Endotoxin Removal

Endotoxin Activity in a Coliphage Preparation


PREPARATION OF ENDOTOXIN-FREE BACTERIOPHAGES
JANUSZ BORATYŃSKI, DANUTA SYPER, BEATA WEBERDĄBROWSKA,
MARZANNA ŁUSIAK-SZELACHOWSKA, GRYZELDA POŹNIAK and ANDRZEJ GÓRSKI.


Methods of Endotoxin Removal from
Biological Preparations:
Pérola O. Magalhães, André M. Lopes, Priscila G.
Mazzola, Carlota Rangel-Yagui, Thereza C. V. Penna,Adalberto Pessoa Jr.


EndoTrap®: highly specific endotoxin removal without incubation

Endotoxin Removal Products



From: www.plosone.org

“Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials”

Endotoxins

From the Net


Endotoxins (not to be confused with enterotoxin) are toxins associated with certain bacteria. Classically, an "endotoxin" is a toxin which, unlike an "exotoxin", is not secreted in soluble form by live bacteria, but is a structural component in the bacteria which is released mainly when bacteria are lysed.

Lipopolysaccharide and other endotoxins

The prototypical examples of endotoxin are lipopolysaccharide (LPS) or lipo-oligo-saccharide (LOS) found in the outer membrane of various Gram-negative bacteria and is an important cause of their ability to cause disease. The term LPS is often used exchangeably with endotoxin, owing to its historical discovery. In the 1800s it became understood that bacteria could secrete toxins into their environment, which became broadly known as "exotoxin". The term endotoxin came from the discovery that portions of Gram-negative bacteria itself can cause toxicity, hence the name endotoxin. Studies of endotoxin over the next 50 years revealed that the effects of "endotoxin" were, in fact, due to lipopolysaccharide.

The only gram positive bacteria that produces endotoxin is Listeria monocytogenes; however these toxins are more accurately described as "endotoxin-like" and should not be confused with that of those found in the cell wall of gram negative bacteria.

Mechanism

In humans, LPS binds to the lipid binding protein (LBP) in the serum which transfers it to CD14 on the cell membrane, which in turn transfers it to another non-anchored protein, MD2, which associates with Toll-like receptor-4 (TLR4).

CD14 and TLR4 are present in several immune system cells (including macrophages and dendritic cells), triggering the signaling cascade for macrophage/endothelial cells to secrete and pro-inflammatory cytokines and Nitric oxide that lead to "endotoxic shock".

Other than TLR4, components of gram negative cell wall may also activate other pathways which may contribute to the overall endotoxic effect.

Assay for detecting presence of endotoxin

A very sensitive assay for detecting presence of endotoxin is the Limulus Amebocyte Lysate assay, utilizing blood from the Horseshoe crab (The horseshoe crab or Atlantic horseshoe crab (Limulus polyphemus) is a marine chelicerate arthropod. Despite its name, it is more closely related to spiders, ticks, and scorpions than to crabs. Horseshoe crabs are most commonly found in the Gulf of Mexico and along the northern Atlantic coast of North America. A main area of annual migration is Delaware Bay, although stray individuals are occasionally found in Europe).

Very low levels of LPS can cause coagulation of the limulus lysate due to a powerful amplification through an enzymatic cascade.



Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the horseshoe crab, Limulus polyphemus. LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane component of Gram negative bacteria. This reaction is the basis of the LAL test, which is used for the detection and quantification of bacterial endotoxins.

There are three basic LAL test methodologies:
-gel-clot
-turbidimetric
-chromogenic
The primary application for LAL is the testing of parenteral pharmaceuticals and medical devices that contact blood or cerebrospinal fluid. In the United States, the FDA has published a guideline for validation of the LAL test as an endotoxin test for such products .

The LAL cascade is also triggered by (1,3)-β-D-glucan. Both bacterial endotoxins and (1,3)-β-D-glucan are considered "Pathogen-Associated Molecular Patterns", or PAMPS, substances which elicit inflammatory responses in mammals.

The Herxheimer Reaction

From the Net

The Herxheimer reaction (also known as Jarisch-Herxheimer or Herx) occurs when large quantities of toxins are released into the body as bacteria (typically Spirochetal bacteria) die, due to antibiotic treatment or rapid detoxification.

Typically the death of these bacteria and the associated release of endotoxins occurs faster than the body can remove the toxins via the natural detoxification process performed by the kidneys and liver. It is manifested by fever, chills, headache, myalgia (muscle pain), and exacerbation of skin lesions. Duration in syphilis is normally only a few hours but can be much longer, up to months or years, for other diseases. The intensity of the reaction reflects the intensity of inflammation present.

The Herxheimer reaction has shown an increase in inflammatory cytokines during the period of exacerbation, including tumor necrosis factor alpha, interleukin-6 and interleukin-8.

History

Both Adolf Jarisch, an Austrian dermatologist, and Karl Herxheimer, a German dermatologist, are credited with the discovery of the Herxheimer reaction. Both Jarish and Herxheimer observed reactions in patients with syphilis treated with mercury. The reaction was first seen following treatment in early and later stages of syphilis treated with Salvarsan, mercury, or antibiotics. It is seen in 50% of patients with primary syphilis and about 90% of patients with secondary syphilis.

The reaction is also seen in other diseases, such as borreliosis (Lyme disease and tick-borne relapsing fever), bartonellosis, brucellosis, typhoid fever, and trichinellosis and Q fever.