Anthrax toxin can enter living cells and the toxin enzymes, Lethal Factor (LF), and Edema Factor (EF) make known changes.  Because of this activity, anthrax toxins are valuable tools to investigate cell processes.  Some of the work currently accomplished with these toxins can be described by the following selected references: 

Abrami L, Bischofberger M, Kunz B, Groux R, van der Goot FG (2010) Endocytosis of the Anthrax Toxin Is Mediated by Clathrin, Actin and Unconventional Adaptors. PLoS Pathog 6(3): e1000792. PMID:  20221438 

Laws TR, Kuchuloria T, Chitadze N, et al (2016) A Comparison of the Adaptive Immune Response between Recovered Anthrax Patients and Individuals Receiving Three Different Anthrax Vaccines. PLoS One 11(3):e0148713. Published 2016 Mar 23. doi:10.1371/journal.pone.0148713 PMID: 27007118 

Dyer PDR, Shepherd TR, Gollings AS et al (2015) Disarmed anthrax toxin delivers antisense oligonucleotides and siRNA with high efficiency and low toxicity. J. Control. Release 2015, 220, 316–328. PMID: 26546271 

Rabideau, AE, Liao XL, Akcay G, Pentelute BL (2015) Translocation of Non-Canonical Polypeptides into Cells Using Protective Antigen. Sci Rep 5: 11944, PMID:26178180, PMCID: PMC 4503955 

Chen KH, Liu S, Bankston LA, Liddington RC, Leppla SH (2007) Selection of anthrax toxin protective antigen variants that discriminate between the cellular receptors TEM8 and CMG2 and achieve targeting of tumor cells. J Biol Chem 282: 9834–9845. PMID: 17251181, PMCID: PMC2530824 

Chaudhary A, Hilton MB, Seaman S, et al (2012) TEM8/ANTXR1 blockade inhibits pathological angiogenesis and potentiates tumoricidal responses against multiple cancer types. Cancer Cell 21:212–226. PMID: 22340594 PMCID: PMC3289547 

To learn more about how our highly purified Anthrax Toxins were used in research, check out our over 200 Anthrax Toxin citations. 

List Labs GMP LPS Infographic

List Labs is proud to provide the world with our GMP LPS, and in this blog post and infographic, we want to share how it is being used.

You can see our full list of GMP LPS citations here.

I’d like to tell you about some of the obstacles you may encounter as you develop your live biotherapeutic product (LBP) and how List Labs can help you navigate through them.

Harnessing bacteria’s potential for a healthier world is our company mission.  Our history and experience have been devoted to bacteria – what bacteria can produce – cultivating bacteria, purifying proteins, and polysaccharides.  List Lab’s passion is to support innovators with quality bacterial products for research and development of vaccines and medical products and to perform contract development and manufacturing service for transformative therapies such as LBPs.

I am very excited to share that List Labs has partnered with BetterLife Pharma to develop and manufacture Altum Pharmaceutical’s novel and transformative therapeutic for the treatment of COVID-19.  It is an honor to join in the fight against one of the greatest challenges of this decade.

Who is List Labs?

List is a privately held, woman-owned, and operated company in California, and about a quarter of our staff has advanced degrees.  Initially, the core part of our business was manufacturing bacterial products.  Beginning with selling one bacterial product in 1978, we now have over 100 stock products, including a GMP product.  List Labs is a GMP-compliant facility, but we’re more than a collection of state-of-the-art equipment, List is much greater than the sum of its parts. Live biotherapeutic projects are not a cookie-cutter process and we are uniquely qualified to provide insight and flexibility to match the needs and requirements of each individual project.  We typically take on 2 to 3 microbiome projects at a time, providing your project with individual attention and the critical advantage necessary to achieve a successful outcome.

Our cGMP compliant facility has 7 expertly designed manufacturing suites allowing segregation of product campaigns and spore containment if needed. The suites undergo treatment with vaporous hydrogen peroxide prior to GMP manufacturing to ensure the quality of your product.  Due to the design of the facility and equipment options, we have the ability to manufacture several products in parallel.

Leveraging decades of experience cultivating a variety of microorganisms and GMP manufacturing experience for our products and partners, the transition to the microbiome space was a natural extension of our capabilities and expertise. We began a project about 7 years ago with a partner to develop and manufacture a live bacteria product.  One of the first in the burgeoning microbiome field.

We have now worked on dozens of projects for indications in the gut, skin, women’s vaginal and urinary tract health, and CNS.  We manufacture products for Phase I and II clinical trials. And we have produced over 20 different LBP products.   These projects not only include manufacturing but also typically require a lot of development, many of which come straight from the academic bench with very little development past a shake flask or bottle cultivation.  We are a partner who is invested in the success of your project and work as an extension of your team.

Let’s use climbing Mt Everest as an analogy. We are climbing the mountain with you, right alongside you.  It’s a great analogy because it gets harder and harder the higher you get, and what you do at the bottom, your preparation will make or break you at the top.  Aggressive timelines, budget constraints, these are the shear cliffs we can see but so many of the obstacles that await are unseen black ice, or bottomless crevasse’s…  I know it’s a bit dramatic but getting a LBP to market is much the same, full of potential pitfalls.

So, today I want to share with you what some of these pitfalls look like and give you a glimpse of how List Labs can help you avoid becoming one of the many, that never realize the ultimate goal.

Source of Strain

If there is anything you take home from this talk, it should be this. “Choose Wisely!” Your choice of strain is made early in the project and changing mid-ascent even to “quote” the same species has a compounding impact. If you need to change strains, all of your in vivo and in vitro assay will need to be repeated along with all your development.  The result can be very costly and cause a substantial delay to your timeline.  A strain identified as a particular species is not equal to another strain identified as the same species.  Each is unique and the characteristics or phenotypes that are important for your strain and indication may not be representative of another strain although identified as the same species.

But whatever your strain of choice is, it is highly likely that we have worked with it or a similar strain before. This is a sample list, although not exhaustive, of the strains we have worked with and of course not giving away any confidentiality of our clients.

Animal-Free Media Replacement

Another potential obstacle is replacing the media with animal-free alternatives.  Since you need to establish a product that is BSE/TSE-free, many clients choose to switch to animal-free media. This is not trivial as there are many animal-derived components that are difficult to replace and are necessary for the robust growth of the organism.  Shown in the graph below, is an animal-based media compared to an animal-free base media missing a critical animal-derived component which when the client came to us they had been unsuccessful in replacing.  Growth in an animal-free media may impact growth rate, final cell density, phenotype (which may be an important characteristic for your indication), potency, and viability.  All of which has implications to the scale of the process in order to have enough viable cells for your dose requirements.  This directly impacts your Return on investment.

Live Biotherapeutic Products

But we know how to tackle the problem. This is an example using the graph I showed you before where growth rate and final cell density were impacted without a very critical animal-derived component.  Once we identified a suitable replacement, we demonstrated similar or better growth than with the animal-derived media.  Then we realized further improvements in cell density with process improvements resulting in a 5-10X improvement in cell density.

Preserving Viability

Another pitfall is loss in viability of your organism through the process. This is a typical manufacturing process flow for a live biotherapeutic product.  Initially, the strain is cultivated in a seed culture either anaerobically or aerobically and inoculated into the large vessel (such as a stainless steel bioreactor or single-use bioreactor), then harvested by tangential flow filtration, formulated, lyophilized, sieved, and then filled into vials, applicators, or formulated to fill capsules.

Different organisms will have different sensitivities to the process that may impact its viability including the growth phase at which the strain was harvested, the harvest process, and the environment during the harvest, or during the lyophilization, sieving, and encapsulation process. Understanding the viability of the organism throughout the process is important to know where to focus your development efforts.

We understand these risks and what tools we can use to improve the yield of the live organism at the end of your process. We have demonstrated substantial improvements in viability by 2 to 100 fold by optimizing these variables.

Scale Up

Another hurdle to overcome is the scale-up of the process. We often start with a process that is at the tube, bottle, or shake flask scale which requires a substantial amount of scale-up development.  The process development of the cultivation and harvest should be performed in a scaled-down version of the process such that performance will be predictable at scale with the necessary process controls to demonstrate similar performance.  List has 1L bioreactors and scaled-down versions of the harvest process so we can isolate specific variables and understand the impact on the process.  We typically perform 10X scale up from 1L to 10L to the 100L process to minimize the surprises at scale and provide a robust process.  Your timeline and costs for development should include these activities.

QC Analytical Development

An obstacle of the QC Analytical Development of your LBP is the development of the bioburden assays, USP<61>/<62> which is not straightforward with a live organism.  For LBPs these assays need to be tailored and developed for each organism and we have experience working on these assays. We are also able to harness a lot of efficiencies for the customer by not only working on the manufacturing but also working on the analytical development in-house. Analytical assays are necessary for both in-process testing and final QC testing for release.

Is List Labs the right CDMO for your project?

As a novice climber, would you attempt to scale Mt. Everest with anything less than the most experienced Sherpa? We are a passionate and dedicated team with the expertise and experience that is critical to reach the summit. We have been there many times.  We recognize the obstacles and while no two ascents are exactly the same, we have the flexibility to guide you to the top and deliver a quality product for the success of your project.  We look forward to working with you!

If you have any questions, please contact us at services@listlabs.com or through our contact page.

By: Stacy Burns-Guydish, Ph.D., President

Stacy Burns-Guydish, PhD

Check out the Overcoming Obstacles in the development of Live Biotherapeutic Products video!

Botulinum Toxin Infographic


Diphtheria Toxin Infographic


Pertussis Toxin Infographic


On May 14, 2020, a team spanning the University of California San Diego, San Francisco General Hospital, Cook County Health and Hospitals System in Chicago, and Washington University in St. Louis published a milestone study in the New England Journal of Medicine. Cohen et al demonstrated the effectiveness of LACTIN-V, a new Live Biotherapeutic Product (LBP) created by Osel, Inc. for the treatment of bacterial vaginosis (BV).

LACTIN-V is a single-strain, topically-administered LBP containing Lactobacillus crispatus CTV-05, a protective human vaginal bacterium that helps to combat the pathogenic bacteria and dysbiosis observed in recurrent BV and urinary tract infections. Its success marks the first single-strain LBP to show clinically significant efficacy in a randomized, double-blind, placebo-controlled Phase 2b trial in the US.

The study was supported by grants from the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH). List Labs is excited by this landmark discovery, by the prospects of this discovery for women’s health around the world, and for further supporting the use of LBPs as a viable therapeutic.  We had a chat with Osel’s Director of Product Development, Tom Parks, to find out more about the project, it’s challenges, and why the product is successful:

What are dysbiosis and BV?

Dysbiosis is a disruption of the human microbiome: the collection of microorganisms, including bacteria, that naturally live on and in our bodies. The full function of the microbiome isn’t fully understood, but dysbiosis is known to be involved in a wide range of skin disorders, intestinal problems and gum diseases, among many others.

