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List Labs currently has a GMP compliant version of HPT™ Lipopolysaccharide from Escherichia coli O113 in stock. What this means for you:

GMP LPS product in stock at List Labs

Get to Phase one quickly with available GMP products

Time is money. The more time spent waiting for your materials to be manufactured is time that you are not conducting your clinical trials. Contracting a manufacturer to produce GMP products can take a year or more for manufacture and release, but with a GMP compliant product in stock, you can purchase what you need when you are ready.

Help your budget with available GMP products 

There are so many costs associated with research studies and clinical trials, who wouldn’t want to save a little money on their project? The expense of custom manufacturing can be steep – purchasing available GMP compliant products from List Labs can help alleviate some of those costs. Lipopolysaccharide is broadly used in many types of clinical trials such as in the study of tumor Ag-loaded IL-12 secreting semi-mature DC for the treatment of pediatric cancer.1

Lipopolysaccharide currently in use in clinical trials worldwide

List Labs’ GMP compliant version of HPT™ Lipopolysaccharide from Escherichia coli O113 has already been used by organizations around the world in clinical trials. The quality of this product is proven by the successful use in Phases one through three in past or ongoing clinical trials. This unique difference sets our GMP product apart from the competition and saves you the risk of an unknown product.

Contact us today to get your GMP LPS while there’s still product in stock!

See how List Labs’ products have been used in research projects on our citations page.

Reference

  1. Dohnal AM, Witt V, Hügel H, Holter W, Gadner H, Felzmann T. Phase I study of tumor Ag-loaded IL-12 secreting semi-mature DC for the treatment of pediatric cancer. Cytotherapy. 2007;9(8):755-70. Epub 2007 Oct 4. PMID: 17917887

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

In this blog we will unravel the terminology describing bacterial toxins. In general, there are at least three ways that bacterial toxins are described in the literature:

Below are examples of each:

Biological designation

When described by their biological designation a part of the genus or species name is used for the toxin. For example: Clostridium tetani produces Tetanus toxin and Corynebacterium diphtheriae produces Diphtheria toxin.

Origin of the toxin

Exotoxins (e.g. polypeptides) are toxins released by a cell, whereas endotoxins (e.g. lipopolysaccharides) are an integral part of the bacterial cell wall.

Body part damaged by the toxin

Bacteria may cause disease through their toxins that enter the body via the respiratory tract, gastrointestinal tract, genital tract, and the skin. Enterotoxins mostly affect the gastrointestinal tract. “Entero” comes from the Greek word “enteron” meaning intestine.

Bacterial enterotoxins include examples of exotoxins produced by some strains of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli).Staphylococcal enterotoxin acts on intestinal neurons to induce vomiting; E. coli producing Shiga toxin causes serious dysentery and can lead to hemorrhagic diarrhea and kidney failure.

You will also see other terms used to designate toxins…

Superantigens: toxins that cause over-reaction

Antigens are characterized by their ability to activate T-cells and other immune system cells; while the T-cell response is a normal part of the immune process, over-activation of T-cells can cause an inflammatory response that can result in shock and multiple organ failure.

Pore-forming toxins that open host cell membranes

Pore-forming toxins (PFT) are toxin proteins with the ability to spontaneously self-assemble forming transmembrane pores in the membrane of target cells. Staphylococcal alpha toxin, also known as alpha-haemolysin, makes specific pores in target cells which are part of the pathology of infection and a valuable tool in construction of nanopores. Tetanolysin is another pore forming toxin produced by C. tetani which can make cells permeable to materials for experimentation.

Intracellular toxins

These toxins have two-part structures and are termed AB toxins. The A stands for “active”, the B for “binding”, for the ways that the two structures cooperatively cause cell damage. In most cases, the B structural element attaches to the cell membrane and provides an entry point for the other part, the A-enzyme component that causes damage to the inside of the cell through its enzymatic activity.

Some AB toxins have more than one B moiety: for example, the cholera toxin has five B proteins that provide entry for the A moiety, so it is designated AB5. The A moiety is initially a coiled chain but once inside the cell it uncoils, where its enzymatic activity kills the enteric cell.

Ligand-receptor interactions

The actions of exotoxins and endotoxins depend on a process whereby a part of their molecular structure, a ligand, can bind or otherwise interact with a structure on the host cell being attacked, a receptor. Thus, this ligand–receptor interaction is crucial to most diseases produced by bacterial toxins.

Lethal dose 50%

Bacteria cause disease by toxin production, invasion and inflammation. All toxins damage or disrupt the functions of the host cells. The term that describes the level of danger presented to the host by a toxin is “Lethal Dose 50%”, abbreviated LD50; the lower the LD50, the lower the amount of toxin to cause death.

 

By: Karen Crawford, PhD.
President, List Labs

Staphylococcal bacteria

Staphylococcal bacteria

Staphylococcal enterotoxin type B (SEB) is a powerful player in the family of toxins; in scientific terms, a superantigen.  This enterotoxin binds to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells and specific V-β chains of the T-cell receptors.  This interaction between the three molecules leads to up-regulation of markers and proliferation of T-cells; additionally, it causes a massive release of proinflammatory cytokines including tumor necrosis factor (TNF), interleukins IL-1, IL-6 and interferon-gamma (INF-gamma) (1,2). SEB can form a complex with and activate T cell receptors even in the absence of MHC Class II antigens, making it a useful tool in stimulating T cells (3).

 

SEB Toxin’s Associations with Human Diseases

SEB is associated with staphylococcal food poisoning, along with TSST-1, is part of the toxic shock syndrome (4) and very likely has a role in human diseases such as atopic dermatitis (5) allergy and rhinitis (6) and the development of autoimmune diseases (7).  A mouse model to simulate Toxic Shock Syndrome has been created by exposing mice to both SEB and lipopolysaccharide (8).

Staphylococcal enterotoxin B is on the Centers for Disease Control and Prevention Select Agents & Toxins list, because of high toxicity and the potential to be aerosolized for wide dissemination; however, the quantity which a principal investigator can possess without registration is sufficient for research. Despite the toxicity and potential danger, SEB is a useful tool in research.

 

Some papers utilizing SEB toxin are described below:

Busbee et al (9) cultured splenocytes in 96-well plates in the presence and absence of SEB.  Supernatants were collected and analyzed for cytokine levels using ELISA kits purchased from Biolegend (San Diego, CA) for determining interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin-2 (IL-2), and IL-6.

Herter et al (10) investigated T cell movement between lymph nodes and sites of inflammation.  In this study, SEB is used extensively as a positive control, stimulating an immune response in the mouse kidney and in various cultured cells.

Janik and Lee (11) has used SEB in mice to develop an understanding of the inhibitory effect SEB may have on pre-existing immunity to pathogens unrelated to the superantigen.  These studies demonstrated that SEB in BALB/c mice selectively targets memory CD4 T cells.

