By: Mary N. Wessling, Ph.D. ELS

List Biological Laboratories’ (List Labs) catalog of products is related to furthering research in human health and preventing disease, most commonly as the starting materials for vaccine research & development or production around the world. Vaccines are mainly identified for their capacity to prevent diseases that the body’s innate defensive mechanisms (the skin and specialized cells in the blood, for example) can’t resist unaided. However, there are many other uses for these purified materials in medical research, and you will likely encounter wording on our website that is not part of everyday vocabulary for non-scientists. This article is intended to provide a basic understanding of some of the more frequently used terms and aid you in selecting the products most essential to your projects.

Toxin vs. Toxoid

For starters, what is the difference between “toxin” and “toxoid”. Broadly defined, anything that can cause harm to an organism is a toxin. However, for List Labs’ products and in biological usage, a toxin refers to a bacterial or viral product that has harmful effects when it enters the body (List Labs’ toxins are in a highly purified form). A toxoid is a chemically altered toxin that has reduced or no toxicity and is used for its remaining antigenic activity, which can stimulate an immune response.

Take, for example, cholera, a disease produced by Vibrio cholerae bacteria, possibly through contact with body fluids from a person ill with cholera or through contaminated water supply. Cholera causes severe diarrhea, and untreated, it can be fatal. However, the purified List Labs’ cholera toxin by chemical modification becomes a toxoid that lacks toxic activity but retains structures that make it useful for immunization of research animals or stimulation of immune cells in vitro.

How do Toxoids Impact the Immune Process? 

To understand how some List Labs products work, an overview of the immune process is helpful. During the course of a day, we frequently touch, ingest, or breathe in something that has potential to harm the body. Our cells react to this invader: is this a threat, or not, and if so, how serious is the threat?

What is the Innate Immune Response? 

The innate immune response is the first order of defense in the immune process. There are many different cell types in our body. Some of these cells are equipped by their structural and biochemical components to destroy dangerous microbial invaders–pathogens–quickly. The inflammation that we experience from minor infections is often a sign of this process as cells from the blood destroy the pathogen. This happens quickly, within hours.

What is the Adaptive Immune Response?

Another cellular response system requires a longer time to react to the threat. These cells react by changing from an inactive form to one that will start a more complex defensive process: this is the second step, the adaptive immune response. There are two different classes of cells that comprise the adaptive immune response; they differ by the structures that give them their ability to bind antigens– the invading bacteria and viruses. Both these cells are called lymphocytes; individually, they are the B-lymphocytes (B-cells) and T-lymphocytes (T-cells). Both originate from stem cells in the bone marrow; B and T refer to the place in the body where they mature. T-cells mature in the thymus into several subclasses of T-cells that circulate in the blood and lymph. “Killer” T-cells recognize foreign antigens on cell surfaces (e.g. from viral infection or malignancy). “Helper” T-cells induce B-cells to produce antibodies. “Suppressor” T-cells dampen or regulate the immune response to prevent over-reaction. B-cells mature in the bone marrow and migrate to secondary lymphoid tissues (e.g. spleen and lymph nodes). When they encounter foreign antigens and/or helper T-cells, they are stimulated to divide and expand clonally to produce antibodies and differentiate into plasma cells.

What is Immune Memory? 

After the B and T-lymphocytes react to an antigen, two results are possible. The first, and desirable result, is that the invader is identified and defeated, leaving behind what might be called its criminal record: immune memory. When the antigen comes creeping back in the future, the adaptive immune system recognizes it and attacks. The second possibility is an over-reaction and lack of cessation of the adaptive immune process that is harmful to the body: an autoimmune condition.

Antigens, Epitopes and Vaccines

Where do vaccines come into this process? An antigen is a substance that causes the body to mount an immune response against it. Antigens include toxins, bacteria, viruses, or other substances that the body recognizes as foreign or not “self”. Vaccines have structural features similar to structures of the toxin or invading pathogen that can elicit adaptive immunity.

An epitope is a specific molecular region on the surface of an antigen, typically one of many on the antigen, that elicits an immune response and is capable of binding with the specific antibody produced by the response. A toxin has many epitopes that can be recognized by the immune response. The epitopes that are required for toxicity have been altered chemically in toxoids or by specific genetic mutations in inactive mutants; however, many epitopes are retained and can stimulate an adaptive or memory immune response that will be effective against the toxin.

Toxins and Toxoid Products for Research

Below is a list of toxin and toxoid or inactive mutant pairs of products available to support your research.