Bacterial vaginosis is an ecological disorder of the vaginal microbiota in which hydrogen peroxide-producing lactobacilli are displaced by predominantly anaerobic bacteria (e.g. Prevotella and  Mobiluncus species), Gardnerella vaginalis, and Atopium vaginae. It is the most common vaginal infection worldwide among women of reproductive age, and affects about 30% of women in the US. Treatment usually consists of a course of an antibiotic such as metronidazole lasting about five days. The treatment itself is usually effective, but in about 75% of cases BV returns within a year, often within just a few weeks.

Why is treating BV so challenging?

The tendency of BV to recur is due to failure to re-establish a Lactobacillus-dominated vaginal microbiome. Since the only treatment is another course of metronidazole or a similar antibiotic, this can lead to a cycle of re-infection. In addition to the discomfort and negative impact on the quality of life, BV is a risk factor for a wide range of sexually transmitted infections, including HIV/AIDS. It is also a risk factor for premature birth and other reproductive health complications.

Currently there is no approved treatment to prevent the recurrence of BV. Women have tried a range of home remedies, from yogurt to tea tree oil. As might be expected, these are often ineffective, and may have side effects of their own.

Osel has developed a treatment for BV that breaks the cycle of dysbiosis. LACTIN-V is a live biotherapeutic product (LBP) containing the hydrogen peroxide-producing strain Lactobacillus crispatus CTV-05, which is a protective member of the native microbiome. Administered after the normal course of antibiotics (once a day for five days, and then twice a week for 10 weeks), LACTIN-V helps to restore the normal vaginal environment and prevent re-emergence of the organisms that cause BV. In the study reported in NEJM, BV recurrence was significantly less in women treated with Lactin-V (30%) compared to the placebo group (45%) (P=0.01).

What goes into manufacturing an LBP?

An LBP is a therapeutic agent based around a live microorganism, intended to restore a balance in the microbiome disrupted by a disease condition. A contract development and manufacturing organization (CDMO) can provide essential expertise for developing an LBP, but finding the right CDMO is tricky since many contract manufacturing organizations do not have experience with maintaining viability of the microorganism through the entire manufacturing process or are flexible for the process nuances of an LBP product. In addition, many organisms of interest as LBPs are anaerobic to some extent (for example, Lactobacillus are facultative anaerobes), and/or spore-forming. Working with such organisms takes special expertise and facilities to provide necessary containment and segregation, maintain viability, maximize yield, and avoid pitfalls.

Osel’s Tom Parks explains, “Relevant live biologic product experience and GMP manufacturing capabilities are obviously important. Flexibility to handle a number of product formats is also helpful. Since the field is relatively new, companies that manufacture LBPs are few and far between.”

Are you looking for a CDMO for your LBP project?  List Labs has 40 years of experience manufacturing bacterial products and many different LBP products for Phase 1 and 2 clinical trials for indications in the gut, skin, vaginal mucosa, and CNS. List Labs has the expertise for handling and cultivating anaerobic bacteria and spore formers.  We are uniquely qualified to provide insight and the flexibility to match the needs and requirements of each individual project, such as filling different product formats.  We are a passionate and dedicated partner who works as an extension of your team to ensure the success of your project.  If you are interested in an LBP project, contact us at sales@listlabs.com

The purity of your recombinant proteins is critical to the success of your research. A number of contaminants can affect both in vitro and in vivo systems. Some of the results are predictable, but others are not. All can affect your experimental data.

This article will focus on one of the most common contaminants: endotoxins or lipopolysaccharides. List Labs provides certificates of analysis showing the very low levels of endotoxins in our products.

What Are Endotoxins?

Lipopolysaccharides, also known as endotoxins, are a class of complex hydrophobic molecules found in the cell membranes of Gram-negative bacteria such as Escherichia coli. They are released in large quantities following cell death and during cell division, so they are a common component of recombinant protein production.

The general structure of an endotoxin is one or more Lipid A molecules bonded to one end of a short polysaccharide oligomer. The oligomer has polysaccharide side chains that carry O-antigen. Endotoxins are generally not inactivated by heat and must be removed during purification.

Endotoxin Effects

Endotoxins have a variety of deleterious effects on mammalian systems. These can vary widely even in similar systems. In fact, at least one case is known in which two insensitive T-cell lines were cloned from an endotoxin-sensitive parent line. One factor seems to be the presence of the CD14 receptor protein on the surface of the affected cells. Higher expression of CD14 seems to correlate with greater endotoxin sensitivity. In especially sensitive systems, even picomolar concentrations of endotoxin can lead to anomalous experimental results.

Effects in vitro

Documented effects in vitro include:

Effects in vivo

In live animals, endotoxins produce an inflammatory response in almost all tissues that are exposed to them. The pyrogenic nature of endotoxins produces effects ranging from fever to fatal septic shock.

All of the above effects, both in vivo and in vitro, may be synergistic with other contaminants or (in live animals) endogenous products. This, combined with widely varying cell sensitivity, make the experimental effects of endotoxin contamination difficult to predict.

Purification to Remove Endotoxins

The standard method of purifying recombinant proteins and removing endotoxins is affinity chromatography, using affinity tags on the target proteins, eluting the bound target, and then cleaving the tags in post-processing. Affinity chromatography is the method of choice at List Labs and gives our recombinant products exceptional purity.

Conclusion

Endotoxin contamination is a potentially serious problem in recombinant proteins, with highly variable and difficult to predict experimental effects. Even low levels of contamination may produce anomalous effects, which may vary across different cell lines and test subjects. Only reliable purification can prevent contamination. List Labs provides certified products with known purity and very low levels of endotoxin.

References

Sigma Aldrich, “Cell Culture FAQs: Bacterial Endotoxin Contamination;”

https://www.sigmaaldrich.com/technical-documents/articles/biology/what-is-endotoxin.html

  1. Dawson, “The Significance of Endotoxin to Cell Culture and Biotechnology;” LAL Update, March 1998, Vol 16, No. 1, p. 1-4; Associates of Cape Cod Incorporated; https://www.acciusa.com/pdfs/newsletter/updt0398.pdf
  2. Schwarz, M. Schmittner, A. Duschl, and J. Horejs-Hoeck, “Residual Endotoxin Contaminations in Recombinant Proteins Are Sufficient to Activate Human CD1c+ Dendritic Cells;” PLoS One. 2014; 9(12): e113840; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4257590/

The List Labs Citation Database is a robust tool for researchers. It offers thousands of papers showing how List Labs products are used experimentally. Researchers can search keywords specific to their fields of study and learn how others in that field have used our toxins and antigens.

In this article, we will explore experimental applications for two of our most popular products: Pertussis Toxin (Products #180, #181, and #184) and Cholera Toxin (Product #100B). This article is not exhaustive – we will focus on recent research – but it offers a survey of ways in which List Labs is helping to get science done.

Experimental autoimmune encephalomyelitis (EAE) is an induced autoinflammatory condition of the central nervous system. It is used in rodents as a model of demyelinating diseases such as multiple sclerosis and of T-cell-mediated autoimmune disease in general. Inducing EAE usually uses isolated myelin proteins or homogenate along with pertussis toxin to open the blood-brain barrier and allow T-cells access to the CNS. Many citations in the database note the use of those products.

But that’s far from the only way researchers have found to use List Labs’ pertussis toxin. An international team of researchers found that pertussis toxin reduces cellular damage following ischemic strokes. Another used it in their study of myocarditis. A Michigan team used pertussis toxin to develop an innovative high-molecular-weight mass spectrometry application. And of course, several research groups used it to develop assays and treatments for whooping cough.

The applications for cholera toxin are equally diverse. Besides treatments for cholera, researchers recently used the toxin to study the role of bone morphogenetic proteins and mesenchymal stem cells in breast cancer, as well as TRAIL therapy to treat such cancers. Other cancer researchers looked at hemocyanin as a treatment for bladder cancer, using List’s cholera toxin.

Investigators studied vaccines against Helicobacter pylori, which is known to cause peptic ulcers and is a risk factor for gastric cancer.

Food allergies are a serious and growing problem. Our citation database lists studies that used cholera toxin to aid in the study of allergies to several common foods (especially peanuts). Cholera toxin was also studied as an adjuvant for intranasal vaccines and used to investigate the role of immunoglobulin E (IgE) in anaphylactic shock—an extreme and potentially fatal allergic reaction.

Cholera toxin was even used to study the cellular mechanisms of Yersinia pestis, the pathogen that causes bubonic plague.

This is only a brief survey of recent research using two of List Labs’ more popular products. It illustrates both the wide range of applications for List’s bacterial toxins and the utility of the List citation database as a tool to facilitate your literature surveys.

 

What is Coronavirus (COVID-19)?

Coronavirus (COVID-19) is a disease caused by a virus (SARS-CoV-2) that can trigger what doctors call a respiratory tract infection, and is transmitted from person-to-person.

What are the Symptoms of Coronavirus (COVID-19)?

Coronavirus (COVID-19) symptoms can range from mild to severe. Though, elderly people and people with underlying health conditions like heart or lung disease, or diabetes seem to be at a much higher risk of developing severe illness from COVID-19.

Recent Research: Experimental Applications of Pertussis Toxin and Cholera Toxin

The List Labs Citation Database is a robust tool for researchers. It offers thousands of papers showing how List Labs products are used experimentally. Researchers can search for keywords specific to their fields of study and learn how others in that field have used our toxins and antigens.

In this article, we will explore experimental applications for two of our most popular products: Pertussis Toxin (Products #180, #181 and #184) and Cholera Toxin (Product #100B). This article is not exhaustive – we will focus on recent research – but it offers a survey of ways in which List Labs is helping to get science done.

Pertussis Toxin Applications

Experimental autoimmune encephalomyelitis (EAE) is an induced autoinflammatory condition of the central nervous system. It is used in rodents as a model of demyelinating diseases such as multiple sclerosis and of T-cell-mediated autoimmune disease in general. Inducing EAE usually uses isolated myelin proteins or homogenate along with pertussis toxin to open the blood-brain barrier and allow T-cells access to the CNS. Many citations in the database note this use of those products.

But that’s far from the only way researchers have found to use List Labs’ pertussis toxin. An international team of researchers found that pertussis toxin reduces cellular damage following ischemic strokes. Another used it in their study of myocarditis. A Michigan team used pertussis toxin to develop an innovative high-molecular-weight mass spectrometry application. And of course several research groups used it developing assays and treatments for whooping cough.

Cholera Toxin Applications

The applications for cholera toxin are equally diverse. Besides treatments for cholera, researchers recently used the toxin to study the role of bone morphogenetic proteins and mesenchymal stem cells in breast cancer, as well as TRAIL therapy to treat such cancers. Other cancer researchers looked at hemocyanin as a treatment for bladder cancer, using List’s cholera toxin.

Investigators studied vaccines against Helicobacter pylori, which is known to cause peptic ulcers and is a risk factor for gastric cancer.