 

References

  1. Marrack P, Blackman M, Kushnir E, Kappler J (1990)The toxicity of staphylococcal enterotoxin B in mice is mediated by T cells.J. Exp. Med. 171: 455–464.
  2. Krakauer T and Stiles BG (2013) The staphylococcal enterotoxin (SE) family: SEB and siblings Virulence 4: 759-773. PMID: 23959032
  3. Hewitt CR, Lamb JR, Hayball J, Hill M, Owen MJ, O’Hehir RE (1992) Major histocompatibility complex independent clonal T cell anergy by direct interaction of Staphylococcus aureus enterotoxin B with the T cell antigen receptor. J Exp Med. 175:1493–1499. PMID: 1588277 
  4. Kashiwada T, Kikuchi K, Abe S, Kato H, Hayashi H, Morimoto T, Kamio K et al (2012) Staphylococcal enterotoxin B toxic shock syndrome induced by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). Intern. Med. 51: 3085–3088. PMID: 23124156
  5. Breuer K, Wittmann M, Bosche B, Kapp A, Werfel T (2000)Severe atopic dermatitis is associated with sensitization to staphylococcal enterotoxin B (SEB). Allergy 55: 551–555. PMID: 10858986
  6. Pastacaldi C, Lewis P, Howarth P (2011)Staphylococci and staphylococcal superantigens in asthma and rhinitis:  systematic review and meta-analysis. Allergy 66: 549–555. PMID: 21087214
  7. Principato M, Qian BF (2014)Staphylococcal enterotoxins in the etiopathogenesis of mucosal autoimmunity within the gastrointestinal tract.Toxins 6: 1471–1489. PMID: 21535520
  8. Huzella LM, Buckley MJ, Alves DA, Stiles BG, Krakauer T (2009) Central roles for IL-2 and MCP-1 following intranasal exposure to SEB: A new mouse model. Vet. Res. Sci. 86:241–247. PMID: 18793785
  9.  BusbeePB, Nagarkatti M, Nagarkatti PS (2014) Naturalindoles, indole-3-carbinol and 3,3′-diindolymethane, inhibit T cell activation by staphylococcal enterotoxin B through epigenetic regulation involving HDAC expression. Toxicol Appl Pharmacol. 274: 7–16 PMID: 24200994
  10. Herter JM, Grabie N, Cullere X, Azcutia V, RosettI F, Bennett P, Herter-Sprie GS, Eylaman W, Luscinakas FW, Lichtman AH and Mayadas TN (2015) AKAP9 regulates activation-induced retention of T lymphocytes at sites of inflammation. Nature Communications6, Art. No.: 10182. PMID: 26680259
  11. Janik DK, Lee WT (2015) Staphylococcal Enterotoxin B (SEB) Induces Memory CD4 T Cell Anergy in vivoand Impairs Recall Immunity to Unrelated Antigens. J Clin Cell Immunol. 6(4):1-8. PMID: 26807307

 

List Labs Citations PageBy: Rachel Berlin, Marketing Manager

The List Labs website hosts a library of scientific article abstracts related to the research performed using our products called the Citations Page. Visitors can search this library to learn how others have used List Labs’ reagents in their research. This valuable resource is updated monthly with new articles from a wide variety of publications. Check out a few recent articles below:

Botulinum Neurotoxin

Carrier Proteins

Clostridium difficile toxin

Lipopolysaccharide (LPS)

Diphtheria toxin

Don’t see the reagent you’re interested in? You can search the citations by product, year, publication, or by the type of cell, animal, assay, protein or research. Check it out today!

List Labs' founder Linda Shoer

List Labs’ founder Linda Shoer

The origin of businesses is often an interesting story. List Biological Laboratories is no exception. The company was founded in 1978 by Linda Shoer. Linda was an entrepreneurial scientist in Silicon Valley, who’d been relocated with her husband from Boston. She had an idea for a company and leveraged an initial order into a loan from a bank, fearless that her vision would be successful. That order and loan served as the starting point for List Laboratories. The first product was a Cholera Toxin.

List Labs Develops Full Range of Bacterial Toxins and Contract Manufacturing Services

Linda had a clear plan for the company and it involved the development of a product line devoted exclusively to Bacterial Toxins and related products. List Labs was the first to commercialize many bacterial toxins for research including C. difficile Toxins and Pertussis Toxins.

Linda was well connected and comfortable networking with colleagues and proposing new business ideas or ventures.  She got the company involved in contract manufacturing and consulting early on. In the 90’s List Labs was instrumental in the manufacturing of a very popular injectable consumer product to smooth facial wrinkles.   Upon her death, she left the business to the current management team; a team that has now worked together for over 20 years.

List Labs – Still Women-Owned and Cutting Edge

Shoer’s presence is still strongly felt and the company has always remained a women owned and operated business. In an era of takeovers and transition, List Laboratories has remained true to its founding and focus. Today, the List Labs catalog offers over 100 products including Toxins, Peptides, Antibodies and Lipopolysaccharides. Many of the employees have worked together for decades.

In 2008, the company built out a new lab, complete with state of the art equipment.   List has produced several batches of high purity proteins used to test vaccines. Additionally, the company specializes in the production, shipment and handling of dangerous goods. In the last several years List Labs has worked on a variety of microbiome projects, custom fills, development work and special Select Agent projects on various subtypes of Botulinum Toxin. We have also provided GMP product for many phase 1 and 2 clinical trials. We enjoy the variety of work and welcome inquiries from new customers.  

Today, List Labs takes great pride in its reputation for high quality products and exceptional customer service. The company works with businesses and organizations worldwide on custom projects or contract manufacturing opportunities as well as selling a broad array of toxins and related products. List Labs heart is in the science and the discovery of innovative solutions. Their office is located in Campbell, CA, in the Silicon Valley. If you have questions about any of our products or services, contact us today!

By: Md. Elias, Ph.D, Senior Scientist

What are Endotoxins?

Endotoxins (aka lipopolysaccharide, LPS or lipoglycan) are part of the outer membrane of Gram-negative bacteria consisting of a lipid moiety and a polysaccharide moiety, the latter is composed of an inner core, outer core and O-antigen joined by covalent bonds1. In animals the lipid part of endotoxin (known as lipid A) often elicits strong immune responses mediated by Toll-like receptor 4 complex (TLR4/MD2/CD14) on the surface of immune cells2. Uncontrolled activation of such immune responses is often associated with production of inflammatory mediators. This may lead to capillary leak syndrome, which causes damage and dilation of the endothelial layer of blood vessels, a decrease in cardiac function and an increase in body temperature (fever); commonly referred to as fatal septic shock1,2.

 

Why is endotoxin contamination common in labs?

Since bacteria are widely present in nature, coexisting with plants and animals, endotoxins are ubiquitous. Endotoxins are naturally released from dead bacteria or as vesicles/blebs as part of the normal bacterial life cycle3. One of the critical properties of endotoxin is its high heat stability; it is found to be very difficult to deactivate/destroy using normal sterilizing conditions. In fact, steam sterilization, while eliminating live microbes, inadvertently increases the endotoxin level on glassware4. Biochemically, endotoxins are hydrophobic in nature and they have a tendency to stick to other hydrophobic materials such as common plastic lab wares. As a result of these properties, endotoxin contamination is common in laboratory procedures. US and European Pharmacopeia guidelines state that complete destruction of endotoxins requires 30 minutes of dry sterilization at 250℃5.

 

What are the FDA limits on endotoxin concentration?

Humans are found to be much more sensitive to endotoxins than the other animals. While a dose of 1 µg of endotoxins per Kg body weight induces septic shock in humans, mice can tolerate a thousand times higher dose. Since bacterial endotoxins are the most prevalent pyrogenic contaminants, the US Food and Drug Administration (FDA) has set limits on the concentration of endotoxin for human and veterinary parenteral drugs and medical devices. Endotoxin levels are measured as EU/ml where EU stands for endotoxin units. One EU equals approximately 0.1 to 0.2 ng endotoxin/ml of solution, depending on the reference standard used; this is the amount of endotoxin present in 105 to 1010 bacteria. FDA guidelines state that endotoxins unit, rather than weight should be used for testing comparisons because the potency of an endotoxin for causing pyrogenic effects depends on a variety of factors: polysaccharide chain length, aggregation, solubility in biological fluids, bacterial source, associated substances, etc. Current USP endotoxin limits in drugs for parenteral administration is 5 EU/kg of body weight per hour and for intrathecal it is 0.2 EU/kg. Endotoxin limits for medical devices is 0.5 EU/ml or 20 EU/device and for cerebrospinal fluid contacted devices it is 0.06 EU/ml or 2.15 EU/device6-9.

