also prepared with commercial E. coll endotoxin (. coll 026-B26, Dlfco) to assess the accuracy of the production reference standard. The values were always found to agree closely with the stated values. 

Preparation of DNA The plasmid DNA quality is critical for a successful transfection, e.g. prepare using the endotoxin-free mega kit from Qiagen. Acceptable OD ratios should be >1.9. 

Endotoxins can be contaminants in many preparations of biopolymers, such as in DNA preparations. They are a major problem in toxicology because they can cause a vehement, sometimes life-threatening response of the... 

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