Toxin and product numbers	Toxoid	Inactive Mutant
Botulinum Neurotoxin Type A from Clostridium botulinum – 130A, 130B, 9130A	133L
Botulinum Neurotoxin Type B, Nicked, from Clostridium botulinum – 136A, 136B	139
Toxin A from Clostridium difficile – 152C	153
Toxin B from Clostridium difficile – 155A, 155B, 155L	154A
Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae – 150	151	149
Enterotoxin Type B from Staphylococcus aureus – 122	123
Tetanus Toxin from Clostridium tetani – 190A, 190B	191A, 191B

 

 

By: Karen Crawford, Ph.D.
President, List Biological Laboratories, Inc.

 

Many bacterial products are potent immune system activators, helping our bodies identify and defend against microbial invasions.  The innate immune system or non-specific immune system is found in animals as well as in plants, fungi and insects and is employed when pathogens break through the outer barrier of skin, scales, or bark.  It is important for any multicellular organism to be able to resist the bacterial pathogens, which can quickly infect tissues that are undefended. Lipopolysaccharides (List products #201 through #434) are frequently used in medical research to challenge the mammalian immune system and induce a cytokine response, setting off a chain of events in the body.  Cytokines are released, attracting macrophages, which attack and “eat” the foreign bodies, and granulocytes, releasing histamines and toxins that are effective in killing bacteria.  Lippolysaccharides have become an important tool in understanding how the body fights infections¹ as well as for understanding inflammation. The chain of signaling set off by lipopolysaccharides includes G-protein activation². LPS has been used to study neurological inflammation^{3, 4}.

Other bacterial “antigens” make potent immune system activators and have slightly more specific effects. For example, challenge with cholera toxin B subunit (List Products #103B – #104) induces lymphoctes to produce a specific kind of T-cell⁵. Activation of the immune system can be quite different, depending on the specific bacteria and virulence factors.  Somehow our bodies have learned to distinguish which bacteria are harmful and which are not; such as in the case of differential activation of immune cells (eosinophils) by probiotic bacteria compared to pathogens such as C. difficile⁶.  Exotoxins from C. difficile are sold as List Products #152 to #155.

 

1. Vassallo M, Mercié P, Cottalorda J, Ticchioni M, and Dellamonica P(2012) The role of lipopolysaccharide as a marker of immune activation in HIV-1 infected patients: a systematic literature review.  Virology J. 9: 174. PMID: 22925532
2. Sangphech N, Osborne BA, Palaga T (2014) Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19). Immunobiology 219(9):653-60. PMID:  24775271
3. Kozlowski C and Weimer RM (2012) An Automated Method to Quantify Microglia Morphology and Application to Monitor Activation State Longitudinally In Vivo. PLoS One 7(2): e31814. PMID: 22457705
4. Russo I, Amornphimoltham P, Weigert R, Barlati S, Bosetti F (2011) Cyclooxygenase-1 is involved in the inhibition of hippocampal neurogenesis after lipopolysaccharide-induced neuroinflammation.  Cell Cycle 10(15):2568-73. PMID:  21694498
5. Sun JB, Czerkinsky C, Holmgren J (2012) B lymphocytes treated in vitro with antigen coupled to cholera toxin B subunit induce antigen-specific Foxp3(+) regulatory T cells and protect against experimental autoimmune encephalomyelitis.  J Immunology 188(4):1686-97. PMID: 22250081
6. Hosoki K, Nakamura A, Nagao M, Hiraguchi Y, Tokuda R, Wada H, Nobori T, Fujisawa T (2010) Differential activation of eosinophils by “probiotic” Bifidobacterium bifidum and “pathogenic” Clostridium difficile.  Int Arch Allergy Immunology 152 Suppl 1:83-9. PMID: 20523069

By: Suzanne Canada, Ph.D.
Tanager Medical Writing

 

An ongoing barrage of information and mis-information has been dispersed through various media about the dangers of toxins in our environment.  Although everyone agrees that you certainly should avoid ingesting, inhaling, or absorbing toxins into your body; your body has natural ways of removing toxins. Some new fads claiming that you need to “detox” to assist in the process may actually harm you more than help.