 

What is the rabbit pyrogen test?

The rabbit pyrogen test, which was introduced during 1940’s, was very successful in screening water and solutions used to validate parenteral drugs. However, this test is expensive, time consuming and not very quantitative. In the 1970’s an in-vitro assay method was developed based on the observation that horseshoe crab (Limulus polyphemus) amebocyte lysate would clot in the presence of a very low level of endotoxins. This is known as Limulus Amebocyte Lysate or LAL assay. The LAL assay was approved by the FDA during 1970’s to measure the LPS in parenteral drugs, devices and products that come in contact with the blood8. There are at least three forms of the LAL assay, each having different sensitivities: 1) LAL gel clot assay, 2) LAL kinetic turbidimetric assay, and 3) LAL chromogenic assay. The former one can detect endotoxins down to 0.03 EU/ml while the later two can detect endotoxin down to 0.01 EU/ml7,8.

 

What is Low Endotoxin/Lipopolysaccharide recovery (LER/LLR)?

Although LAL is a powerful assay to detect the presence of endotoxin at very low levels, concerns have grown in the recent past when measurable endotoxin concentration was found in decline over time (such as during storage) in products or in-process materials despite the fact that the samples may maintain pyrogenicity in the USP pyrogen test. This phenomenon is termed as low endotoxin/lipopolysaccharide recovery or LER/LLR. It was revealed that LER/LLR phenomenon can occur from masking of endotoxins by pharmaceutical excipients such as widely used polysorbate and citrate or by added/contaminated proteins8,10,11.

As LER has been observed in a range of different sample matrices, the specific mechanism of LER has not been explained; although a number of hypotheses have been proposed. Chen and Vinther suggested that a chelating agent and polysorbate may mask the endotoxins and form the LER/LLR complex that inhibits endotoxin binding to its receptor, Factor C, needed for LAL reaction12. However, results of other studies do not support any specific mechanism. In one study, at low concentration of surfactant (0.0001% v/v polysorbate 20), LAL activity is enhanced to approximately 180%. At increasing concentration of surfactant, the LAL activity went down and reduced to almost zero at about 0.0025% v/v polysorbate 20. Other studies suggest that there are interplays in between endotoxins and the formulation in terms of aggregation, solubilization and masking. Time and temperature are also reported to have effects on LER. The LER phenomenon was reported to occur more rapidly at room temperature than at 2-8℃, and a seven day incubation is sufficient to determine whether a drug exhibits LER or not. LER is also reported when organic compounds such as citrate, acetate and MES buffers including the benzamidine (protease inhibitor) or EDTA or dimethyl sulfoxide was present as excipient in the product12,13,17.

 

What are the current FDA recommendations and guidelines for Low Endotoxin/Lipopolysaccharide recovery?

As the LER/LLR phenomenon in pharmaceutical formulations became more evident from a large number of studies, the FDA became concerned about LER/LLR in drugs and medical devices, and came up with new USP guidelines albeit with old guidelines in place. The USP guidelines recommend that drug producers should perform hold time studies to detect LER/LLR for all new drugs. The hold time studies should be done by adding known quantity of endotoxins to undiluted product and then measure the concentration of detectable endotoxin over time under appropriate storage conditions13. A decline in endotoxin concentration is indicative of LER. In addition to the hold time studies, the USP is proposing a new Reference Standard (RS), Naturally Occurring Endotoxin (NOE) from a well characterized as Gram negative bacteria. Reasons to use NOE as an RS are described in details in a recent review written by Dr. Radhakrishna S. Tirumalai, who is a Principal Scientific Liaison in the Science Division, USP14. The reasons to use NOE in future for LPS quantification are very thoughtful: First, natural endotoxins are vesicles or ‘blebs’ of the outer membrane of gram negative bacteria. Second, cell wall fragments that are generated from naturally dead bacteria are real-life contaminants that might be present in pharmaceutical raw materials, water systems, in process samples and final drug products. Third, chemically extracted LPS which is often called ‘endotoxin’ does not exist in nature and it is biochemically dissimilar to the native endotoxin. Fourth, extracted LPS is stripped off from cell walls, will be absorbed to surfaces, and will form micelles and other aggregates in solution. Fifth, different product formulations and factors such as temperature, pH, salt, detergents, chelating agents also have effects on aggregation. Clearly, extracted LPS may be an inappropriate choice as a RS as it is chemically, biologically, and structurally different from natural gram negative bacterial cell wall fragments. The new USP guidelines also included a recommendation for bacterial strains, along with methodology for preparation, storage and documentation of ‘NOE’ that mimics the ‘real world’ endotoxin contamination14.

 

What are strategies to overcome low endotoxin/low lipopolysaccharide recovery?

A number of strategies for overcoming LER/LLR have been suggested. Sample dilution to 1/1000 showed significant improvement in the recovery of added endotoxin to overcome LER/LLR in endotoxin assay15. Addition of magnesium sulfate in two antibodies formulated with polysorbate 80, citrate or sodium phosphate was shown to mitigate LER/LLR in an endotoxin assay15,16. A freeze thaw regime was reported to mitigate LER/LLR in one study. Other studies have shown that protease treatment to unmask endotoxin worked to mitigate LER in endotoxin assays16,17. Given the different interactions with different products and excipients, it is conceivable that one specific strategy will not work for all and strategies need to be developed for each product16-18.

 

Conclusion:

Since endotoxins are abundant, highly heat stable and difficult to remove, two general strategies are recommended for addressing and mitigating the LER/LLR phenomenon. The first strategy would be to minimize endotoxin contamination at all levels i.e. in the materials that go into a product, in all the process involved in its manufacture, prevention of bio-burden in manufacturing process and ensuring endotoxin removal at relevant process steps. Second, develop strategies to test for LER and develop methods to overcome LER/LLR.

List Labs is one of the leading manufacturers of high quality endotoxins. Our lipopolysaccharides (LPS) and their derivatives (Products#400, #401, #421, #423, #433, #434) are purified from various bacterial sources with proprietary technology. GMP grade endotoxins are available by custom order. These endotoxins are widely used in the field of immunobiology as immune stimulators/modulators in various cell culture work and as adjuvants. In cell culture studies, endotoxin free media and reagents are considered a routine practice to use because endotoxins have been shown to affect/interfere with cell growth and function, and are known to be the source of significant variability. Each of our toxin products is carefully tested by our QC department using FDA licensed LAL assay kit and FDA approved LAL assay methods to measure the level of endotoxins. Details of endotoxin content are mentioned in the certificate of analysis of the product.