Food allergies are a serious and growing problem. Our citation database lists studies that used cholera toxin to aid in the study of allergies to several common foods (especially peanuts). Cholera toxin was also studied as an adjuvant for intranasal vaccines and used to investigate the role of immunoglobulin E (IgE) in anaphylactic shock—an extreme and potentially fatal allergic reaction.

Cholera toxin was even used to study the cellular mechanisms of Yersinia pestis, the pathogen that causes bubonic plague.

This is only a brief survey of recent research using two of List Labs’ more popular products. It illustrates both the wide range of applications for List’s bacterial toxins and the utility of the List citation database as a tool to facilitate your literature surveys.

List Labs has been making botulinum toxins for research for 30 years. 

In that time, we’ve made reagent and GMP products for researchers and pharma companies.  Our products are of the highest quality and purity; they are produced in consistent processes, tested and stabilized by freeze-drying.  Proteins that need to be activated by proteolytic cleavage (nicking) are activated and purified, providing consistent proteins for your research. You do not loose toxin in the nicking process, nor do you run experiments with toxin having a variable amount of nicking.  We offer both pure neurotoxin and toxin complex, fully activated.

For work with the enzymatic light chains, we offer recombinant Light Chains in four different serotypes, LC/A, /B, /C and /D which are non-toxic and may be applied to research using common laboratory practices.  Recombinant heavy chain, binding domains, both GST tagged and un-tagged are available. Our toxoids are made from purified neurotoxin types A and B to give you the most specific antibody production.

We have demonstrated the use of our type A toxin antibodies; one polyclonal raised against the heavy chain is an effective capture antibody for ELISAs and other detection strategies and the other antibody, a mouse monoclonal will specifically bind to type A light chain. This antibody pair will capture and identify small amounts of toxin.

The table below lists the Product #’s for these research reagents; several are offered in different sizes.

TOXINTYPE COMPECE NEUROTOXIN TOXOID CHAINS ANTIBODIES
A 128 130 133 611, 612, 613 730, 731
B 138 138 139 620, 622, 623
C 625
D 146 630
E 140 141

 

If you have questions, please contact us at sales@listlabs.com for more information.

If you are an existing customer, you can place your order with a purchase order at orders@listlabs.com

If you are not yet a customer, fill out the customer app, once approved, we can fill your order.

See information outlining the purchase of controlled toxins on our website.

Orders of Select Agent products must total less than 1mg.  There are no such limitations on antibodies or chains.

List Labs currently has over 3300 citations with more being added every month! Within our citations page, we provide the ability to search and sort from over 100 cataloged items that are offered here.

We are honored to supply researchers worldwide with highly purified bacterial toxins that can potentially be instrumental in helping to change the world!

In this post, we’ve gathered all of our current citations for our Diphtheria product group. Please use these citations as a reference and resource for your potentially life-changing work!

Diphtheria Toxin & CRM

Corynebacterium diphtheriae is a Gram-positive, bacterium that infects epithelial cells of the upper respiratory tract and produces diphtheria toxin.  Diphtheria toxin is proteolytically cleaved forming a two-part toxin, held together by a disulfide bridge.  The amino-terminal carries the toxin’s enzymatic activity, capable of ADP-ribosylation and inactivation of translation elongation factor 2 (EF-2).  The carboxy-terminal domain binds to specific host receptors, the heparin-binding EGF-like growth factor (HB-EGF) on human epithelial cells, and translocates the catalytic domain into the cell.  After binding to the cell receptor, the diphtheria toxin is taken up by endocytosis, the pH of the endocytic vesicle drops, and the translocation region of the toxin helps guide the catalytic domain into the host cytoplasm where it is released.  Within the cytoplasm, the diphtheria toxin catalytic domain ADP ribosylates EF-2, terminating protein synthesis and causing the death of the cell.  Diphtheria toxin is highly potent, and as little as one catalytic domain is thought to cause cell death.  In cell culture, diphtheria toxin inhibits protein synthesis and causes death in cells carrying the HB-EGF receptor.  This toxin has been used to specifically eliminate receptor-expressing cells in transgenic mice.

View List Labs Diphtheria toxins for sale.

View some of List Labs Diphtheria related blogs:

Bacterial Toxin Terminology

Toxoids, Toxins and Vaccine Related Terminology

List Labs Reagents Used in Research – January ’18

Carrier Protein Used in Life-Changing Research

Cell Ablation Using Diphtheria Toxin (DT) is an Important Technique for Studying Regeneration in Living Animals

By: Hans Bigalke

Tetanus toxin (TeNT) and botulinum toxin (BoNT) appear quite different at first glance, however, when we take a closer look at how these toxins function, they are more similar than suggested by the diseases they cause.

Symptoms of Tetanus vs. Symptoms of Botulism

While tetanus causes a body to take a rigid, inflexible state, a very well-described and feared disease since antiquity; botulism reveals itself in limp, uncontrolled muscles, symptoms that mimic those of other diseases, hiding the cause of the disease until the modern age.

Symptoms are strikingly opposite: tetanus is characterized by unrelieved tension or spasticity of the striated muscles and botulism by a limp or flaccid state of the same muscles. In both cases, the muscles can no longer be moved in a coordinated manner, resulting in respiratory paralysis and death.

Tetanus and Botulism Have Similar Basic Origins and Structures

Both diseases can be attributed to toxins created by Clostridia. In general, TeNT is formed by bacteria introduced through injuries such as puncture wounds, placing the bacteria where they can grow in the absence of oxygen. BoNT is also synthesized by bacteria growing under lack of oxygen; however, in contrast to TeNT, botulinum is usually encountered when bacteria multiply and produce toxin in contaminated foods and the toxin is swallowed with the contaminated food. Both toxins enter the bloodstream and are distributed through the body (1, 2, 3, 4).

Botulinum and tetanus neurotoxins are both large proteins composed of two parts, a heavy chain, and a light chain. The light chain represents the active component; it is a protease that cleaves peptides regulating exocytosis of neurotransmitters, rendering the nerve unable to communicate. The heavy chain navigates the toxin into target cells and is responsible for transfers through several membranes.

Although botulinum and tetanus toxins have the same basic structure, tetanus neurotoxin exists solely as a two-part protein neurotoxin; where botulinum toxin is, at least initially, associated with accessory proteins, forming a toxin complex.  This complex can be more than four times larger than the neurotoxin alone (5, 6).

TeNT is taken up by peripheral cholinergic nerve endings and is transported intraaxonally, retrogradely into the soma of the nerve cell (7). It leaves the motor neuron and subsequently enters nerve endings of inhibitory interneurons (8, 9, 10, 11). Within the inhibitory neurons, the tetanus enzyme cleaves vesicular VAMP2, inhibiting the release of the transmitters glycine and GABA (12). With this action, the fine adjustment of the coordination of motor motion is disturbed. Inhibition is no longer possible so excitatory input is passed unfiltered from the spinal cord to the periphery. Minute peripheral sensory stimuli release a pronounced spasm, the clinical indication of tetanus.  A similar muscle spasm is caused by strychnine, a blocker of glycine receptors.

In addition to this central effect, TeNT also has peripheral effects, splitting VAMP-2 in cholinergic nerve endings, leading to flaccid paralysis. However, this effect is triggered only at about 100-1000 fold higher concentrations, amounts of toxin which are not naturally encountered, so that peripheral effects play no role in clinical tetanus. Peripheral effects can be studied experimentally on isolated nerve-muscle preparations.

It turns out that TeNT largely mimics the effect of BoNTs (13). Both TeNT and BoNTs cleave vesicular proteins that trigger fusion of the transmitter-containing vesicles with the plasma membrane. Concentrations of BoNT needed to create paralysis are in general as low as the concentration of TeNT leading to the central effect. The BoNT serotype B not only splits the same protein as TeNT, it even cleaves it in the same place (14). Clearly, the difference between the action of botulinum and tetanus toxins is the location where the light chain is released and destroys the vesicle docking mechanism.  Transport to the different sites of action is carried out by the heavy chains of these toxins. Surprisingly, BoNT/A and E also enter the soma of motor neurons by retrograde transport and eventually interneurons, where they can trigger central effects (15, 16, 17, 18, 19). These effects occur only at high concentrations and are masked by the peripheral paralysis.

Synapses must be actively sending or receiving neurotransmitters to allow endocytosis of both BoNT and TeNT. The reason for this lies in the localization of the receptors for these toxins on the luminal side of the synaptic vesicle. Only after the synaptic vesicle merges and becomes incorporated into the cell membrane do the receptors become accessible to the toxins. However, the dependence of uptake on synaptic activity is only valid if the peripheral effects are involved. Systemic TeNT, which is transported axonally, enters neurons by a different mechanism;  it is endocytosed independent of synaptic activity (10, 20).  TeNT enters vesicles which transport peripheral metabolites via the retrograde route into the soma, for reuse or introduction into other metabolic pathways. TeNT travels on this route as a stowaway.

BoNT serotypes and TeNT are believed to be derived from an ancient toxin that has adapted to different targets in the course of evolution. An adaption allowing the toxin to readily reach a different destination in the nervous system is probably responsible for disguising the toxin.

TeNT FAQs

Question: How similar are the amino acid sequences of TeNT and BoNT?

Answer: The similarity is about 40-50%, depending on the BoNT serotype.

Question: Why is TeNT not absorbed orally?

Answer: A complex of several proteins protects BoNTs from proteolytic degradation in the upper small intestine, this complex is responsible for the oral availability of botulinum toxins. During its further passage in the digestive system, as soon as the pH changes from acid to basic, neurotoxin leaves the complex and is able to enter the circulatory system. SinceTeNT has no protective complex proteins, like all other proteins, it is destroyed in the course of the gastrointestinal passage.

Question: Can BoNT form in poorly perfused human tissue, similar to TeNT?

Answer: Clostridium botulinum also grows in poorly perfused tissue injury and can form and release BoNT. Recently, such intoxications have been observed in drug addicts who use injectables contaminated with clostridial spores (21). BoNT is also formed in the intestine of infants when they consume spore-contaminated food, like honey.

Question: Is TeNT like BoNT also synthesized outside of a living organism, for example, in food?

Answer: At least under laboratory conditions, TeNT is produced from bacteria in fermenters. Whether Clostridium tetani naturally produce the toxin under oxygen deficiency outside living organisms is a good question.  Tetanus toxin, without a protective coating, is more vulnerable to the environment than botulinum toxin complex. It does not survive the digestive process when ingested.  Toxin produced outside of a living organism will likely not survive and would not provide a competitive advantage. From the point of view of the organism which uses toxin to secure food and a livable environment, making toxin which is destroyed would be a waste of energy.

Question: BoNT is used therapeutically to treat pathological muscle cramping and spasticity. Are there any indications for TeNT, e.g. local paralysis after spinal injuries or stroke?