There are certainly many pollutants in the world today that should be minimized or avoided¹. Naturally occurring sources of toxins in the environment include:

• Other people: who can carry human pathogens
• Food poisoning: due to contamination with E. coli, Salmonella, or even Botulism
• Wild animals: that may carry typhus, viruses, cholera (e.g., rats in Manhattan were recently surveyed by researchers at Columbia University and were found to carry many bacteria and viruses)
• Bacillus and Clostridial bacteria, the spores of which exist naturally in soil
• Plant toxins (lectins, tannins, alkaloids) that limit herbivore ingestion and damage
• Air pollution: dust, pollen, fungal spores such as mold and mildew

However, hype about the prevalence of toxins in our homes for the purpose of selling extreme detoxification products and procedures could hurt people both physically and financially. Misinformation has been widely promoted, claiming that these toxins are the source of many health problems such as ADD, autism, chronic fatigue, and even cancer.  In fact, detoxification is sometimes appropriate when prescribed by doctors in the healthcare setting:

“In the setting of real medicine, detoxification means treatments for dangerous levels of drugs, alcohol, or poisons like heavy metals.  Detoxification treatments are medical procedures that are not casually selected from a menu of alternative health treatments, or pulled off the shelf in the pharmacy.  Real detoxification is provided in hospitals when there are life-threatening circumstances.” ³

Increases in the use and promotion of “Detox” diets, products, and procedures has brought them under scrutiny of some health researchers.  In one case, researchers from Georgetown University Medical School looked at 20 studies published in the last decade and found no evidence of benefit to colon cleansing.⁴  An investigative article by Consumer Reports reported that their medical consultants questioned the need for detoxification at all! ⁵  Another evaluation published at WebMD concluded that you could quickly lose a weight using a detox diet, but you will have to endure hunger, weakness, and could experience side effects of low energy, low blood sugar, muscle aches, dizziness, and nausea.  Other health authorities point out that the human body has natural processes to handle the elimination of toxins, no matter what you eat.⁶

How Does Your Body Get Rid of Toxins?

Far from being helpless, the human body has developed many ways to defend itself against toxins in the environment. ⁸  The body defends itself through three major organ systems:

1. The skin and gut, which act as a physical barrier.

2. The kidneys and liver: The primary function of the liver, kidneys, and urinary system is to expel toxins that result from the body’s metabolism of food and drink ⁷.

3. The immune system: Organs including lymph nodes, bone marrow, thymus, and white blood cells resist or eliminate potentially harmful foreign materials or abnormal cells.  Their major targets are bacteria and viruses.  White blood cells (Lymphocytes: B-cells, T-cells, macrophages, etc) are highly specialized cells which recognize and destroy specific targets.

The innate immunity and the complement system consist of 11 plasma proteins produced by the liver, usually activated by pathogens and antibody complexes, which help to eliminate pathogens.  This mechanism includes inflammation, which is the human body’s first defense that destroys invaders, and prepares affected areas for healing and repair.

References:

1. US EPA website: www.epa.gov
2. Paul T., (2014)  New York’s Rat Population Hosts Dangerous Pathogens. Columbia University Medical Center.
3. Gavura S., (2014). The Detox Scam: How to spot it, and how to avoid it. Science-Based Medicine.
4. Raymond J., (2011). Detox danger: Trendy colon cleansing a risky ritual. NBC News.
5. Do you really need to detox?  Consumer Reports, Jan 2009.
6. Zelman K., (2016). The Truth About Detox Diets. WebMD.
7. Liver, Kidney and Urinary System. NetDoctor, 2016.
8. Ritchison G., Blood and Body Defenses II. BIO 301 Human Physiology.

What is Lipopolysaccharide?

Lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, is a potent stimulator of the vertebrate innate immune system.  This innate immune system, mediated by macrophages and dendritic cells, generates a rapid response to infectious agents.  Structural patterns common to diverse LPS molecules are recognized by Toll-like receptors (TLR) and accessory proteins in serum.  LPS released from bacterial membranes is bound to LPS binding protein (LBP) in serum, transferred to CD 14, an LPS receptor glycoprotein, and presented to the TLR-4-MD-2 complex, stimulating production of cytokines.

LPS has a wide range of uses in research and drug development.  It may be used to stimulate immune cells and investigate the innate immune response.  In drug development, structurally modified LPS forms, such as Lipid A, have been used as vaccine adjuvants.  LPS-derived oligosaccharides have been conjugated to carrier proteins in the development of LPS containing human vaccines.  On the other side of the spectrum of uses, LPS stimulation of the inflammation cascade is the cause of sepsis; thus, LPS and the triggered signaling pathways which lead to production of cytokines are targets for drug development.