 

  1. Rietschel, E.T.,Kirikae, T., Schade, F.U., Mamat, U., Schmidt, G., Loppnow, H., Ulmer, A.J., Zähringer, U., Seydel, U., Di Padova, F. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 1994, Feb;8(2):217-25. PMID: 8119492.
  2. Ohto, U.,Fukase, K., Miyake, K., Shimizu, T. Structural basis of species-specific endotoxin sensing by innate immune receptor TLR4/MD-2. Proc Natl. Acad. Sci. U S A. 2012, May 8;109(19):7421-6. PMID: 22532668.
  3. Kulp, A., Kuehn, M.J. Biological functions and biogenesis of secreted bacterial outer membrane  vesicles. Annu. Rev. Microbiol. 2010, 64:163-84. PMID: 20825345.
  4. Hecker, W.,Witthauer, D., Staerk, A. Validation of dry heat inactivation of bacterial endotoxins. PDA J Pharm. Sci. Technol. 1994, Jul-Aug;48(4):197-204. PMID: 7804819.
  5. Nakata, T. Destruction of typical  endotoxins by  dry heat as determined  using LAL assay and pyrogen  assay. J Parenter. Sci. Technol. 1993, Sep-Oct;47(5):258-64. PMID: 8263663.
  6. Gorbet, M.B.,Sefton, M.V. Endotoxin: the uninvited guest.  2005, Dec;26(34):6811-7. PMID: 16019062.
  7. Iwanaga, S. Biochemicalprinciple of Limulus test for detecting bacterial endotoxins. Proc Jpn. Acad. Ser B Phys Biol. Sci. 2007, May;83(4):110-9. PMID: 24019589.
  8. Chen J, Anders VI. Low Endotoxin Recovery (LER) in Common Biologics Products. Parenteral Drug Association Annual Meeting, Orlando, FL, April 2013.
  9. US Food and Drug Administration. Guidance for Industry: Pyrogen and Endotoxins Testing: Questions and Answers. June 2012.
  10. Bolden, J.S.,Warburton, R.E., Phelan, R., Murphy, M., Smith, K.R., De Felippis, M.R., Chen, D. Endotoxin Recovery Using Limulus Amebocyte Lysate (LAL) Assay.  2016, Sep;44(5):434-40. PMID: 27470947.
  11. Karen Z.M. Current USP Perspectives on Low Endotoxin Recovery (LER). Endotoxin detection part IV. A supplement to American Pharmaceutical Review. 2016. Recovery-LER/.
  12. Chen, J., and Vinther, A. Low Endotoxin Recovery (“LER”) in Common Biologics Products. Orlando: Parenteral Drug Association Annual Meeting; 2013.
  13. Karen, Z., Radhakrishna, T., David, H., James, A., Dennis, G., Robert, M., and Donald, S. Endotoxins Standards and Their Role in Recovery Studies: The Path Forward. BioPharma Asia. November/December 2016.
  14. Radhakrishna, S. T. Naturally Occurring Endotoxin: A new reference material proposed by the US Pharmacopeia. American Pharmaceutical Review. Endotoxin supplement 2016.
  15. Burgenson, A.L. Endotoxins from different sources: Variability in reactivity and recoverability. Presented at the Pharmaceutical Microbiology Forum. Bacterial Endotoxins Summit Meeting, Philadelphia, PA. 2014.
  16. Platco, C. Lab Experiences: Low Endotoxin Recovery. Presented at the Pharmaceutical Microbiology Forum. Bacterial Endotoxins Summit Meeting, Philadelphia, PA. 2014.
  17. Williams, K.L. Endotoxin Aggregation & Binding Properties. Recovering Endotoxin Spikes from Products & Container Clousers. Presentation at the Parenteral Drug Association Conference, Berlin, Germany. 2014.
  18. Tim, S. Removal of Endotoxin from Protein in Pharmaceutical Processes. Endotoxin detection part IV. A Supplement to American Pharmaceutical Review. 2016.

By: Md. Elias, Ph.D, Senior Scientist

List Labs is one of the leading manufacturers of high quality adjuvants from bacterial sources. Our highly purified adjuvants for research and development are Tetanus Toxoid (Product #191), Cholera Toxin B Subunit (Product #104), Diphtheria Toxin CRM197 Mutant (Product #149), Adenylate Cyclase Mutant, Cya-AC– (Product #198L), Pertusis Toxin Mutant (Product #184), and LPS and its derivatives (Products #400, #401, #421, #423, #433, #434). GMP grade material is available by custom order.

In immunology, an adjuvant is a component that enhances and/or potentiates the immune responses (humoral and /or cell mediated) to an antigen and modulates it to achieve the desired immune responses. Adjuvants can be used for various reasons: (i) to enhance the immunogenicity of antigens; (ii) to reduce the amount of antigen or the number of immunizations needed for protective immunity; (iii) to improve the efficacy of vaccines in immune-compromised persons; (iv) to increase functional antibody titer; or (v) as antigen delivery systems for the uptake of antigens by the mucosa (1-3). Brief descriptions of List Labs products that have potential uses as vaccine adjuvants or immune modulators are provided below. For more details, please visit www.ListLabs.com.

Tetanus Toxoid (Product #191): Tetanus toxoid is prepared by formaldehyde inactivation of pure neurotoxin (Product #190). There are FDA approved vaccines that use a tetanus toxoid antigen to protect children and adult against tetanus such as DAPTACEL and Tripedia, and others that use it as a carrier in conjugate vaccines against various pathogens. For example, MenHibrix® is an FDA approved vaccine where tetanus toxoid has been conjugated to Neisseria meningitidis serogroup C and Y capsular polysaccharides and Hib capsular polysaccharide. Several other tetanus toxoid conjugated vaccines are in research and investigation stages such as Type III group B streptococcal polysaccharide-tetanus toxoid conjugate vaccine (4). Information on our entire family of Tetanus products can be found at https://listlabs.com/products/tetanus-toxins-&-related-products/.

Cholera Toxin B subunit (Products #103B and #104): Cholera toxin B subunit (CTB) is the cell binding domain of cholera toxin protein complex. The holotoxin consists of a single A subunit bearing ADP-ribosyl-transferase activity surrounded by five B subunits that bind to GM1 ganglioside receptors on mammalian cell surfaces and facilitate entrance of the A subunit into cells. The non-toxic CTB has been shown to be an efficient mucosal adjuvant and carrier molecule for the generation of mucosal antibody responses and/or induction of systemic T-cell tolerance to linked antigens. Due to the ubiquitous presence of the GM1 ganglioside receptor on eukaryotic cell membranes, CTB has been extensively used as a conjugate and non-conjugate vaccine adjuvant in a wide variety of model systems.

A CTB-urease conjugated vaccine has been shown to prevent infection by Helicobacter pylori, a bacterium that infects greater than 50% of world population and can cause a variety of gastrointestinal diseases (5). A series of studies have been carried out to develop CTB carrier based vaccines to prevent HIV-1 (6) and West Nile Virus infections (7). CTB has been used as a component of a skin patch for transcutaneous immunization against hepatitis B virus in a mouse model (8). Besides the adjuvant activity, recent studies show that CTB can suppress immunopathological reactions in allergy and autoimmune diseases such as Crohn’s disease (9). Information on our entire family of Cholera products can be found at https://listlabs.com/products/cholera-toxins/.

Diphtheria Toxin CRM197 Mutant (Product #149): CRM197 is a non-toxic mutant of diphtheria toxin lacking the ADP-ribosylation activity (10). CRM197 results from a naturally occuringsingle base change (glutamic acid to glycine) in the toxin gene which is immunologically indistinguishable from the native diphtheria toxin. CRM197 functions as a carrier for polysaccharides and haptens making them immunogenic (11, 12). It is utilized as a carrier to develop conjugate vaccines for diseases such as pneumococcal and meningococcal infections. MenACWY-CRM is an approved vaccine to protect adults and adolescents against disease caused by meningococcal serogroups A, C, W-135 and Y. Information on our entire family of Diphtheria products can be found at https://listlabs.com/products/diphtheria-toxins/.

Adenylate Cyclase Toxoid, Cya-AC– (Product #198L): A genetically modified adenylate cyclase toxin (ACT) lacking adenylate cyclase activity (CyaA-AC–) has been produced (13). Although the catalytic activity is destroyed, CyaA-AC– is still cell invasive and able to induce an immune response to co-administered pertussis antigens (14, 15).  CyaA-AC– has been shown to promote delivering of vaccine antigens into the cytosol of major histocompatibility complex (MHC) class I antigen-presenting cells (16). CyaA-AC– has been used as a tool to deliver antigens to T-cells in anti-cancer immunotherapeutic vaccines (17, 18).

