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By: Karen Crawford, PhD.
President, List Labs

Staphylococcal bacteria

Staphylococcal bacteria

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

 

SEB Toxin’s Associations with Human Diseases

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

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

 

Some papers utilizing SEB toxin are described below:

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

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

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

 

References

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

 

By: Rachel Berlin, Marketing Manager

Microbiome Research ReagentsMicrobiome research is uncovering the enormous potential for developing drugs, such a live biotherapeutic products, from the microbiome.  This burgeoning field is the future of medicine.

List Labs is excited and proud to support microbiome research by providing reagents to scientists studying the human microbiome. Below is a list of microbiome research studies that have used List Labs’ research reagents such as Athrax, Pertussis, Cholera and Difficile Toxins.

Pertussis Toxins Used in Microbiome Research:

Difficile Toxins Used in Microbiome Research: 

Cholera Toxins Used in Microbiome Research: 

Anthrax Products

 

In addition to producing products for microbiome research, List Labs also provides contract GMP manufacturing of live biotherapeutic products for phases 1-3 of clinical trials. For more information on microbiome and live biotherapeutics- check out this blog post or watch our informative video. See how scientists have used our products in their research on our citations page.

Contact us to discuss your next project!

Botulinum Toxin Protein

3D Rendering of Botulinum Toxin Protein

By: T.J. Smith

Following a botulism outbreak due to contaminated ham that severely sickened 10 and resulted in the death of three people in Ellezelles, Belgium, a review and case study on botulism was published by a researcher named Emile Van Ermengem (Van Ermengem 1897).  While Van Ermengem was not the first to study this syndrome, his article supplied critical information defining botulism as a type of food poisoning having specific paralytic symptoms.   He determined that the illness was an intoxication, not an infection, and that its cause was a bacterial toxin.  He was also able to isolate and characterize the organism responsible for the toxin as an anaerobic spore-forming bacillus, which he named Bacillus botulinus (later renamed Clostridium botulinum).  “Botulinus” is the latin word for sausages, and this nomenclature was used due to the historic link between botulism and improperly processed sausages, particularly blood sausages.  His careful and painstaking research provided the foundation for future studies on botulism, its causes and treatments.

During this time, botulism was thought to be related specifically to improperly processed meat, such as sausages and ham, and caused by a single monospecific toxin.  Within the next decade both of these theories were proven wrong.  In 1904, Dr. G. Landmann isolated a botulinum toxin-producing bacterial strain from preserved bean salad which had caused 11 deaths in Darmstadt, Germany (Landmann 1904).  This was the first reporting of botulism due to a source other than meat or fish.  In 1910, Leuchs showed that the Ellezelles strain from Van Ermengem and the Darmstadt strain from Landmann were immunologically distinct toxins, providing the first evidence that all botulinum neurotoxins were not the same (Leuchs 1910).  This marked the beginning of a century of study related to botulinum neurotoxin diversity which included clinical case reviews and morphological, immunological, and, most recently, genetic studies.

As the bacteria responsible for botulinum neurotoxins initially seemed to be nearly identical in morphology and cultural characteristics, early delineations were the result of serological studies, which were apparently the “hot new thing” of the day.  Antisera produced using a particular bacterial strain was tested against other strains to determine relationships among both the toxins and the bacteria that produced them.  The assays were both qualitative and quantitative and included agglutination, immuno-absorption, and neutralization techniques (Schoenholz and Meyer 1925).  They were originally targeted to both the toxins and the bacteria that produced them, but the emphasis quickly shifted to neutralization of toxins using specific antisera.  These antisera, which were predominantly of equine origin, were developed for treatment purposes as well, and they continue to be the only approved treatment for botulism to this day.  In 1919, Georgina Burke produced antisera from three strains isolated in California, Oregon, and New York, and she was able to show that the toxins from the two West Coast strains were immunologically identical, while the New York toxin was distinct (Burke 1919).  She identified these toxins as type A (West) and Type B (East).  Later studies of U. S. strains by K. F. Meyer and B. Dubovsky substantiated her findings (Meyer and Dubovsky 1922).