Answer: Theoretically one could imagine such applications. In the developed world, however, the population is fully immunized against TeNT, so that injected toxin is immediately neutralized by specific antibodies. A similar situation occurs when antibodies are formed during therapy with BoNT and the BoNT becomes ineffective.

Question: Can TeNT like other bacterial toxins be used as a tool in research?

Answer: Several opportunities are offered by tetanus for research.  TeNT serves as an aid to the study of axonal transport and has the potential to be used as a carrier for other proteins or substances that are to be channeled into the spinal cord.  TeNT binds exclusively to neurons and as a result, is an excellent neuronal marker. For this purpose, either the toxin itself or the binding C-fragment can be equipped with a tag like FITC or detected by standard immunology.  Finally, tetanus toxoid is an excellent carrier for antigens used to develop vaccines (22).

Question: Is the receptor known for TeNT?

Answer: The receptor tetanus toxin is unknown. However, the toxin has two pockets in the binding domain that could recognize different receptors. It is suggested that the receptor responsible for peripheral paralysis is located on the inside of synaptic vesicles like the receptors for the other clostridial neurotoxins and that the receptor that transports the toxin axonally is accessible to the toxin independently of exocytosis. TeNT like BoNT/A is bound to polysialo-gangliosides that reside on the outer side of the plasma membrane of neurons.

Question: Is the disease tetanus still a health problem?

Answer: With the help of immunization of the population against the disease, tetanus occurs only rarely and in unimmunized people. The WHO recommends boost injections every ten years. Tetanus is quite a problem in developing countries. In some states in Africa for example, many infants die from Clostridium tetani infections that occur when umbilical cords are cut with contaminated tools.

References:

  1. Popoff MR, Bouvet P 2009 Oct; “Clostridial toxins“ Future Microbiol. 4(8):1021-64. PMID: 19824793
  1. Rossetto O, Pirazzini M, Bolognese P, Rigoni M, Montecucco C. 2011 Dec; “An update on the mechanism of action of tetanus and botulinum neurotoxins” Acta Chim Slov. 58(4):702-7 PMID: 24061118
  1. Binz T, Rummel A. 2009 Jun; “Cell entry strategy of clostridial neurotoxins” J Neurochem. 109(6):1584-95. PMID: 19457120
  1. Pirazzini M, Rossetto O, Eleopra R, Montecucco C 2017 Apr, “Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology“ Pharmacol Rev 69(2):200-235 PMID: 28356439
  1. Gu S, Rumpel S, Zhou J, Strotmeier J, Bigalke H, Perry K, Shoemaker CB, Rummel A, Jin 2012 Feb “Botulinum neurotoxin is shielded by NTNHA in an interlocked complex” 24;335(6071):977-81. PMID: 22363010
  1. Benefield DA, Dessain SK, Shine N, Ohi MD, Lacy DB 2013 Apr; “Molecular assembly of botulinum neurotoxin progenitor complexes“ Proc Natl Acad Sci USA 110(14):5630-5 PMID: 23509303
  1. Erdmann G, Wiegand H, Wellhöner HH. 1975 “Intraaxonal and extraaxonal transport of 125I-tet- anus toxin in early local tetanus” Naunyn Schmiedebergs Arch Pharmacol. 290(4):357- 73 PMID: 53793
  1. Surana S, Tosolini AP, Meyer IFG, Fellows AD, Novoselov SS, Schiavo G. 2018 Jun “The travel diaries of tetanus and botulinum neurotoxins” Toxicon 1;147:58-67. PMID: 29031941
  1. Bercsenyi K, Giribaldi F, Schiavo G. 2013; “The elusive compass of clostridial neurotoxins: deciding when and where to go?” Curr Top Microbiol Immunol. 364:91-113 PMID: 23239350
  1. Lalli G, Bohnert S, Deinhardt K, Verastegui C, Schiavo G. 2003 Sep; “The journey of tetanus and botulinum neurotoxins in neurons” Trends Microbiol. 11(9):431-7. PMID: 13678859
  1. Schwab ME, Thoenen H. 1976 Mar “Electron microscopic evidence for a transsynaptic migration of tetanus toxin in spinal cord motoneurons: an autoradiographic and morphometric study” Brain Res. 26;105(2):213-27 PMID: 1260442
  1. Brunger AT, Rummel A. 2009 Oct; “Receptor and substrate interactions of clostridial neurotoxins” Toxicon. 54(5):550-6 PMID: 19268493
  1. Schmitt A, Dreyer F, John C. 1981; “At least three sequential steps are involved in the tetanus toxin-induced block of neuromuscular transmission” Naunyn Schmiedebergs Arch Pharmacol. 317(4):326-30. PMID: 6119629
  1. Schiavo G, Benfenati F, Poulain B, Rossetto O, Polverino de Laureto P, DasGupta BR, Montecucco C. 1992 Oct “Tetanus and botulinum-B neurotoxins block neurotransmitter release by pro- teolytic cleavage of synaptobrevin” Nature. 29;359(6398):832-5. PMID: 1331807
  1. Wiegand H, Erdmann G, Wellhöner HH. 1976; “125I-labelled botulinum A neurotoxin: pharmaco- kinetics in cats after intramuscular injection” Naunyn Schmiedebergs Arch Pharmacol. 292(2):161-5 218
  1. Restani L, Giribaldi F, Manich M, Bercsenyi K, Menendez G, Rossetto O, Caleo M, Schiavo G. 2012 Dec; “Botulinum neurotoxins A and E undergo retrograde axonal transport in primary motor neurons” PLoS Pathog. 8(12) PMID: 23300443
  1. Wiegand H, Wellhöner HH. 1977 Jul; “The action of botulinum A neurotoxin on the inhibition by antidromic stimulation of the lumbar monosynaptic reflex” Naunyn Schmiedebergs Arch Pharmacol. 298(3):235-8. PMID: 895899
  1. Restani L, Novelli E, Bottari D, Leone P, Barone I, Galli-Resta L, Strettoi E, Caleo M. 2012 Aug; “Botulinum neurotoxin A impairs neurotransmission following retrograde transynaptic transport” 13(8):1083-9. PMID: 22519601
  1. Caleo M, Restani L. 2018 Jun “Direct central nervous system effects of botulinum neurotoxin” Toxicon. 1;147:68-72 PMID: 29111119
  1. Bohnert S, Schiavo G. 2005 Dec “Tetanus toxin is transported in a novel neuronal compartment characterized by a specialized pH regulation” J Biol Chem. 23;280(51):42336-44. PMID: 16236708
  1. Gonzales y Tucker RD, Frazee B. 2014 Dec; “View from the front lines: an emergency medicine perspective on clostridial infections in injection drug users” Anaerobe. 30:108-15 PMID: 25230330
  1. Aba YT, Cissé L, Abalé AK, Diakité I, Koné D, Kadiané J, Diallo Z, Kra O, Oulaï S, Bissagnéné E. 2016 Aug; “[Neonatal and child tetanus morbidity and mortality in the University hospitals of Abidjan, Côte d’Ivoire (2001-2010)]” Bull Soc Pathol Exot. 109(3):172-9 PMID: 27177642

List Labs offers citations on our website for easy use by researchers.  At present we have over 3,000 citations from publications around the world, with emphasis on the last 5 years.  We provide information on how to purchase the referenced products and the ability to sort from among the over 100 catalog items we offer here.

We are very appreciative of the work done using our products and the many ways they have been featured in research that’s potentially instrumental in changing the world.

We have gathered citations for our Pertussis product group.  We hope you enjoy this infographic and find it useful.

 

List Labs Pertussis toxins 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. pertussissource 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.

View List Labs Pertussis toxins for sale.

View some of List Labs Pertussis blogs:

A fast, sensitive, specific and accurate detection method to determine active infectionAnthrax detection method

Dr. Nancy Shine
(408) 874-1305
NShine@ListLabs.com

[Campbell, CA, 11/29/2018]

• Method to detect Anthrax before it’s deadly
• Anthrax is a problem for livestock
• Test method is both specific and sensitive for Anthrax
• List Labs is looking to partner on this newly discovered method

Since the intentional release of anthrax spores leading to lethal inhalational
anthrax in 2001, the need for rapid and sensitive detection of infection has been critical.
Unfortunately, early symptoms of infection are similar to those of common illnesses.
While the symptoms are not remarkable, the Bacillus anthracis bacteria enter the
patient’s blood stream and rapidly multiply. This expanding population of bacteria
produces deadly proteins which will eventually overcome the patient. Classical
techniques to detect and identify bacteria in blood take too long. We have devised a
rapid method for detecting one of the proteins produced in the infection. This protein,
anthrax lethal factor, is produced early in infection in a quantity sufficient for detection
making it possible to rapidly determine that a patient is infected and to initiate therapy. A
quick diagnosis is essential for successful treatment of the disease.

Anthrax is not only a bioterrorism threat. There are many areas in the world
where anthrax is endemic. Efforts have been focused on surveillance in countries where
livestock are infected. Contact between infected animals and humans leads to disease.
A quick diagnosis depends on the availability of a rapid, sensitive and simple test.

This paper reports the design of a sensitive and specific test for anthrax infection.
A reagent that specifically detects the presence of low amounts of lethal factor from
anthrax infection is described. This study sets a new standard for a sensitive, simple,
and specific method to detect anthrax infection.

List Labs is looking to partner with an organization that can take this biotechnology to the level of application in the field. Please contact Dr. Shine if you’re interested in partnering.

About List Biological Labs, Inc.

Established since 1978, List Biological Labs, Inc. specializes in native toxins, recombinant proteins, bacteria, Biotherapeutics and GMP products. We develop assays, perform contract manufacturing and produce our own GMP LPS product.

List Labs produces toxins for the research community, including: C. difficile toxin A and toxin B, shiga toxins, cholera toxin, anthrax toxins (PA, LF, and EF), pertussis toxin, diphtheria toxin, CRM197, tetanus toxin, staphylococcal enterotoxin B, botulinum toxins as well as several types of lipopolysaccharides (LPS) or endotoxin for purchase by the research community.

List Labs scours the internet monthly, looking for citations referencing our products. We are highly intrigued by, and love to investigate the different ways researchers use our products. Scientists world-wide have used our toxins for research and have produced interesting results.

List Labs History of working with Botulinum Neurotoxin

List Labs has developed sensitive assays used by pharmaceutical companies, research universities and government agencies to detect Botulinum Toxin Type A in complex samples and to screen for potential inhibitors. List Labs also has a bifunctional assay for Botulinum Type A which measures SV2c receptor binding enzymatic activity.

List Labs is a well-known quality provider of bacterial toxins for research. We harness our vast experience, expertise, state of the art equipment and facilities to bring researchers some of the purest products available.

You can purchase List Labs Botulinum Neurotoxin research reagents here, and view our full catalog of products here.