Pertussis Toxin Mutant (Product #184): List Labs produces Pertussis Toxin Mutant, a genetically inactivated form of pertussis toxin where mutations were introduced to abolish the catalytic activity of the S1 subunit while the toxin complex still retains the cell binding ability (19). A pertusis toxin mutant has been used as an adjuvant or as a carrier to promote an immune response. These studies indicated that pertussis toxin mutant possesses adjuvant properties with the ability to encourage both local and systemic responses, to promote T helper cell responses to co-administered antigens and to favor the production of Th1/Th17 cells, important in mediating host immunity to infectious pathogens (20). Pertusis toxin binds to the cell receptor, TLR4 which activates Rac and subsequently causes various effects depending on the type of cell treated (21). The toxin or binding oligomer induces dendritic cell maturation in a TLR4-dependent manner (22). Information on our entire family of Pertussis products can be found at https://listlabs.com/products/pertussis-toxins-&-virulence-factors/.

LPS and its derivatives: List Labs provides LPS and various derivatives: highly purified HPTTM LPS from Escherichia coli O113 (Product #433); Ultar Pure Escherichia coli O111:B4 LPS (Product #421); Escherichia coli O55:B5 LPS (Product #423); Ultra pure LPS from Salmonella Minnesota R595 (Product #434); Lipid A Monophosphoryl from Salmonella Minnesota R595 (Product #401) and highly purified HPTTM LPS from Bordetella pertusis strain 165 (Product #400). For other LPS products please go to our product website. These LPS products are widely used as vaccine adjuvants and immune stimulators.

LPS is a potent stimulator of the vertebrate innate immune system mediated by macrophages and dendritic cells and 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 responses.  In drug development, structurally modified LPS forms, such as monophophoryl lipid A (MPLA) have been used as adjuvants in a wide range of vaccine formulations. MPLA, a TLR4 agonist has been formulated with liposomes, oil emulsions, or aluminium salts for several vaccines such as malaria vaccine (known as RTS,S) that is comprised of MPLA and a detoxified saponin derivative, QS-21 (3). Information on our entire family of Lipopolysaccharides can be found at https://listlabs.com/products/lipopolysaccharides/.

List Labs specializes in producing high quality adjuvants for vaccine development and is interested in partnering with others on new projects.  See some of our special projects or contact us for more information.

  1. Lee S.,Nguyen M.T. Recent advances of vaccine adjuvants for infectious diseases. Immune Netw. 2015, 15(2): 51-7. PMID: 25922593
  2. Petrovsky N., Aguilar J.C. Vaccine adjuvants: current state and future trends. Immunol Cell Biol.2004, 82(5): 488-96. PMID: 15479434 
  3. Alving C.R., Peachman K.K.,Rao M., Reed S.G. Adjuvants for human vaccines. Curr Opin Immunol. 2012, 24 (3):310-5. PMID: 22521140 
  4. Baker C.J., Rench M.A., McInnes P. Immunization of pregnant women with group B streptococcal type III capsular polysaccharide-tetanus toxoid conjugate vaccine. 2003. 21(24)3468-72. PMID: 12850362
  5. Guo L., Li X., Tang F., He Y., Xing Y., Deng X., Xi T. Immunological features and the ability of inhibitory effects on enzymatic activity of an epitope vaccine composed of cholera toxin B subunit and B cell epitope from Helicobacter pylori urease A subunit. Appl Microbiol Biotechnol. 2012, 93(5):1937-45. PMID: 22134639
  6. Matoba N., Kajiura H., Cherni I., Doran J.D., Bomsel M., Fujiyama K., Mor T.S. Biochemical and immunological characterization of the plant-derived candidate human immunodeficiency virus type 1 mucosal vaccine CTB-MPR. Plant Biotechnol J.2009, 7(2):129-45. PMID: 19037902
  7. Tinker J.K., Yan J., Knippel R.J., Anayiotou P., Ornell K.A. Immunogenicity of a West Nile virus DIII-cholera toxin A2/B chimera after intranasal delivery. Toxins (Basel).2014, 6(4):1397-418. PMID: 24759174
  8. Anjuere F., George-Chandy A., Audant F., Rousseau D., Holmgren J., Czerkinsky C. Transcutaneous immunization with cholera toxin B subunit adjuvant suppresses IgE antibody responses via selective induction of Th1 immune responses. J Immunol.2003, 170(3):1586-92. PMID: 12538724
  9. Sun J.B., Czerkinsky C.,Holmgren J. Mucosally induced immunological tolerance, regulatory T cells and the adjuvant effect by cholera toxin B subunit. Scand J Immunol. 2010, 71(1):1-11. PMID: 20017804
  10. Pappenheimer Jr. A.M., Uchida T., Harper A.A. An immunological study of the diphtheria toxin molecule. 1972, 9(9):891-906. PMID: 4116339
  11. Gupta R.K., Siber G.R. Reappraisal of existing methods for potency testing of vaccines against tetanus and diphtheria. 1995, 13(11): 965-6. PMID: 8525688
  12. Benaissa-Trouw B., Lefeber D.J, Kamerling J.P., Vliegenthart J.F., Kraaijeveld K., Snippe H. Synthetic polysaccharide type 3-related di-, tri-, and tetrasaccharide-CRM (197) conjugates induce protection against Streptococcus pneumoniae type 3 in mice. Infect Immun.2001, 69(7):4698-701. PMID: 11402020
  13. Simsova M., Sebo P., Leclerc C. The adenylate cyclase toxin from Bordetella pertussis–a novel promising vehicle for antigen delivery to dendritic cells. Int J Med Microbiol. 2004, 293(7-8):571-6. PMID: 15149033
  14. Macdonald-Fyall J., Xing D., Corbel M., Baillie S., Parton R., Coote J. Adjuvanticity of native and detoxified adenylate cyclase toxin of Bordetella pertussistowards co-administered antigens. 2004, 22(31-32):4270-81. PMID: 15474718
  15. Cheung G.Y., Xing D., Prior S., Corbel M.J., Parton R., Coote J.G. Effect of different forms of adenylate cyclase toxin of Bordetella pertussis on protection afforded by an acellular pertussis vaccine in a murine model. Infect Immun.2006, 74(12):6797-805. PMID: 16982827
  16. Osicka R., Osicková A., Basar T., Guermonprez P., Rojas M., Leclerc C., Sebo P. Delivery of CD8(+) T-cell epitopes into major histocompatibility complex class I antigen presentation pathway by Bordetella pertussis adenylate cyclase: delineation of cell invasive structures and permissive insertion sites. Infection Immunity, 2000, 68(1): 247-256. PMID: 10603395
  17. Dadaglio G., Morel S., Bauche C.,  Moukrim Z., Lemonnier F.A., Van Den Eynde B.J., Ladant D., Leclerc C.  Recombinant adenylate cyclase toxin of Bordetella pertussisinduces cytotoxic T lymphocyte responses against HLA*0201-restricted melanoma epitopes. Int Immunol. 2003 15(12):1423-30. PMID: 14645151
  18. Fayolle C., Ladant D., Karimova G., Ullmann A., Leclerc C. Therapy of murine tumors with recombinant  Bordetella pertussisadenylate cyclase carrying a cytotoxic T cell epitope. J Immunol. 1999, 162(7):4157-62. PMID: 10201941
  19. Brown D.R.,Keith J.M., Sato H., Sato Y. Construction and characterization of genetically inactivated pertussis toxin. Dev Biol Stand. 1991, 73:63-73. PMID: 1778335
  20. Nasso M., Fedele G., Spensieri F., Palazzo R., Costantino P., Rappuoli R., Ausiello C.M. Genetically detoxified pertussis toxin induces Th1/Th17 immune response through MAPKs and IL-10-dependent mechanisms. J Immunol. 2009, 183(3):1892-9. PMID: 19596995
  21. Nishida M.,Suda R., Nagamatsu Y., Tanabe S., Onohara N., Nakaya M., Kanaho Y., Shibata T., Uchida K., Sumimoto H., Sato Y., Kurose H. Pertussis toxin up-regulates angiotensin type 1 receptors through Toll-like receptor 4-mediated Rac activation. J Biol Chem. 2010, 285(20):15268-77. PMID: 20231290
  22. Wang ZY., Yang D., Chen Q., Leifer C.A., Segal D.M., Su S.B., Caspi R.R., Howard Z.O., Oppenheim J.J. Induction of dendritic cell maturation by pertussis toxin and its B subunit differentially initiate Toll-like receptor 4-dependent signal transduction pathways. Exp Hematol. 2006, 34(8):1115-24. PMID: 16863919