In the following decades several additional serotypes were identified.  In 1922, Dr. Ida Bengtson reported a toxin from a C. botulinum strain that was not neutralized by either type A or type B antisera, which she designated type C (Bengtson 1922).  The bacterial strain was isolated from fly larvae that proved to be causative agents in the intoxication of chickens that had ingested these larvae.  This illustrates that botulism is not restricted to humans but rather can be seen in a wide variety of animals as well.  In fact, differential sensitivities of the toxins in animals has formed a background for discerning various toxin types.  In addition, catastrophic losses due to botulism have been noted in domestic fowl, cattle, horses, and even minks and foxes, prompting the development and use of vaccines in these animals for protection.   H. R. Seddon isolated a culture that apparently produced type C toxin from an outbreak in cattle in Australia (Seddon 1922).  The toxin could be neutralized by Bengtson’s antisera, however, the reverse was not true.  This “one-way” neutralization was the first of several anomalies that were discovered when serotyping botulinum neurotoxins.

In 1929, Meyer and Gunnison showed that the toxin from a culture isolated by Theiler and associates in South Africa was immunologically distinct from types A, B, or C.  This toxin, which was also related to intoxication in cattle, was designated type D (Meyer and Gunnison 1929).

In the following decade, several botulism cases were noted that were related to ingestion of fish.  While Russian scientists were the first to note these unusual cases of botulism, it was Dr. Janet Gunnison who determined the toxins were a new type, and Dr. Elizabeth Hazen who published initial reports on type E botulism cases (Gunnison, Cummings et al. 1936, Hazen 1937).  Outbreaks due to dried, smoked, or fermented fish, fish eggs, whale blubber, and seal or walrus meat are common, but there have been rare type E cases related to other foods as well.

The first case due to type F was linked to an outbreak involving duck paste on Langeland Island, Denmark, in 1958 (Moller and Scheibel 1960).  Reported cases due to type F are rare and have been restricted to humans so far.  Type G was isolated from a cornfield in Argentina in 1969 as part of a soil sampling study conducted by Dr D. F. Gimenez and Dr. A. S. Ciccarelli (Gimenez and Ciccarelli 1970).  This type is unusual in that there are no direct reports of intoxications due to type G in people or animals.  However, a study of autopsy materials related to sudden deaths due to unknown causes in Switzerland identified type G producing organisms among the samples (Sonnabend, Sonnabend et al. 1981).  Why type G is only found in Argentina or Switzerland is a mystery.

As of 1970, there were seven known immunologically distinct botulinum toxin types.   However, as we will discover, this was just the beginning of our understanding of the diversity that is seen within botulinum neurotoxins.

About List Labs

List Labs offers over 100 reagents including Botulinum Toxins. These products are used in a wide variety of scientific research studies. You can read about some of them on our citation page. Contact us today to discuss your next project.

About the Author

Theresa Smith has studied botulinum neurotoxins for over 25 years, specializing in toxin countermeasure research, and is considered a leading expert regarding diversity in botulinum neurotoxins as well as the organisms that produce these toxins.

References

Bengtson, I. (1922). “Preliminary note on a toxin-producing anaerobe isolated from the larvae of Lucilia caesar.” Pub Health Repts 37: 164-170.

Burke, G. S. (1919). “Notes on Bacillus botulinus.” J Bact 4: 555-571.

Gimenez, D. F. and A. S. Ciccarelli (1970). “Another type of Clostridium botulinum.” Zentralbl Bakteriol Parasitenk Infektionskr Hyg Abt 215: 221-224.

Gunnison, J. B., et al. (1936). “Clostridium botulinum type E.” Proc Soc Exp Biol Med 35: 278-280.

Hazen, E. L. (1937). “A strain of B. botulinus not classified as type A, B, or C.” J Infect Dis 60: 260-264.

Landmann, G. (1904). “Uber die ursache der Darmstadter bohnenvergiftung.” Hyg Rundschau 10: 449-452.

Leuchs, J. (1910). “Beitraege zur kenntnis des toxins und antitoxins des Bacillus botulinus.” Z Hyg Infekt 76: 55-84.