The newest LPS from List Labs:

List Labs provides LPS types referenced in the studies below, E. coli O111:B4, Product # 421 and E. coli O55:B5, Product # 423.  We have also added a highly purified LPS from E. coli O113, Product #433, a valuable tool in immunology research.  Additionally, to support work with whooping cough vaccines, we now provide LPS from Bordetella pertussis, Product #400.  New product descriptions follow:

#433, HPT™ LPS, highly purified from Escherichia coli O113

HPT^TM Lipopolysaccharide (LPS) serotype O113, Highly Purified Toxin, is produced by methods ensuring the greater purity of the product.  This process uses a hot phenol extraction and proprietary chromatographic methods that effectively remove traces of protein and nucleic acid while maintaining consistently high activity reported in units of endotoxin.  Removal of these intrinsic proteins is important in that they may activate TLR 2 if present.  If there is any concern that signaling pathways are triggered by protein contaminants, this is a good LPS to use.  This LPS type was used for the National Reference Endotoxin and for the Second International Standard for Endotoxin.

#400, HPT™ LPS, highly purified from Bordetella pertussis strain 165

List Labs has developed new products in the Bordetella pertussis family due to the whooping cough outbreaks and the renewed interest in evaluation of vaccines.  B. pertussis LPS, product # 400, is isolated from native cultures of B. pertussis strain 165, and as such has an abbreviated structure, comprised of lipid A and a core oligosaccharide without an O-specific polysaccharide side chain.  In isolated B. pertussis LPS, some congeners have a trisaccharide in place of the O-chain and some do not.  HPT^TM, Highly Purified Toxin, is prepared by hot phenol extraction and proprietary chromatographic methods that effectively remove traces of protein and nucleic acid while maintaining a consistently high concentration of endotoxin units.

For more information on LPS from List Labs click here.

Use our useful Citation Finder to see List Labs lipopolysaccharides used in research.

Other citations include:

Kubler-Kielb J (2011) Conjugation of LPS-Derived Oligosaccharides to Proteins Using Oxime Chemistry. Bioconjugation Protocols, Methods in Molecular Biology 751:317-327. PMID: 21674340.

To determine if a potential drug could attenuate the consequences of exposure to LPS, a mouse model of LPS induced sepsis was created through injection of 10 mg/kg E. coli O111:B4 LPS.

Chang Y-C,Tsai M-H, Sheu W, Hsieh S-C and Chiang A-N (2013) The Therapeutic Potential and Mechanisms of Action of Quercetin in Relation to Lipopolysaccharide-Induced Sepsis In Vitro and In Vivo. PLoS One 8(11):e80744. PMC3834323.

In a study of the activation of coagulation, Pawlinski et al created a mouse model of endotoxemia with a single intraperitoneal injection of 5 mg/kg of E. coli O111:B4 LPS.

Pawlinski R, Wang JG, Owens AP 3rd, Williams J, Antoniak S, Tencati M, Luther T, Rowley JW, Low EN, Weyrich AS and Mackman N (2010) Hematopoietic and Nonhematopoietic Cell Tissue Factor Activates the Coagulation Cascade in Endotoxemic Mice. Blood 116(5):806–814. PMC2918334.

LPS induces a model of inflammatory pain in the mouse paw.  With the use of mutant mice, Calil et al were able to identify the signaling pathway involved in this pain model.

Calil IL, Zarpelon AC, Guerrero AT, Alves-Filho JC, Ferreira SH, et al. (2014) Lipopolysaccharide Induces Inflammatory Hyperalgesia Triggering a TLR4/MyD88-Dependent Cytokine Cascade in the Mice Paw. PLoS ONE 9(3):e90013. PMC3940714.

Mühlbauer et al carried out experiments in cell culture using 0.5 to 1 µg/ml of E. coli O111:B4 to demonstrate the induction of the intracellular pattern recognition receptor Nod2.

Mühlbauer M, Cheely AW,Yenugu S and Jobim C (2008) Regulation and Functional Impact of Lipopolysaccharide Induced Nod2 Gene Expression in the Murine Epididymal Epithelial Cell Line PC1. Immunology 124:256-264. PMC2566630.

Systemic administration of LPS exacerbates the formation of brain lesions in brains of mice.  These lesions play a key role both in acute brain disorders such as stroke, traumatic brain injury, and in chronic neurodegenerative disorders such as Alzheimer disease, Parkinson disease, or amyotrophic lateral sclerosis.

Degos V, Peineau S, Nijboer C, Kaindl AM, Sigaut S, Favrais G, Plaisant F, Teissier N, Gouadon E,Lombet A, Saliba E,Collingridge GL, Maze M, Nicoletti F,  Heijnen C,  Mantz J, Kavelaars A, and Gressens P (2013) GRK2 and Group I mGluR Mediate Inflammation-Induced Sensitization to Excitotoxic Neurodegeneration. Ann Neurol. 73(5):667-678. PMC3837433.