View our Botulinum neurotoxin citations infographic below:

By: Mary N. Wessling, Ph.D. ELS

List Biological Laboratories, Inc.’s products are being used to confront one of the most pressing problems in health care today: stopping the worldwide spread of illness caused by Clostridium difficile (CD, C Difficile Toxin), a gram-positive, spore-forming anaerobic bacillus. The spores are highly resistant to adverse environmental conditions and are frequently among the contaminants in food products, where they germinate [1]. The CD pathogen causes severe diarrhea and pseudomembranous colitis, among other dangerous gastrointestinal ailments. At present, the estimate is 500,000 cases in hospitals and long-term care facilities, with an annual mortality rate of 15,000 to 30,000 in the US and worldwide [2]. The estimated annual cost of treating CD infections ranges from $436 million to $3.2 billion per year in the US alone [3].

C Difficile Toxin in Hospitals

Historically, CD was mainly a problem for hospitals and long-term care facilities, but infections may spread rapidly into the community, especially among persons who have required antibiotic treatments that kill off competing strains in the intestines and allow CD to multiply. CD is a rapidly evolving bacterium, with hypervirulent strains contributing to the increase in mortality. At present, 234 unique genomes have been identified that cause most of the hospital outbreaks in the US and Europe. The current testing of stool samples to confirm diagnosis requires up to 3 days to differentiate between dangerous CD infection (CDI) and less harmful causes of diarrhea. A delay in diagnosis of CD presents a difficulty in treatment, making the development of more rapid diagnostic techniques a high priority.

C Difficile Toxin Types A & B

Clostridium difficile produces two major toxins, C Difficile Toxin A aka TcdA (Product #152) and C Difficile Toxin B aka TcdB (Product #155), the latter being the more virulent. These toxins inactivate the Rho-GTPase through glycosylation, and the structural bases for their activities have been clarified by X-ray diffraction, biochemical assays, and molecular dynamics [4].  The List Labs Toxin A and B products are playing an important role in the search for a more rapid and accurate methods to diagnose CDI. To meet the demands of those more sensitive and exacting methods, List’s difficile toxins are of higher purity than previously available products.  The purity of List Labs’ toxins enables diverse creative and fascinating scientific approaches that adapt and modify known analytical techniques to CDI testing. What follows (in chronological order of publication) are recent studies that relied on List Labs’ products for their results.

C Difficile Infection Studies

Molecular diagnostic techniques are increasingly being used to quantify the seriousness of infection and to distinguish CDI from other causes. The study by Moura et al [2] used List Labs’ products (purified TcdA and TcdB) for a proteomic analysis that identified and quantified the protein factors involved in CD toxin production through an enhanced mass spectrometric (MS) method. This method provided a basis for development of improved MS methods that demand only small samples and contribute to a better understanding of toxin-mediated illnesses, their prevention and therapy.

In the same year, Lei and Bochner [5] used List Labs’ Toxins A and B in phenotype microarrays (PMs) under different culture conditions. Noting that with the evolution of the CD genome, multiple lineages evolved independently; they examined how the Toxin B cytopathic effect caused cell rounding and used that to measure the virulence of CD under different conditions. Using the PMs, they developed a 1-day test that compared the pure List Labs’ toxins and unpurified toxins to see parallels, which could be measured by colorimetry. This provided a more rapid test than the 3-day diagnostic tests currently in use.

In 2014, Huang et al [6] used CD toxin B to approach the problem of distinguishing between infection, colonization, and live and dead CD organisms. Their real-time microelectronic sensor-based analysis had high sensitivity and relied on reading the impedance of cells applied to microelectrodes to detect specific cellular processes through quantification over time. The method is rapid: the authors reported that 80% positive results were obtained within 24 hours. Concentration of the CD in stool measured by their method correlated with clinical severity, providing a method to monitor the progress of CDI in patients.

In 2014, an innovative study by Leslie et al [7] used human intestinal organoids (HOIs) derived from stem cells to model the disruption of barrier functions in the human intestine by CD. The HOIs were generated by directed differentiation of human pluripotent stem cells, which were then further differentiated into intestinal tissue. These HOI’s were subjected to the C. difficile strain, TcdA or TcdB. Increased toxicity under conditions favorable to production of toxins by CD was measured by presence of cell rounding using fluorescent dextran. Injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable CD.

In 2015, Hong et al [3] used an extension of a method they had developed previously combined with ELISA, eventually applying single-stranded DNA molecular recognition elements (MRE) to microchips. They used List Labs’ lyophilized toxin B, reconstituted and immobilized on magnetic beads; after incubation with an ssDNA library, 80 randomly selected clones were sequenced and analyzed. The goal was to identify ssDNA MREs that bind to toxin B; eventually, fluorescence was used to examine the structure of one selected MRE bound to toxin B. This very complex process yielded an MRE bound with high specificity with toxin B in human fecal matter; it demonstrated a proof-of-concept diagnostic application.

In quite another approach to preventing the morbidity and mortality attributed to CD, a study by Zilbermintz et al [8] showed that the antimalarial drug amodiaquine has a protective effect against CD. The drug was one of a group of existing FDA-approved compounds screened to extend their use to as broad-spectrum, host-oriented therapies. Amodiaquine interferes with the functioning of the host protein cathepsin B targeted by CD and other pathogens as well. The aim of their approach is to find therapies that circumvent the effect of pathogen mutations that lead to drug resistance. All toxins used in the study were purchased from List Biological Laboratories. Though not a new diagnostic technique, this discovery promised an approach to CDI using existing pharmacology, which then could reduce the immense cost of treating CD patients [8].

 

References:

  1. Xiao Y et al. Clostridial spore germination versus bacilli: genome mining and current Food Microbiol. 2011 Apr;28(2):266-74. doi: 10.1016/j.fm.2010.03.016. Epub 2010 Apr 1. PMID: 21315983 
  2. Moura H et al. Proteomic analysis and label-free quantification of the large Clostridium difficile toxins. Int J Proteomics. 2013;2013:293782. doi: 10.1155/2013/293782. Epub 2013 Aug 27. PMID: 24066231 
  3. Hong KL et al. In vitro selection of a single-stranded DNA molecular recognition element against Clostridium difficile toxin B and sensitive detection in human fecal matter. J Nucleic Acids. 2015;2015:808495. doi: 10.1155/2015/808495. Epub 2015 Feb 5. PMID: 25734010
  4. Yin JC et al. Structural insights into substrate recognition by Clostridium difficile sortase. Front Cell Infect Microbiol. 2016 Nov 2a2;6:160. eCollection 2016. PMID: 27921010
  5. Lei XH , Bochner BR. Using phenotype microarrays to determine culture conditions that induce or repress toxin production by Clostridium difficile and other microorganisms. PLoS One. 2013;8(2):e56545. doi: 10.1371/journal.pone.0056545. Epub 2013 Feb 20. PMID: 23437164
  6. Huang B et al. Real-time cellular analysis coupled with a specimen enrichment accurately detects and quantifies Clostridium difficile toxins in stool. J Clin Microbiol. 2014 Apr;52(4):1105-11. doi: 10.1128/JCM.02601-13. Epub 2014 Jan 22. PMID: 24452160
  7. Leslie JL et al. Persistence and toxin production by Clostridium difficile within human intestinal organoids result in disruption of epithelial paracellular barrier function. Infect Immun. 2015 Jan;83(1):138-45. doi: 10.1128/IAI.02561-14. Epub 2014 Oct 13. PMID: 25312952
  8. Zilbermintz L et al. Identification of agents effective against multiple toxins and viruses by host-oriented cell targeting. Sci Rep. 2015 Aug 27;5:13476. doi: 10.1038/srep13476. PMID: 26310922 

By: Suzanne Canada, Ph.D.

Diphtheria toxin is an important tool used for selective killing (ablation) of cells for research purposes.  Using this technique, dubbed “toxin receptor–mediated cell knockout” when it was first used [1], researchers can selectively remove a specific type of cell in a live mouse without having to generate transgenic “knockout” animals, which can be more time-consuming.  The animals are engineered to express a diphtheria toxin (DT) receptor on the surface of a specific cell type.  These animals are normal until exposed to DT, which acts as a potent inhibitor of protein synthesis and kills only those cells that express the DT receptor.  This technique is a powerful tool to explore the role of specific cell types in disease, and is being used to study both the recovery of pituitary cells and the role of T-cells in inflammatory colitis.

The pituitary gland plays an important role in the endocrine system, which presides over growth and development, stress response (adrenal glands), and metabolism (thyroid gland).  Willems and colleagues [2] have been studying the regeneration of the pituitary—research that could lead to methods or therapies to heal pituitary deficiencies.  Transgenic mice that express a DT receptor on the membrane of the growth hormone (GH) cells were treated with DT, which selectively killed those cells.  The researchers then monitored the ability of these ablated cells to regenerate.  Using this technique, they found that stem cells in the pituitary participate in the regeneration process.  Younger mice had a greater ability to recover from injury to the pituitary than older mice.  However, if the injury was prolonged (11 days compared with 3 days) the ability for stem cells to react and aid in recovery could be delayed or even blocked. These researchers may find how stem cells could be activated to boost regeneration of a damaged pituitary gland.

Cell regeneration also plays an important role in the digestive system: researchers are studying how T-cells regulate inflammation in the gut.  Increases in activated T-cells are associated with active flare-ups of ulcerative colitis and Crohn’s disease [Kappler, 2000].  To that end, Boschetti and colleagues [3] used DT to selectively deplete CD4+, CD25+, and Foxp3+ regulatory T-cells [T-regs] in the gut of transgenic mice.  Using that process, the researchers were able to ablate >95% of the T-regs. The proliferation and recovery of the various T-cell subsets in the lymph nodes and colon was monitored using flow cytometry.  By monitoring the recovery of the T-regs, the researchers found that inflammation causes regulatory T-cells to move to the colon lamina propria, and that those cells could suppress proliferation of CD4+ effector cells in vitro.  Although the Foxp3+ T-regs could not completely prevent colitis in the mice, they did reduce the severity of inflammation in the gut.

This technique is a powerful approach to selectively remove certain cells in mice and other model systems where the animals do not naturally have a DT receptor.  The DT from List Labs is recommended for this purpose because its high purity produces the best desired effect.

To read about even more uses of Diphtheria Toxin and other List Labs products, browse our Citations page.