Meyer, K. F. and B. Dubovsky (1922). “The distribution of the spores of B. botulinus in the United States. IV.” J Infect Dis 31: 559-594.

Meyer, K. F. and J. B. Gunnison (1929). “South African cultures of Clostridium botulinum and parabotulinum. XXXVII with a description of Cl. botulinum type D, N. SP.” J Infect Dis 45: 106-118.

Moller, V. and I. Scheibel (1960). “Preliminary report of an apparently new type of Cl. botulinum ” Acta Path Microbiol Scand 48: 80.

Schoenholz, P. and K. F. Meyer (1925). “The serologic classification of B. botulinus.” J Immunol 10: 1-53.

Seddon, H. R. (1922). “Bulbar paralysis in cattle due to the action of a toxicogenic bacillus, with a discussion on the relationship of the condition to forage poisoning (botulism).” J Comp Path Ther 35: 147-190.

Sonnabend, O., et al. (1981). “Isolation of Clostridium botulinum type G and identification of type G botulinal toxin in humans: report of five sudden unexpected deaths.” J Infect Dis 143: 22-27.

Van Ermengem, E. (1897). “A new anaerobic bacillus and its relation to botulism (originally published as “Ueber einen neuen anaeroben Bacillus und seine beziehungen zum botulismus” in Zeitschrift fur Hygiene und Infektionskrankheiten, 26:1-56) ” Clin Infect Dis 4: 701-719.

 

 

By: Rachel Berlin, Marketing Manager

1978 was a great year for scientific advancement – NASA hired the first women astronauts, the first test tube baby was born and List Labs was founded. Linda Shoer, our founder, saw an opportunity when she realized there was a need for a commercial supplier of Cholera Toxin for studies in signal transduction and neuronal track tracing. On May 18, 1978, Linda started something more than a company, she started a family- the List Labs family.

List Labs 40th Anniversary

List Labs’ catalog has grown to include over 100 products, which have been used in thousands of scientific research projects over the years. More recently we have added services to our offerings; this trend started in the early 90’s when List Labs manufactured Botulinum Toxin (Botox) for Allergan. Since then our services have expanded to include cGMP grade manufacturing, live biotherapeutics products, scalable process development for fermentation, purification and lyophilization, enhanced QC testing and more! We’ve also added to our team of experts with employees in Microbiology, Production, QC, QA, Sales and Marketing, Shipping, Administration and Finance.

List Labs carries on the values instilled by Linda Shoer and continues to grow our team, our capabilities and experience. We are excited to see what the next 40 years bring!

By: Stacy Burns-Guydish, Ph.D., Senior Director, Production

The terms “Microbiome” and “live biotherapeutics” have been repeated frequently in the last few years in scientific circles. Researchers are understanding more and more about the various microbiomes in the human body and how they affect our overall health. From this research, microorganisms have been identified that may be beneficial to our health and could be used as a therapeutic, otherwise known as a live biotherapeutic products. This article explains an overview of some microbiome and live biotherapeutics basics.

What is Microbiome? 

The human microbiome is the collection of trillions of microbes living in and on the human body. Scientists believe that it plays a role in many basic life processes and are important to our health.  Perturbation of the microbiome has been associated with a growing number of diseases including inflammatory bowel disease, allergies, asthma, autism, and cancer.

What are some different types of human microbiomes? 

Microbiomes are found, for example, in our gut, skin, vagina, and mouth.  Each of these sites have a different consortia of microorganisms.  Beneficial microorganisms have been identified in each of these niches.  Researchers are studying the various human microbiomes to better understand their importance in health and disease.

What is a Live Biotherapeutic Product (LBP)? 

A live biotherapeutic product contains a live microorganism that is used for the prevention, treatment, or cure of a disease or condition. As the characterization of the human microbiome and its link to human health has become better understood, microorganisms have been identified which may have a health benefit.  The use of these microorganisms as a live biotherapeutic product in clinical application shows great promise.  Several clinical trials are underway to evaluate their potential as a therapeutic.

What equipment and facilities are necessary for the production of a Live Biotherapeutic Products?