By:
Debra Booth, VP of Operations
Linda Eaton, Ph.D., VP of Research & Development
Stacy Burns-Guydish, Ph.D., Senior Director of Microbiology
PJ Nehil, Sales & Distribution Coordinator

Dear Researchers Everywhere,

List Labs recently exhibited at the 2016 BIO International Convention. As a producer of bacterial products for research, as well as a provider of custom laboratory services, we were excited to meet with current and potential customers. We were eager to gain some insight into the current and future state of biotechnology and the up and coming field of microbiome research. But you never know exactly how you’ll feel until you spend the time in the conference exhibit hall. We were very pleased to see that we are a part of an industry that is moving forward at the pace of a start up, fueled by the novel ideas and intellect of many scientists.

Last week, thousands of people filled the Moscone Convention Center in San Francisco. The event was brilliantly organized and the venue was strategically arranged. The various pavilions were organized by state or country and some by specialty. It was a great opportunity to network and identify new sources for projects and services. Attendees were CEO and business development folks interested in learning more about what exhibitors have to provide. At our booth, we talked about immunotherapy, live biotherapeutics, contract manufacturing, GMP production, and more. It was a pleasure to shake hands with many customers, distributors, and colleagues and discuss ways we can partner with them to move their research forward.

We also had the opportunity to meet with vendors, in shipping, supplies and services. These vendors are critical to delivery of our biological and therapeutic products, and we benefited from learning about their new offerings as strategic partners. The enthusiasm was palpable from both exhibitors and attendees. At this event, we didn’t just meet industry veterans. We met many young scientists and job seekers looking for their first break. Some even came directly to us to hand-deliver their resumes. The event had a job expo, fueling another layer of energy and opportunity for exhibitors. We were resident in the California Pavilion where we learned that the State of California has a Biosciences Training Program, which will help companies pay for new employment training. Community colleges around the country are encouraging students to contribute to the future of biotechnology through clinical and regulatory apprenticeships. It’s great to see that science is providing opportunity for students in so many ways.

In closing, we found the 2016 BIO International Convention to be highly productive for our company. For those of you who didn’t get a chance to meet us at the convention, it’s very easy to reach us online and on social media. We would love to connect with you on LinkedIn, tweet with you on Twitter, like each other on Facebook and Google+. You can also check these accounts if you’re curious about your next opportunity to meet us at a conference. We even have a YouTube channel and a blog where you can learn more about us. We’d love to see your YouTube videos and read your blog if you have them as well. The future of biotechnology looks bright and we’re more excited than ever to be a part of it. We will definitely attend more events like this and we hope to see you at BIO 2017 in San Diego!

Regards,
Debra, Linda, Stacy, & PJ

By:
Karen Crawford, Ph.D., President
Eva Purro, Director of Quality Assurance
Dom C. Ouano, Marketing

While most of List Labs’ products are intended as research reagents (Research Grade), several can be produced as GMP products for use in humans. Below are key differences between the two.

Research Only VS Preclinical/Clinical/Human Use

Reagent grade products for research only are labeled “not for human use” but are produced using good laboratory practices. These reagents are readily available on our website and any quantity can be purchased. Products intended for human use are produced under cGMP (current Good Manufacturing Practices, see the Code of Federal Regulations 21 CFR 211) and are provided to clients with a customized contract.

reagent vs gmp products

cGMP = Higher Production Standards

Producing compounds under cGMP regulations is a more costly process compared to reagent grade. cGMP compliance includes all aspects of production: documented training programs, QA issued production records, dedicated production suite preparation, testing and release of raw materials, analytical method qualification dedicated supplies, and validated cleaning methods. In addition, a Drug Master File may be submitted to the FDA, which can be cross referenced by our GMP customers.

See more about our cGMP production capabilities.

One example of a product produced as both reagent grade and GMP grade is HPT™ E. coli O113 LPS. Although a chemist may not be able to tell the difference between the reagent grade and the cGMP material, the difference is in the compliance to the GMP’s as described above. Our reagent grade material is produced with good laboratory procedures, however it is not compliant to GMP. Consequently, reagent grade E. coli LPS is not for human use and cGMP LPS may be applied “for human use” per FDA approval. cGMP for human use is not so much a property of the E.coli LPS as it is describing the environment and procedures surrounding the preparation of the compound.

For example….

Our LPS from E. coli O55:B5 or E.coli O113, Products #203, #423 and #433, are reagent grade products and are often used in research, particularly for inducing the maturation of Dendritic Cells.

We also provide cGMP LPS from E.coli O113 on a contract basis, which is made compliant to GMP and is appropriate for FDA approved use in humans.

Learn more about special projects including bulk drug substance & active pharmaceutical ingredient development, we have developed in partnership with our clients or contact us with any further questions or inquiries regarding this or any of our other products and services.

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

While you go about your day, you are surrounded by micro-organisms.  Although most of us spend a lot of time washing up and trying not to think about the propensity of creatures that share our personal space, scientists have been studying them.  Due to their great progress, we are reaching an understanding of how these bacteria and fungi affect our bodies’ functions [1]. The evidence indicates that this inner-ecosystem can not only cause disease if perturbed, but also influence our overall health!  Organisms including Escherichia coli, Helicobacter pylori, Streptococcus thermophilus, and species of Clostridia, Lactobacillus, and Bacterioides inhabit our gut. Corynebacterium jeikeium as well as Staphylococcus species live on our skin, and other Streptococci as well as Neisseria and Candida albicans inhabit our mouth and upper respiratory system [2].  The makeup and diversity of organisms has been found to be strongly influenced, not only by what you eat [3], but also by who you live with [4, 5].  With greater understanding of this rich soup of life that we carry with us, the microbiome has become the new frontier in cutting-edge drug development [6].

In the last three years, research into the molecular basis of microbial influence has blossomed.  The first and most obvious application for this information was in treating C. difficile infections; which result from overgrowth of the opportunistic pathogen after an antibiotic regimen or hospital stay.  Researchers found that fecal transplants from a healthy individual were an effective way to treat this potentially fatal infection [7, 8]. The role of intestinal microbes in Inflammatory Bowel Disease (IBD) has been established [9] in the last year or two.  Based on this knowledge, possible treatments for IBD, such as ulcerative colitis and Crohn’s disease, are in development. Other publications point to microbes’ role in inflammation of the skin and respiratory tract, including acne [10]and asthma[11].  More excitement has been generated as investigators have found links to other chronic diseases including diabetes [12, 13], hypertension [14, 15], and chronic liver disease [16].  Preliminary investigations suggest a connection between overall gut microbial composition and obesity [17].  Some studies in mouse models have even linked the microbiome to the neurological conditions of Alzheimer’s [18] and autism [19, 20].

