Endotoxin Fingerprinting

The pyrogenicity and immunogenicity of endotoxin are closely related to their chemical structure. Differences in the lenghth and composition of the O-antigen and core oligosaccharide can have significant effects on their biological activity. The number and length of fatty acid chains in the Lipid A portion are also important. Maybe the most dramatic example of how endotoxin structure effects potency is the phosphates on the disaccharide. Mono-phosphoryl endotoxin is completely inert.

A group of scientists in Hungary recently published a new technique which allows fast and quantitative detection of endotoxin species. The technique involves fluorescent labeling of endotoxin followed by electrophoresis on a microchip. The procedure has been used to establish an electrophoretic profile of many bacterial strains. Comparing results to these profiles, the procedure can quickly elucidate bacterial strain with both purified and crude endotoxin samples. This technology provides the ability of, not only, quantitative endotoxin detection but the identification of the source, which may be of equal importance.

Capillary Electrophoresis Chips for Fingerprinting Endotoxin Chemotypes and Subclasses

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Chronic Fatigue Syndrome and the Microbiome

Myalgic encephalomyelitis (ME), also known as chronic fatigue syndrome (CFS), is a condition which is characterized by the onset of severe fatigue, usually between the ages of 20-40. It is often associated with other traits such as impaired memory or concentration, myalgia, arthralgia, headaches and sleep disorders. The cause of CFS is unknown, but viral infection, bacterial infection, genetic disorders and psychological disorders have all been suggested.

A recent study from Colombia University may elucidate the cause of CFS. Noting that about 90% of CFS patients also suffer from irritable bowel syndrome (IBS), these researchers postulated that the gut microbiome may be involved. By analyzing fecal samples they discovered that specific intestinal bacterial species were associated with CFS and that the abundance of these species correlated with disease severity. Surprisingly, using an immune profiling immunoassay to monitory 61 different bloodstream immune markers, they did not find significant correlations between CFS and control patients. This may suggest that all of the CFS patients were chronic. The identification of bacteria involved in CFS allows the ability to sub-type patients based on fecal microbiome and to identify bacterial targets for therapeutics.

Chronic Fatigue Syndrome Associated with Abnormal Gut Microbes

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Cancer Killing Bacteria

This blog has previously highlighted the use of genetically modified bacteria as therapeutics, including a previous post detailing its effectiveness in treating patients impaired in proper nitrogen regulation. Here we focus on recent work from Korea in which a non-virulent form of Salmonella was engineered to express Vibrio vulnificus flagellin B (FlaB). Using human cell culture they demonstrated that this Salmonella resulted in infiltration of tumors with immune cells with activation of M1 macrophages and inhibition of M2 macrophages. Using knockout cell lines, they also showed that this activity required TL4 signaling. Finally, using mice they showed significant decreases in tumor size in about half of the animals treated. This is an exciting step forward in using genetically modified bacteria in cancer immunotherapeutics.

GM Salmonella Destroys Cancer

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New Advances in Endotoxin Detection

As previously discussed in this newsletter, LAL is the gold standard for endotoxin detection. However, due to limitations, such as assay interference, alternative non-enzymatic alternatives are being investigated regularly.

We recently highlighted an alternative test from China dependent on the release of fluorescent mCherry protein due to activation of NF-kB. Since that time two other alternative approaches have been published. The first, from the University of Urbino in Italy, also used NF-kB expression, this time in MM6 cells, to simulate an innate immune response. The initial tests showed linearity and correlation with LAL but did not provide a clinically relevant minimum detection. The second approach, a collaboration from India, shows promise of a dipstick-type technology that can provide broad endotoxin quantification quickly and without the need for machinery. In this test, spotted endotoxin is detected by binding to polymyxin B and visualized via conjugated gold nanoparticles. The test identified concentrations as low as 10 pg/ml and showed correlation with hematological data.

The continuous push for alternatives to the LAL assay highlight the difficulties associated with an enzyme-based assay. For additional information in overcoming endotoxin masking and/or LAL inhibition/enhancement problems visit our website for information on EndoPrep and ESP.

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Altered Gut Microbes Can Cause Obesity

Previous reports have linked the gut microbiome to obesity. A recent study has taken this a step further and provided a mechanism for how this may occur. Scientists at Yale, led by Dr. Gerald Shulman, made the observation that acetate stimulates insulin secretion by activation of the parasympathetic nervous system. They made the complimentary observation that rodents that were fed a high-fat diet produced elevated levels of acetate. Next, they used fecal transplantation from high-fat diet rodents to low-fat diet rodents to demonstrate an increase in acetate, and subsequently in insulin, caused by the gut microbiome. These results suggest the gut microbiome regulates insulin release via acetate activation of the parasympathetic nervous system in a positive feedback loop.