 

References

  1. Michiko Saito , Takao Iwawaki , Choji Taya , Hiromichi Yonekawa , Munehiro Noda , Yoshiaki Inui , Eisuke Mekada , Yukio Kimata , Akio Tsuru & Kenji Kohno (2001) Diphtheria toxin receptor|[ndash]|mediated conditional and targeted cell ablation in transgenic mice. Nature Biotechnology 19, 746–750. PMID: 11479567
  2. Willems, C; Fu, Q; Roose, H; Mertens, F; Cox, B; Chen, J; Vankelecom, H (2016) Regeneration in the Pituitary After Cell-Ablation Injury: Time-Related Aspects and Molecular Analysis.  Endocrinology 157 705-21. PMID: 26653762
  3. Boschetti, G; Kanjarawi, R; Bardel, E; Collardeau-Frachon, S; Duclaux-Loras, R; Moro-Sibilot, L; Almeras, T; Flourié, B; Nancey, S; Kaiserlian, D (2016) Gut Inflammation in Mice Triggers Proliferation and Function of Mucosal Foxp3+ Regulatory T Cells but Impairs Their Conversion from CD4+ T Cells. J Crohn’s and Colitis advanced access publication 30 June 2016.  PMID: 27364948 
  4. Kappeler A1, Mueller C. (2000)The role of activated cytotoxic T cells in inflammatory bowel disease.  Histol Histopathol. 2000 Jan;15(1):167-72. PMID: 10668207

By: M. Wessling

A recently published study by Wen et al [1] provides an encouraging look at a possible therapy for the chronic neuro-inflammatory disease multiple sclerosis (MS). What is even more interesting is that this experimental autoimmune encephalitis myelitis (EAE) study, which used the List Labs Product #180 Pertussis Toxin in mice to induce autoimmune symptoms, supports observations that MS patients and their physicians have accumulated for decades—the relationship between sunlight and MS progression. It also supported a 2015 study by Thouvenot et al [2] that showed a relationship between the degree of disability in fully ambulatory patients with the relapsing-remitting form of MS and Vitamin D deficiency, related in some cases to insufficient exposure to sunlight.

Challenges associated with MS treatments

Introducing and developing new effective treatments for MS is challenging because the disease has different levels and rates of progression in different patients. In fact, there is evidence that 25% to 50% of persons with MS choose not to take existing disease-modifying therapies, even though the risks and benefits are well known.[3]  MS is also approximately 1.5 times more prevalent in women than men, and more frequent in more northerly regions, and  MS is now being recognized more frequently in children than before.[4] Indeed, the prevalence of MS is prone to uncertainty; mostly since MRI, which is used to diagnose MS is not available in poorer regions. Another confounding factor is that in some traditions there may be a perceived cultural association between the symptoms at onset—fatigue, blurring of vision, motion impairments—and moral culpability.

Scientific research for MS treatments is progressing

However, recent years have brought much progress in the analytical scientific methods that show changes in cell structure in in vivo samples from patients with MS. Oxidative stress was identified as a major factor in the cell apoptosis that leads to neurodegenerative diseases, such as MS. The study by EM Kuklina [5] used blood samples from MS patients to conclude that melatonin plays a definitive role as an effective regulator of immune reactions that act through the subset of T lymphocytes producing IL-17 (Th17). As the Th17 subset plays a key role in MS pathogenesis, the results suggest that melatonin could nullify the immunomodulating hormone effects toward Th17.

The Wen EAE study bridges the gaps between scientific theory and human observation using genetically characterized mice under carefully controlled conditions. This study therefore could lead to new and effective therapies for MS. Going beyond previously published studies on the effects of melatonin, these authors examined the effects of treatment with melatonin and its precursor, N-Acetylserotonin (NAS), on neurodegenerative symptoms that parallel those in human MS. The EAE results showed clinical improvement that included reduced inflammatory markers and free radical generation, and sparing of axons, oligodendrocytes, and myelin. Further, the proposed mechanism would explain how the decline in motor symptoms in the NAS and melatonin-treated mice is linked to the role of Th1 cytokines in MS progression.

While it’s very different from our Pertussis Toxin, another List Labs product used in Multiple Sclerosis research is Epsilon Toxin (Product #126A). Read about it here: Epsilon Toxin: A Fascinating Pore Forming Toxin That Crosses The Blood Brain Barrier

See other uses of List Labs products in recent research on our Citations page, featuring thousands of articles with abstracts.

 

References

  1. Wen J, Ariyannur PS, Ribeiro R, Tanaka M, Moffett JR, Kirmani BF, Namboodiri M, Zhang Y. Efficacy of N-Acetylserotonin and Melatonin in the EAE Model of Multiple Sclerosis. J Neuroimmune Pharmacol 2016 Aug. 25 [Epub ahead of print]. PMID: 27562847
  2. Thouvenot E, Orsini M, Daures J-P, Camu W. Vitamin D Is Associated with Degree of Disability in Patients with Fully Ambulatory Relapsing-Remitting Multiple Sclerosis. Eur J Neurol 2015, 22: 564-569. PMID: 25530281
  3. National Multiple Sclerosis Society. Multiple Sclerosis FAQ’s. New Research Fall 2015. Acquired 10/30/2016.
  4. Alonso A, Hernán MA. Temporal Trends in the Incidence of Multiple Sclerosis: A Systematic Review. Neurology 2008; 71:129-135. 2008. PMID: 18606967
  5. Kuklina EM. [Melatonin as an Inducing Factor for Multiple Sclerosis.] Zn Nevrol Psikhiatr Im S S Korsaakova. 116 (5):102-5. 2016. [Article in Russian]

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

Concerns over outbreaks of potentially serious childhood diseases are in the news again in California with an increase in whooping cough infections in 2014, caused by the pathogen Bordetella pertussis, and a measles outbreak in December 2014 to February 2015.  Since outbreaks and epidemics may be infrequent, many of us do not realize that vaccine development is an ongoing process. Public health experts monitor changes in the predominant strains and pathogen virulence continuously.  Although the sudden upswing in cases is alarming for those with young children, fuller understanding of the cyclic nature of these outbreaks fosters refinements to public health practices as well as the vaccines.

Recent increases in the number of cases of pertussis infections are due to eroding immunity in the population of immunized individuals, as well as changes in the prevalent virulent strains.  Outbreaks of new strains or new agents occur every 6 months or so [1]. For example, every 4 or 5 years, a new increase in pertussis infections is observed [2]. However, development of safe and effective vaccines is a process that takes up to 15 years [1].  Therefore, health agency efforts must be ongoing and constant funding must be in place to have these vaccines ready when the public needs them.

Although we are observing outbreaks of whooping cough and measles in the past year, the rates of infection are still much lower than what was observed in the pre-vaccine era.  In the pre-vaccine era (prior to the 1940s in the USA), 157 serious pertussis infections occurred yearly per 100,000 cases, including ~15 infant deaths/100,000 cases [2].  The highest incidence recorded was 260,000 cases in 1934 [3]. Since the broad adoption of vaccinations, whooping cough is much rarer during most years; upswings were observed in California in 2010 and again in 2014.  In 2010, the most serious cases were seen among infants under one year old.  The experts then realized that they could best control the rate of serious infections in infants by immunizing mothers at G27-36 weeks or immediately after the birth [4], and by making sure everyone in the expectant family was also immunized.  Furthermore, the 2014 outbreak of whooping cough was highest among 15 year olds (137.8 cases/100,000) [2].  By gathering immunization records and analysis of blood samples for anti-pertussis antibodies among those who had an infection, the doctors deduced that eroding resistance to pertussis corresponded with the use of the acellular pertussis vaccine.

The acellular pertussis vaccine was developed in the 1990’s in response to a high rate of side effects (fever) in those receiving the whole cell vaccine.  The acellular vaccine uses a few especially prominent antigens (pertussis toxoid, filamentous heamagglutinin, and pertactin, among others) that are specific to all pertussis bacteria. In the late 1990s, the FDA recommended replacement of the whole cell vaccine with the acellular vaccine which doesn’t cause as much fever and discomfort following vaccination boosters.

Investigations of why the acellular pertussis vaccine is not conferring resistance as durable as others, such as the tetanus or diphtheria vaccines, are ongoing.  Some vaccine experts point to evidence that resistance to whooping cough isn’t as durable with the acellular vaccine [5, 6]; however, other analyses conclude that the genes encoding antigens targeted by acellular pertussis vaccines are changing at higher rates than other surface-protein encoding genes of the pathogen [7, 8].  A meta-analysis of different immunization schedules found that resistance depended on time since last immunization or exposure [9], with resistance dipping by 5 years after the last booster shot.  Some researchers have found that having more anti-pertussis antigens in the vaccine conferred a higher level of protection [10], and new antigens for the vaccine are under evaluation (e.g., LpxL 1 [11]).

Several virulence factors of B. pertussis are available for research purposes from List Labs including pertussis toxin (Products #179, #180 and #181), pertactin (Product #187), fimbriae 2/3 (Product #186), adenylate cyclase toxin (Product #188 and 188L),  filamentous heamagglutinin (#170) and lipopolysaccharide (Product #400). Inactivated toxins, known as toxoids, are frequently used in the vaccines.  List Labs offers toxoids of C. difficile toxins (Products #153 and #154), diphtheria toxin (Product #151), Staphylococcus aureus enterotoxin B (Product #123), tetanus toxin (Product #191) and botulinum neurotoxin types A and B (Product #133 and #139, respectively).  List Labs’ vaccine carrier proteins are provided for research use only; however, GMP material may be produced on a contract basis.  More information is available on our website: www.listlabs.com.

 

References

  1. Rappuoli, R., Vaccines, Emerging Viruses, and How to Avoid Disaster. BMC Biology, 2014. 12: p. 100. PMID: 25432510
  2. Winter, K., et al., Pertussis epidemic–California, 2014. MMWR Morb Mortal Wkly Rep, 2014. 63(48): p. 1129-32. PMID: 25474033
  3. CDC, Pertussis Vaccination: Use of Acellular Pertussis Vaccines Among Infants and Young Children Recommendations of the Advisory Committee on Immunization Practices (ACIP) Morbidity and Mortality Weekly Review, 1997. 46: p. 1-25. PMID: 9091780
  4. Raya, B.A., et al., Immunization of Pregnant Women Against Pertussis: The Effect of Timing on Antibody Avidity. Vaccine, 2015. 33(16):1948-52 PMID: 25744227
  5. Silfverdal, S.A., et al., Immunological Persistence in 5 y olds Previously Vaccinated with Hexavalent DTPa-HBV-IPV/Hib at 3, 5, and 11 Months of Age. Hum Vaccin Immunother, 2014. 10(10): p. 2795-8.
    PMID: 25483640
  6. Hara, M., et al., Pertussis outbreak in university students and evaluation of acellular pertussis vaccine effectiveness in Japan. BMC Infect Dis, 2015. 15(1): p. 45. PMID: 25656486
  7. Sealey, K.L., et al., Genomic Analysis of Isolates From the United Kingdom 2012 Pertussis Outbreak Reveals That Vaccine Antigen Genes Are Unusually Fast Evolving. J Infect Dis, 2014. 212(2): p. 294-301. PMID: 25489002
  8. Torjesen, I., Proteins Targeted by Pertussis Vaccine Are Mutating Unusually Quickly, Study Finds. BMJ, 2014. 349: p. g7850. PMID: 25552634
  9. McGirr, A. and D.N. Fisman, Duration of Pertussis Immunity After DTaP Immunization: A Meta-analysis. Pediatrics, 2015. 135(2): p. 331-343. PMID: 25560446
  10. Tefon, B.E., E. Ozcengiz, and G. Ozcengiz, Pertussis Vaccines: State-Of-The-Art and Future Trends. Curr Top Med Chem, 2013. 13(20): p. 2581-96. PMID: 24066885
  11. Brummelman, J., et al., Modulation of the CD4(+) T cell response after acellular pertussis vaccination in the presence of TLR4 ligation. Vaccine, 2015. 33(12): p. 1483-91. PMID: 25659267