With all this research going on, you need a great resource like LIST Biological Laboratories, with experience and expertise with microbial products spanning over 25 years. LIST has several products available that can serve as positive controls for your microbial research.  Potent toxins from C. difficile are available (LIST products #157, #158), as well as antibodies that aid in their detection (LIST products #753, #754). Lipopolysaccharides are also available, which cause inflammation and activation of immune signaling cascades, and are extracted from bacterial cell walls of E. coli O111:B4, O55:B5, O157:H7, J5 and K12; Salmonella typhimurium, Salmonella minnesota and Bordetella pertussis. Other acute immune system activators such as Staphylococcal toxins (LIST products #120, #122) and Shiga toxins (LIST products #161 & #162) are also available.

In case the assortment of purified bacterial products on hand are insufficient for your research needs, LIST also provides contract manufacturing for biotherapeutics, as well as microbial purification services.

References

  1. Human Microbiome Project, C., A framework for human microbiome research. Nature, 2012. 486(7402): p. 215-21. PMID: 22699610
  2. Human Microbiome Project, C., Structure, function and diversity of the healthy human microbiome. Nature, 2012. 486(7402): p. 207-14. PMID: 22699609
  3. David, L.A., et al., Diet rapidly and reproducibly alters the human gut microbiome. Nature, 2014. 505(7484): p. 559-63. PMID: 24336217
  4. Yatsunenko, T., et al., Human gut microbiome viewed across age and geography. Nature, 2012. 486(7402): p. 222-7. PMID: 22699611
  5. La Rosa, P.S., et al., Patterned progression of bacterial populations in the premature infant gut. Proc Natl Acad Sci U S A, 2014. 111(34): p. 12522-7. PMID: 25114261
  6. Donia, M.S., et al., A Systematic Analysis of Biosynthetic Gene Clusters in the Human Microbiome Reveals a Common Family of Antibiotics. Cell, 2014. 158(6) p1402 – 1414. PMID: 25215495
  7. Seekatz, A.M., et al., Recovery of the gut microbiome following fecal microbiota transplantation. MBio, 2014. 5(3): p. e00893-14. PMID: 24939885
  8. Scott, K.P., et al., Manipulating the gut microbiota to maintain health and treat disease. Microb Ecol Health Dis, 2015. 26: p. 25877. PMID: 25651995
  9. Huttenhower, C., A.D. Kostic, and R.J. Xavier, Inflammatory bowel disease as a model for translating the microbiome. Immunity, 2014. 40(6): p. 843-54. PMID: 24950204
  10. Christensen, G.J. and H. Bruggemann, Bacterial skin commensals and their role as host guardians. Benef Microbes, 2014. 5(2): p. 201-15. PMID: 24322878
  11. Martin, C., et al., Host-microbe interactions in distal airways: relevance to chronic airway diseases. Eur Respir Rev, 2015. 24(135): p. 78-91. PMID: 25726559
  12. Tang, D., et al., Comparative investigation of in vitro biotransformation of 14 components in Ginkgo biloba extract in normal, diabetes and diabetic nephropathy rat intestinal bacteria matrix. J Pharm Biomed Anal, 2014. 100: p. 1-10. PMID: 25117949
  13. Sato, J., et al., Gut dysbiosis and detection of “live gut bacteria” in blood of Japanese patients with type 2 diabetes. Diabetes Care, 2014. 37(8): p. 2343-50. PMID: 24824547
  14. Pluznick, J., A novel SCFA receptor, the microbiota, and blood pressure regulation. Gut Microbes, 2014. 5(2): p. 202-7. PMID: 24429443
  15. Pluznick, J.L., Renal and cardiovascular sensory receptors and blood pressure regulation. Am J Physiol Renal Physiol, 2013. 305(4): p. F439-44. PMID: 23761671
  16. Minemura, M. and Y. Shimizu, Gut microbiota and liver diseases. World J Gastroenterol, 2015. 21(6): p. 1691-702. PMID: 25684933
  17. Al-Ghalith, G.A., P. Vangay, and D. Knights, The guts of obesity: progress and challenges in linking gut microbes to obesity. Discov Med, 2015. 19(103): p. 81-8. PMID: 25725222
  18. Bibi, F., et al., Link between chronic bacterial inflammation and Alzheimer disease. CNS Neurol Disord Drug Targets, 2014. 13(7): p. 1140-7. PMID: 25230225
  19. De Angelis, M., et al., Fecal microbiota and metabolome of children with autism and pervasive developmental disorder not otherwise specified. PLoS One, 2013. 8(10): p. e76993. PMID: 24130822
  20. Pequegnat, B., et al., A vaccine and diagnostic target for Clostridium bolteae, an autism-associated bacterium. Vaccine, 2013. 31(26): p. 2787-90. PMID: 23602537

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 inflammation3, 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

 

Vaccines have been used to help control diseases for more than 200 years and are the common practice for children and adults. Childhood vaccination has substantially reduced the morbidity and mortality from infectious diseases in much of the developed world, and influenza vaccinations have reduced the impact of seasonal influenza infections.1 However, medical researchers are constantly looking for ways to improve the vaccines that are already used, and develop new ones.

Opportunities for improvement of vaccines abound. For example, although much attention is given to child vaccinations, a reservoir of infection could be eliminated through promotion of adult booster shots such as pertussis booster shots for expectant mothers and close family members, to help protect susceptible newborns. In addition, some diseases that have vaccines currently available still flourish in areas of the world where infrastructures for vaccination are poor and are too costly or cannot be delivered in their current forms.1 Researchers are still trying to develop vaccines for other important diseases, such as HIV/AIDS, malaria and leishmaniasis. Vaccines are also being developed for bacterial pathogens, such as Vibrio cholerae O1 and enterotoxigenic Escherichia coli (ETEC) that are responsible for a high proportion of diarrheal disease and death in adults and children in many countries in Africa and Asia.2

By modifying the factors included in the vaccine, researchers balance the effectiveness of the immune response with the side effects. Previously, whole cell vaccines containing whole organisms that had been chemically inactivated were the norm, but the side effects of fever and discomfort following injections were much more common. Many of the vaccines used today, including those for measles and some influenza vaccines, use live, attenuated viruses. Others use killed forms of viruses, pieces of bacteria (lipopolysaccharides), or inactivated forms of bacterial toxins, known as “toxoids.” Killed viruses, lipopolysaccharides and toxoids can evoke an immune response that protects against future infection.3 Acellular vaccines were introduced in the late 1990’s that contain either three or five key bacterial proteins and have been quite effective in protecting infants and children under four with a much lower rate of side effects.

List Labs offers several virulence factors which are used in vaccine testing.  For testing C. difficile vaccines; available reagents are C. difficile Toxin A (Product #152), C. difficile Toxin B (Product #155), C. difficile Toxoid A (Product #153), C. difficile Toxoid B (Product #154), and both subunits of the Binary Toxin (Products #157 and #158).  Numerous Bordetella pertussis virulence factors are available for use in testing including: Products #179, #180 or #181 Pertussis Toxin, Product #170 FHA, Product #186 Fimbriae, Product #187 Pertactin, Product #188 and #189 Adenylate Cyclase and Product #400 Highly Purified B. pertussis LPS.  Anthrax vaccine testing maybe carried out using Protective Antigen (Product #171) with Lethal Factor (Product #172) in a toxin neutralization assay.  Although these factors are not suitable for testing on humans, they are excellent research tools.