How Altered Gut Microbes Cause Obesity

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Designer Bacteria as Therapeutics

The role the microbiome plays in human disease, such as metabolic, gastrointestinal and neurological disorders, has been well-established. This has led to therapies that, just a few years ago, seemed radical, including fecal transplantation. Currently, several groups are working to design orally-administered “smart” bacteria that can perform deficient enzymatic roles in specific disease.

One example is a genetically-altered E. coli strain designed by Synlogic in Cambridge. This version of E. coli degrades ammonia into arginine in patients unable to regulate their nitrogen level. The strain has been designed to only work in the human gut by requiring low-oxygen, a condition in the gut, and thymidine, which is not found in high levels in the digestive tract. Other uses for “smart” bacteria include anti-cancer therapeutics and food treatment for phenylketonurics.

Companies Bet on Designer Bacteria as New Way to Treat Disease

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Low Endotoxin Recovery

Low Endotoxin Recovery (LER) is the product-specific inhibition of the bacterial endotoxin test not due to assay interference. The prevailing thought is that endotoxin detection is dependent on supramolecular structure and excipients in pharmaceutical products can destablilize endotoxin complexes, leading to samples containing endotoxin monomers that are not detected by standard assays. Such “false negatives” could lead to significant pyrogenic reactions if aggregation were to take place in vivo. Sample treatments (such as divalent cations, dispersants and proteases) to induce the aggregated state have been shown to have promise, though, results appear to be sample-specific.

Facing Up to the Challenge of Low Endotoxin Recovery

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Can Bacteria be the Fountain of Youth?

Over the past decade the importance of the bacterial microbiome has become clear with links to gastrointestinal, inflammatory, metabolic and neurological disorders being observed routinely. A new study, published in the journal Current Biology, extends this into longevity. Scientists gathered samples from people in the Sichuan province in China, known for an abundance of active individuals over 90. Using Random Forest analysis of microbiome data they determined that these individuals have a high degree of microbiome diversity and increased numbers of bacteria known to be beneficial to gut health. This provides the framework to develop probiotic therapies aimed at healthy aging.

The Gut Microbiome Holds the Key to Longevity

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Cell-Based Endotoxin Detection

The gold standard for endotoxin detection is the LAL assay. However, due to its enzymatic nature and the dependence on harvesting a wild species, an alternative method could prove to be valuable. Scientists in China and the US recently published a live cell-based option.

The test relies on a specific cell line (293/hTLR4A-MD2-CD14) transfected with all pertinent endotoxin receptor elements and a red fluorescent reporter protein (mCherry) under the control of NF-kB. After transfection, the cells that are exposed to endotoxin trigger a signaling cascade that leads to activation of NF-kB, and therefore, production of mCherry protein. Fluorescence is read in a 96-well format.

The report demonstrates specificity to endotoxin, reliance on the TLR-4 pathway, dose-dependant signal which allows the production of a standard curve, correlation with cytokine (TNFa, IL-8) expression and sensitivity to a wide range of endotoxin species.

High-Throughput Living Cell-Based Optical Biosensor for Detection of Bacterial Lipopolysaccharide (LPS) Using a Red Fluorescent Protein Reporter System

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A Review on Endotoxemia

The October issue of the Journal of Leukocyte Biology includes an excellent review article on the issues surrounding endotoxemia and in detecting endotoxin in biological fluids. As is often the topic with this newsletter, the author begins by noting the relationship between circulating endotoxin and problems such as atherosclerosis, alcoholic liver disease, autoimmunity, metabolic syndrome, renal injury, obesity, depression, HIV disease and type 2 diabetes. In addition, the report highlights the variation in reports of the amount of circulating endotoxin in various disease and the correlation with cytokine levels.

The article highlights the heterogeneity of endotoxin, including how differences in acyl chains and phosphates on the Lipid A moiety can affect interactions with MD-2-TLR4 and thus potency. It also covers routes of endotoxin into the circulation and interactions with proteins (e.g., lipoproteins, LBP, albumin) in the bloodstream. The majority of the paper deals with methods of detecting endotoxin in biological samples and the limitations of each method. This includes LAL, ELISA and EAA.

Endotoxemia – Menace, Marker or Mistake?

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