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: Suzanne Canada, Ph.D.
Tanager Medical Writing

An exciting report was released in October about a new class of targeted anti-tumor drugs, in which genetically engineered stem cells were used to deliver cytotoxins to brain tumors.1 Brain cancers known as glioblastomas (GBM) are notoriously difficult to treat because the tumors often re-grow after surgery and because most standard cancer therapies cannot pass the blood-brain barrier. Those cancer therapies that can reach the tumors must be delivered at high doses which can be toxic to the entire body, without specifically targeting the GBM tumor. In this case, a research team at Massachusetts General Hospital (MGH) in Boston used stem cells, added to mouse brain tumors after surgery, to deliver Pseudomonas exotoxin directly at the site of the tumor itself. 2

Although this research is cutting-edge and an exciting development for GBM patients, the idea of using toxins attached to targeting molecules such as antibodies or specific ligands has long been explored as a way of fighting diseases, especially cancer. One popular approach has been to use antibodies linked to toxins to aid in targeting the therapy. (See Chari 2008 and Goldmacher 2011 for reviews).3, 4 An example is the approach taken by group of researchers looking for ways to increase the effectiveness of Herceptin®, a monoclonal antibody that is best known for targeting HER-overexpressing malignant breast cancer tumors. Antibody was coupled to both diphtheria toxin and multi-walled carbon nanotubes. They found that both conjugates were more effective in specifically killing HER-2 expressing cells than Herceptin® alone.5

An elegant approach to targeting toxins is to activate the toxin by cleavage at the site of therapy. This is precisely the approach used by Schafer and colleagues.6 Their model system exploited the fact that metalloproteinases are commonly overexpressed on the surface of squamous cell cancers. Anthrax toxin was engineered to be activated by cleavage by urokinase plasminogen activator (uPA) on the cell surface and metalloproteinases. This approach seemed to work on xenografted human head and neck squamous cell carcinoma (HNSCC) cell lines by inducing apoptotic and necrotic tumor cell death. However, cultured cancer cell lines were found to be insensitive to the engineered toxin, so the researchers concluded that the regulation of two-fold activation was not straightforward as anticipated.

Shiga toxin– produced by an organism responsible for bacterial dysentery – has properties that could be harnessed for cancer research7. A group of researchers took advantage of the binding of the Shiga toxin B pentamer to the glycosphingolipid globotriaosylceramide (Gb3) on the cell surface. After binding, the Shiga toxin complex is internalized by eukaryotic cells where the Shiga toxin A moiety can exert its toxic effect. Gb3 is reportedly over-expressed in throat, gastric, and ovarian cancers—and researchers hope that this overexpression pattern could be used to attain more targeted therapy. Specific binding of GB3 by the Shiga toxin B pentamer could also be exploited for imaging of these tumors and for delivering a genetically engineer Shiga toxin A chimera that would only be activated in cancer cells.

In their quest for new and more effective therapies, researchers have noted that bacterial toxins are examples of highly toxic, but also targeted and regulated systems that have co-evolved with the eukaryotic hosts (humans).8, 9 In the words of Fabbri et al., “Knowledge of their properties could be used for medical purposes.”  List Biological Laboratories, Inc. provides purified bacterial toxins for research purposes, including Anthrax toxins (Product # 169, 172, & 176), Shiga toxins (Product # 161 & 162), Diphtheria toxins (Product # 149, 150, & 151), and others.

 

  1. Paddock C., (2014) Stem cells that release cancer-killing toxins offer new brain tumor treatment. Last accessed: 06 January 2015.
  2. Stuckey DW, Hingtgen SD, Karakas N, Rich BE, Shah K (2015) Engineering toxin-resistant therapeutic stem cells to treat brain tumors Stem Cells 33(2):589-600. doi: 10.1002/stem.1874. PMID: 25346520
  3. Chari RV (2008) Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc Chem Res 41(1):98-107. PMID: 17705444
  4. Goldmacher VS, Kovtun YV (2011) Antibody-drug conjugates: using monoclonal antibodies for delivery of cytotoxic payloads to cancer cells Ther Deliv 2(3):397-416. PMID: 22834009
  5. Oraki KM, Mirzaie S, Zeinali M, Amin M, Said HM, Jalaili A, Mosaveri N, Jamalan M (2014) Ablation of breast cancer cells using trastuzumab-functionalized multi-walled carbon nanotubes and trastuzumab-diphtheria toxin conjugate Chem Biol Drug Des 83(3):259-65. PMID: 24118702
  6. Schafer JM, Peters DE, Morley T, Liu S, Molinolo AA, Leppla SH, Bugge TH (2011) Efficient Targeting of Head and Neck Squamous Cell Carcinoma by Systemic Administration of a Dual uPA and MMP-Activated Engineered Anthrax Toxin. PLoS ONE 6(5): e20532. PMID: 21655226 
  7. Engedal N, Skotland T, Torgersen ML, Sandvig K (2011) Shiga toxin and its use in targeted cancer therapy and imaging Microb Biotechnol 4(1):32-46. PMID: 21255370
  8. Barth H, Aktories K, Popoff MR, Stiles BG (2004) Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins Microbiol Mol Biol Rev 68(3):373-402.
    PMID: 15353562
  9. Fabbri A, Travaglione S, Falzano L, Fiorentini C (2008) Bacterial protein toxins: current and potential clinical use Curr Med Chem 15(11):1116-25. PMID: 18473807

Fundastic believes in being objective and transparent when it comes to funding and financing for small businesses. The following is an interview between Fundastic writer Sarah Tang and List Labs President Dr. Karen Crawford.
View the original post here.

By: Sarah Tang
Fundastic Business Owner Stories

Dr. Karen Crawford is the president of List Labs, a manufacturing and contracting lab that was the first of its kind to commercialize many bacterial toxins for research. Since its establishment by Linda Shoer in 1978, List Labs has been female owned and operated. First a scientist, then a businesswoman, Dr. Crawford brings an analytical mindset to List Lab’s unique business operations.

The Start

How did you start your business?

Linda Shoer had her PhD, post-doctorate, and she wanted a career in science. Her sisters were also PhD scientists, and one was working at a pharmaceutical company which at the time was developing a vaccine against cholera. Shoer saw cholera toxin as a profitable product. Cholera toxin was not only needed for vaccine development but it was also useful in cell research. She approached a distributor and made a deal that if she produced a batch they would buy the product. Cholera toxin was List Lab’s first product.

I joined List Labs in 1989. My PhD involved growing bacteria and to learn how viruses replicated. I began my career teaching science. I moved to California when I had my two boys and at that stage of my life I wanted to do something related to children. I volunteered and became a science teacher in the Saratoga area. I left when I saw school funding decreasing. My kids had grown-up by then, too. So I interviewed with List Labs. Shoer’s work seemed truly interesting. They were producing many different products with a small team of about 10. We’re about 24 now, producing 100 different products.

How did you fund your business in the beginning?

Shoer took a small loan from local bank. It was just her in the beginning. She kept the operations small. Her first employee was the landlord’s granddaughter and they were just a few blocks away from our current location in a small 3-room lab.

Running the Business

How did you learn to run the business?

As a scientist I need to understand a problem and think about how to address it. It is the same for business, but in managing a company you work at a different level. My personality is about getting into the details to learn a lot about one thing. In business you become a person who knows a little about a lot. I need to know about insurance coverage, finances, dealing with personnel. But luckily because I have good people working for me I don’t have to know a lot about those things, just something.

First Customer?

Sigma, a distributor, was our first customer. Our customers are vaccine companies, universities, hospitals and government research. The product focus of our business is driven by the customers and they have changed dramatically since 1978. There was a point when almost everything we did was to support anthrax vaccine development. We were in the process of making non-toxic anthrax products to test vaccines when some people released anthrax spores wanting to create havoc. There was a lot of government funding going in that direction so our business shifted its focus to the anthrax product line.  While switching focus to meet the needs of customers we have maintained a steadily growing product line.

Now people are into other things. For instance there’s a lot of money going into research with emerging viruses right now. The government wants bio-security, companies want to develop vaccines, and universities want to figure out how things work, and we have to understand all these points of view.

What’s the biggest mistake you made in the first year?

Not making the business modular. I brought the business into the current building which is bigger. A bigger facility means you need more business. There’s a lot of controlling factors on the design of a biological containment facility, and we wanted a design that could support different kinds of projects. If the business had been more modular you could take a piece out and put it to rest when you don’t have the business to fill it. But we built the facility as a single unit so we have to maintain the whole thing and that becomes a financial responsibility.

What’s the smartest thing you did in the first year?

Moving the business to a bigger facility – it’s the bad and the good. Overall it’s great. It gives us a lot more opportunities that wouldn’t come with a smaller less well designed facility.

What’s the most rewarding thing about running your own business?

This work is really interesting. It was always my dream to go into research and in this business; I support research in labs throughout the world.

What’s the most challenging thing about running your own business?

Keeping projects coming in to fill our capacity. Marketing is our solution to that. Marketing used to be word of mouth. People would cite us in papers under their list of materials, and if someone wanted to try the same or similar experiment they would order from us. It used to be your network was the people you meet at meetings, people from your school and the people in the lab with you. We didn’t have the social media we have today which makes networking easier.

What’s the most surprising thing about running your own business?

I’m always surprised when people gather up forces and help me achieve something that I see as a goal. That’s always a delight. Somehow I feel like, oh my goodness, first off this has to be done and, two,  I’ve got to do it myself. But then someone steps up and helps me do it.

What business owner or entrepreneur do you admire most?

It would have to be Linda Shoer, the one who started the business; she was fearless. She was able to go out and do cold-call kind of introductions to get business. She did quite well that way. She was also good at making relationships with people that could help her. She had a good way of getting people to feel that she needed their help and they would help her. I think women can be especially good at that, appearing to need help.

What I’ve Learned

What do you wish you had known before you had started your business?

Establish good contacts– people who can do things for you, because you can’t do everything yourself. When I first started Linda had a handful of people she could always call. There was the the electrician, the accountant, the lawyer you could always call. The business has become more complicated as we’ve grown but we always have people we can call on.

If you could go back to when you were starting your business, what advice would you give yourself?

If I were to do another business like this I would make it more modular. It’s hard to imagine it when you have a business that depends upon a lot of infrastructure. This is a pretty unusual business and it’s hard to be ready when the business expands and contracts. It’s not anything I’ve seen done, to have a facility that’s a big shell and having little functional pieces that can be put together, but I think it can be done.