Inactive toxins are quite useful in making antibodies or in capturing antibodies from a vaccinated population on ELISA plates.  Three of our inactivated toxins, which carry mutations in the toxin active site, are B. pertussis Adenylate Cyclase Toxoid, Pertussis Toxin Mutant, Product #184 and CRM197, a non-toxic Mutant Diphtheria Toxin, Product #149.  Toxoids made by formaldehyde treatment of toxins include versions of C difficile Toxins (Products #153 and #154), Diphtheria Toxoid (Product #151), Staphylococcus aureus Enterotoxoid B (Product #123), Tetanus Toxoid (Product #191) and Toxoids of Botulinum Neurotoxins A and B (Product #133 and #139, respectively).

 

  1. Hammond B., Sipics M., Youngdhal K., (2013). From the History of Vaccines, a project of the college of physician of Philadelphia. ISBN: 9780988623101
  2. Svennerholm AM., (2011) From Cholera to Enterotoxigenic Escherichia coli (ETEC) vaccine development.  Indian J Med Res. 133(2): 188–194. PMID: 21415493
  3. Leitner DR., Feichter S., Schild-Prüfert K., Rechberger GN., Reidl J., Schild S., (2013) Lipopolysaccharide modifications of a cholera vaccine candidate based on outer membrane vesicles reduce endotoxicity and reveal the major protective antigen. Infect Immun 81(7):2379-93. PMID: 23630951

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

HPTTM 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.  HPTTM, 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.

 

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

Pertussis Infections (aka Whooping Cough) Increasing

Rates of pertussis infection, commonly referred to as whooping cough, are on the upswing. Whooping cough is a highly contagious respiratory infection with flu-like symptoms including a blocked or runny nose, sneezing, mild fever and the distinctive, hack-like cough. Prior to the availability of effective whole-cell vaccines, before the mid-1940’s, whooping cough was almost entirely a childhood disease. However, up to half of all cases in the USA are among older children, 7 to 19 years old (CDC Pertussis Surveillance and Reporting, 2013).

Why Are There More Instances of Whooping Cough?

Factors in the resurgence of whooping cough include the level of immunity in a given population, the switch from a whole-cell B. pertussis vaccine to an acellular vaccine (which offered reduced side effects), trends in antigenic variation (e.g.,  pertactin expression), and the cyclical nature of B. pertussis infections. “Herd immunity” occurs when more than 95% of a given population has up-to-date vaccinations; without this substantial immunized group, outbreaks become more likely, which affect young infants most severely.

Analysis of the genotype profile in strains isolated in Korea in 2011-2012 indicate that genotypic changes in currently circulating strains are strongly associated with the recent increase of pertussis cases (Kim, 2014). One factor in the resurgence may be the rise of pertactin deficient strains of B. pertussis, possibly as an adaptation (Lam 2014).

Whole Cell Vaccines vs. Acellular Vaccines

Previously whole cell vaccines containing chemically-inactivated pertussis cells were the norm, though fever was a known side-effect. Acellular vaccines were introduced in the late 1990’s and have become the standard of care.  Acellular vaccines are derived from either three or five key virulence factors from the B. pertussis organism, including pertussis toxin and pertacin. While use of the whole-cell pertussis vaccine resulted in a historic low in 1976 of 1,010 cases reported in the USA, 25,616 new cases of pertussis were reported in 2005, with cyclical incidence rates peaking every 3 to 4 years.

Bordetella Pertussis Used in Research

Bordetella pertussis produces a number of proteins that not only contribute to its pathogenicity, but also are used in research as well (Carbonetti, 2010).  The adenylate cyclase toxin (ACT), which is believed to directly penetrate human phagocytes, causes a disruption their normal function by direct production of intracellular cyclic AMP (Kamanova 2008). Pertussis toxin has been used in development of a pertussis diagnosis assay. The recommended pertussis diagnostic tests, culture and real-time PCR assays, lack sensitivity at later stages of the disease. An IgG anti-pertussis toxin ELISA (PT-ELISA) has been introduced as an immunoassay to be used for sero-diagnosis or vaccine evaluation (Kapasi, 2012). Culture and RT-PCR assays detected more cases of pertussis in infants, whereas PT-ELISA detected more cases in adolescents and adults. Serology involving the pertussis toxin is a cost-effective and complementary diagnostic, especially among older children, adolescents, and adults during the late disease phase. More research is required in understanding the long-lasting protective response resulting from vaccination.  Pertussis virulence factors from List Biological Laboratories are effective antigens for serology assays.

Pertussis toxin is a protein-based AB5-type exotoxin with unique qualities that plays a key role in B. pertussis pathogenesis. The exotoxin comprises six subunits (named S1 through S5, where each complex contains two copies of S4) (Kaslow, 1994; Locht, 1995). The subunits are arranged in A-B structure: the A component is enzymatically active and is formed from the S1 subunit, while the B component is the receptor-binding portion and is made up of subunits S2–S5 (Locht, 1995). The subunits are encoded by ptx genes encoded on a large PT operon that also includes additional genes that encode Ptl proteins. Together, these proteins form the PT secretion complex (Weiss, 1993). PT has also become widely used as a biochemical tool to ADP-ribosylate GTP-binding proteins in the study of signal transduction.

List Labs Provides B. Pertussis for Research

List Biological Laboratories provides B. pertussis virulence factors: Pertussis Toxin, Pertussis Toxin Subunits, Filamentous Hemagglutinin (FHA), Fimbriae 2/3, Pertactin (69 kDa protein), Adenylate Cyclase Antigen and B. pertussis Lipopolysaccharide (LPS), all derived from the native B. pertussis source for research and diagnostic purposes.  Recombinant Adenylate Cyclase from B. pertussis is now offered in a new, highly purified form, ideal for studies with an active enzyme.  Pertussis toxin mutant is a relatively non-toxic protein which may be used in place of the toxin for serology. Additionally, pertussis toxin mutant is a vaccine carrier.  These toxins are purified by a tried-and-true method which ensures their activity to high quality standards.

 

References

CDC Pertussis (Whooping Cough) Surveillance and Reporting: http://www.cdc.gov/pertussis/surv-reporting.html

Carbonetti NH (2010). “Pertussis toxin and adenylate cyclase toxin: key virulence factors of Bordetella pertussis and cell biology tools”. Future Microbiol 5(3): 455–69.

Kamanova J, Kofronova O, Masin J, Genth H, Vojtova J, Linhartova I, Benada O, Just I, Sebo P. (2008) Adenylate cyclase toxin subverts phagocyte function by RhoA inhibition and unproductive ruffling. J Immunol 181(8):5587-97.

Kapasi A, Meade BD, Plikaytis, B, Pawloski L, et al. (2012) Comparative study of different sources of pertussis toxin (PT) as coating antigens in IgG Anti-PT Enzyme-linked immunsobent assays.  Clin Vaccine Immunol 19(1):64.

Kaslow HR, Burns DL (June 1992) Pertussis toxin and target eukaryotic cells: binding, entry, and activation  FASEB J 6 (9): 2684–90.

Kim SH, Lee J, Sung HY, Yu JY, Kim SH, Park MS, Jung SO (2014) Recent Trends of Antigenic Variation in Bordetella pertussis Isolates in Korea. J Korean Med Sci 29(3):328-33.

Lam C, Octavia S, Ricafort L, Sintchenko V, Gilbert GL, Wood N, et al (2014) Rapid increase in pertactin-deficient Bordetella pertussis isolates, Australia. Emerg Infect Dis 20(4):626-33.

Locht C, Antoine R (1995) A proposed mechanism of ADP-ribosylation catalyzed by the pertussis toxin S1 subunit. Biochimie 77 (5): 333–40.

Weiss A, Johnson F, Burns D (1993) Molecular characterization of an operon required for pertussis toxin secretion  Proc Natl Acad Sci USA 90 (7): 2970–4.

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