 

About the Author — Sarah is a recent graduate of UC Berkeley where she learned to love the diverse personalities of mom-and-pop stores. She likes intriguing storefronts, creative specialty stores, and well-designed business websites.

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

 

Three anthrax toxin components—Protective antigen (PA), edema factor (EF) and lethal factor (LF) are available for research purposes from LIST Biological Laboratories, separately at a high level of purification. At least two out of three of these components are necessary to enter a mammalian cell and exert a toxic effect.

With the aim of developing antitoxin therapies, scientists have been investigating the structure of PA, EF, and LF, and the complexes that they form with mammalian cell surface receptors, in hopes of finding the best way to disrupt or block the toxicity. Previously, NIAID-supported scientists have shown that protective antigen can bind edema factor and lethal factor at the same time, forming a greater variety of toxin complexes than were formerly known.1 They also had produced a three-dimensional molecular structure of the anthrax protective antigen protein bound to one of the receptors (CMG2) it uses to enter cells.2 More recently, a group of students in Kansas used Jmol and 3D printing technology to model and Anthrax toxin heterotrimer (PA, EF and LF) which forms a pore in the mammalian cell surface.

In an in vitro disease model, researchers constructed an artificial membrane bilayer using lipid and demonstrated that the blood of animals carrying anthrax infections was able to disrupt this membrane, a model of the cell membrane.  Membrane disruption requires acidification, and therefore the membrane remains intact until the pH is lowered.  When the pH is lowered to the required level for toxin complex binding, the membrane is disrupted by the anthrax toxin already embedded in it.4

Anthrax researchers have explored ways to protect healthcare workers and others who may have been exposed or are likely to be infected. One group of scientists has investigated the feasibility of RNA silencing technology (siRNA) to block expression of the anthrax toxin PA receptors on the cell surface, two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2).  Blocking expression of the receptors was reported to provide almost complete protection against the LF intoxication in mice, and also protected against LF effects in human kidney cells as well as macrophage-like cells.5 

Methods of vaccination have been under investigation for some time, as one of the most likely methods to provide lasting protection against anthrax infection.  In another 2014 publication, researchers at the University of Texas have reported success in vaccinating guinea pigs against anthrax infection using vaccines based on DNA-protein antigen components as well as another based on recombinant protein components.  After immunization, the animals were challenged with a lethal dose of B. cereus G9241 aerosol.  Complete protection against lethal challenge was observed in all guinea pigs that had a detectable pre-challenge serum titer of toxin neutralizing antibodies.6

List Biological Laboratories, Inc. offers EF (Product number 167A), LF (176), and PA (171) as well as the antibodies for their detection (773, 769 and 772, and 771, respectively). Refer to the website: https://www.listlabs.com/product-information/anthrax-toxins/ for more information.

 

References

  1. NIAID website: http://www.niaid.nih.gov/topics/anthrax/pages/default.aspx
  2. CDC website: http://www.cdc.gov/anthrax/
  3. Andrews J, Chick A, Chao T, Chogada V, Douglas A, Florack A, McCormick S, Kessler E, Tuel K, Whalen J and Fisher M (2014) The Anthrax Toxin Heterotrimer: Explorations of the Protective Antigen and Edema and Lethal Factors (LB98). FASEB J 28:LB98 
  4. Nablo BJ, Panchal RG, Bavari S, Nguyen TL, Gussio R, Ribot W, Friedlander A, Chabot D, Reiner JE, Robertson JW, Balijepalli A, Halverson KM, Kasianowicz JJ (2013) Anthrax toxin-induced rupture of artificial lipid bilayer membranes J Chem Phys 139(6):065101. PMID: 23947891
  5. Arévalo MT, Navarro A, Arico CD, Li J, Alkhatib O, Chen S, Diaz-Arévalo D, Zeng M  (2014) Targeted silencing of anthrax toxin receptors protects against anthrax toxins.  J Biol Chem 289(22):15730-8. PMID: 24742682
  6. Palmer J, Bell M, Darko C, Barnewall R, Keane-Myers A. (2014) Protein- and DNA-based anthrax toxin vaccines confer protection in guinea pigs against inhalational challenge with Bacillus cereus G9241. Pathog Dis 72(2):138-42. PMID: 25044336

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:

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.

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

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

 

The world of microbial pathology is often understood as a system of various organisms who are trying to survive by using plants or animals as “hosts”. The dynamics of these systems can described the pathogen as trying to establish a living space for itself, while the “host” does its best to evict these unwelcome tenants.  One of the most commonly known pathogens is Staphylococcus, a highly adaptable and ubiquitous opportunistic pathogen.  The staphylococcal enterotoxins are small heat-stable proteins used by Staphylococcus strains to help them in their attempt to colonize.

One of the most notable and yet poorly understood properties of Staphylococcal enterotoxin B (SEB) is its potent ability to stimulate immune T-cell proliferation.  It doesn’t make sense that a bacterium that is intent on colonization would find it advantageous to promote an immune response.  On the other hand, it could have been coincidence or selective adaptation that the mammalian immune system learned to recognize one of its most ubiquitous enemies. After many of years of observation by immunologists that lymphocytes proliferated when enterotoxin was added to whole blood in test tubes, John Kappler dubbed them superantigens (Kappler, 1989).

The following references use SEB as a control for stimulate T-cells:

Use our handy citations finder to search for our Staphylococcal toxins used in research.

The significance of T-cell stimulation by SEB has been the topic of much hypothesis and discussion. The site of SEB binding to T-cells has been investigated to a molecular level (Li, 1998; Saline, 2010).  SEB and other enterotoxins are used in investigations of innate immunity to bacterial infections, signaling by toll-like receptors, and the modulation of inflammatory responses, especially cytokine stimulation (Kumar, 2010; Ortega, 2010; Vidlak, 2011; Edwards,  2012).  The function, origin, and role in adaptation that t-cell stimulation serves will be a topic of ongoing scientific debate. In any case it is clear that SEB and other bacterial superantigens are very good at bypassing the antigen recognition by T-cells (Sundberg, 2002; Kumar, 2013).  Some investigation into the biochemical level of SEB recognition by T-cells has indicated that this protein shares antigenic sequences with known self-antigens in mammals (White, 1989).  This observation supports the theory that activation of T-cells plays a role in noninfectious diseases like autoimmune responses (Edwards, 1996; Kumar, 1997; Li, 1996; Sundberg, 2002), which might make SEB an appropriate stimulator when studying these mechanisms of disease.  List Labs provides for SEB produced in a native Staphylococcus aureus, for research purposes that provides potent stimulation of the immune system and cytokine production.

  1. Edwards CK., Zhou T., Zhang J., Baker TJ., De M., Long RE., Borcherding DR., Bowlin TL., Bluethmann H., Mountz JD., (1996) Inhibition of superantigen-induced proinflammatory cytokine production and inflammatory arthritis in MRL-lpr/lpr mice by a transcriptional inhibitor of TNF-alpha. J Immunol 157(4): 1758-1772. PMID: 8759766
  2. Edwards LA., O’Neill C., Furman MA., Hicks S., Torrente F., Pérez-Machado M., Wellington EM., Phillips AD., Murch SH., (2012) Enterotoxin-producing staphylococci cause intestinal inflammation by a combination of direct epithelial cytopathy and superantigen-mediated T-cell activation. Inflamm Bowel Dis 18(4): 624-640. PMID: 21887731
  3. Kappler J., Kotzin B., Herron L., Gelfand EW., Bigler RD., Boylston A., Carrel S., Posnett DN., Choi Y., Marrack P., (1989) V beta-specific stimulation of human T cells by staphylococcal toxins. Science 244(4906): 811-813. PMID: 2524876
  4. Kumar S., Colpitts SL., Ménoret A., Budelsky AL., Lefrancois L., Vella AT., (2013) Rapid alphabeta T-cell responses orchestrate innate immunity in response to Staphylococcal enterotoxin A. Mucosal Immunol 6(5): 1006-1015. PMID: 23321986
  5. Kumar S., Ménoret A., Ngoi SM., Vella AT., (2010) The systemic and pulmonary immune response to staphylococcal enterotoxins. Toxins (Basel) 2(7): 1898-1912. PMID: 22069664
  6. Kumar V., Aziz F., Sercarz E., Miller A ., (1997) Regulatory T cells specific for the same framework 3 region of the Vbeta8.2 chain are involved in the control of collagen II-induced arthritis and experimental autoimmune encephalomyelitis. J Exp Med 185(10): 1725-1733. PMID: 9151697
  7. Li H., Llera A., Tsuchiya D., Leder L., Ysern X., Schlievert PM., Karjalainen K., Mariuzza RA., (1998) Three-dimensional structure of the complex between a T cell receptor beta chain and the superantigen staphylococcal enterotoxin B. Immunity 9(6): 807-816. PMID: 9881971

 

List Biological Laboratories, Inc. is pleased to announce a key addition to our Clostridium difficile Toxin A and Toxin B product line. Our new product, 155L is a liquid formulation of Toxin B. This product allows for the highest activity level of any of our C.difficile offerings and the most stability.  It already shows stability for several months when stored at 2-8°C and we will continue to update.

This product is offered in a 50 µg size for $450.

We are very excited about the liquid formulation and the flexibility it will allow in the lab. It is now available from stock, and ships on Blue Ice throughout the U.S. and Canada and worldwide with special arrangements.

Clostridium difficile Toxin A
We have enjoyed tremendous success with our Toxin A. We have determined that the best activity and stability post-reconstitution (up to 2 months) is in the 100 µg size, #152C. The price for this product has been lowered from $535 to $300 based on the dramatic improvements in our yield and the substantial growth in overall sales. We are happy to pass this savings on, making it a terrific value for our C.difficile customers.

Clostridium difficile Toxin B
Products 155A and 155B have a newly formulated buffer which allows for highly active Toxin B. After reconstitution, this product is stabile for days when stored at 2-8°C and is now available from stock.

Related Products
We offer a variety of Binary Toxins and Antibodies to complement your C.difficile research. We have Binary Toxins, both A and B subunit. Check our website for more information.

Bulk Product Orders
Should you have need of a substantial quantity of C.difficile in bulk, please let us know what quantity and formulation you desire. With sufficient quantity, we can often tailor to your specific needs. We generally have bulk available for all C.difficile offerings.  We also offer other types of bulk reagents for purchase.

Coming Soon-Pipeline
Clostridium difficile produces and secretes a glutamate dehydrogenase (cdGDH). This enzyme is highly conserved among different ribotypes, and antibodies to cdGDH are often included in detection kits for C. difficile infection (CDI), increasing sensitivity. While cdGDH is available recombinantly, we have isolated and purified the cdGDH from a native strain of C. difficile. In addition to the enzyme, chicken antibodies will be generated and made available soon.

We welcome your feedback on these changes and would love to hear how the new formulations and new products work for you. Please contact us with your comments and feedback.

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.