Many of the microorganisms identified for the manufacture of live biotherapeutic products are obligate or strict anaerobes and spore forming organisms.  These type of organisms present unique challenges to the emerging microbiome therapeutic space.  In particular, many of the microorganisms are anaerobes which cannot be exposed to air and thus expertise is required in handling and cultivating the organism.  Facilities and equipment important for the cGMP manufacturing of these organisms include:

List Labs is your partner for Live Biotherapeutic Products

List Labs has manufactured several live biotherapeutic products for phase clinical trials. With 40 years of experience, List Labs is distinctly qualified to help you with your next microbiome project and affords the flexibility required to achieve the strictest timelines and goals. We understand that each project is unique and we draw from our vast experience to deliver you a custom solution that meets your needs. Contact us today to find out how we can help you!

 

By: Rachel Berlin, Marketing Manager

2018 Translational Micriobiome Conference

List Labs is proud to be exhibiting at the 4th Annual Translational Microbiome Conference April 18-20 at the Boston Marriott Long Wharf.

List Labs Microbiome and Live BiotherapeuticsList Labs has extensive experience with microbiome and more specifically live biotherapeutic projects including vaginal, gut, skin and the central nervous system indication and tumor treatment – many of which have completed Phase I and Phase II trials. Stop by our booth and find out how we can help you with your next microbiome project. Learn about how scientists have used List Labs’ products for their microbiome research on our citations page.

Interested in scheduling a meeting with us during the conference? Contact us!

By: Rachel Berlin, Marketing Manager

List Labs at ASM BiothreatsList Labs is proud to be exhibiting at ASM Biothreats February 12-14th. The conference will be held in Baltimore, Maryland at the Hilton Hotel.

Thought leaders in academia, industry and government will gather to present and discuss the latest developments in the emerging field of biothreats. This year’s conference has an expanded program to include tracks on high consequence pathogen research, biological threat reduction, product development, and policy.

List Labs will be exhibiting in booth #29 and Nancy Shine will be presenting her poster on Sensitive Detection of Anthrax Lethal Factor in Plasma Using a Specific Biotinylated Fluorogenic Substrate during poster session 1 on Wednesday, February 14th from 10:30 AM- 11:30 AM in space #020. Come learn about our products that assist in the biological threat reduction such Botulinum Neurotoxins, Anthrax Lethal Factor, FRET Peptides, Shiga Toxins, Tetanus Toxins, and more! All of our research reagents are available for purchase on our website.

Visit Nancy and Karen in the List Labs booth #29, or contact us to schedule a time to meet with them at the show.  Click here for more information or to register for this conference.

List Labs attending ASM Biothreats

By: Shawn Lyles, Marketing Manager

List Labs will be attending the 54th annual Interagency Botulism Research Coordinating Committee (IBRCC) this year from October 27-30th. The conference will be held in Ellicott City, MA at the Turf Valley Resort and Conference Center. This international forum presents state-of-the-art research on botulinum toxin and the deadly disease of botulism. This important conference provides the opportunity for federal and non-federal agencies to coordinate in the effort against botulism in all of it’s forms.

The following List Labs employees will be attending the conference:

Want to schedule a meeting with our team during the show? Contact us today!

List Labs currently has nearly 50 botulinum related products including recombinant light chain, heavy chain, antibodies and specific substrates in stock. See how scientists have used List Labs’ reagents in their research projects on our citations page.

 

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: Karen Crawford, Ph.D., President

Dear Microbiome Researchers,

I just returned from the Second Annual Translational Microbiome Conference in Boston and my head is spinning with the possibilities. Suggested connections between the microbial community living on/in our bodies and health are expanding from the health of our gut to asthma and beyond. Many in the field consider the Microbiome another organ, the most easily replaced or improved organ in the human body.

As we become increasingly aware that antibiotics both cure and create problems, it is encouraging to think that beneficial bacteria could be introduced and become a stable beneficial addition to our microbiome. Larry Weiss of AOBiome, a skin microbiome company, presented a product which can be obtained on the internet called Mother Dirt; a bottle with friendly bacteria originally derived from the soil, to spray on our bodies, replacing chemically-derived skin treatments such as soap and deodorant. Ammonia-utilizing bacteria in Mother Dirt convert naturally occurring nitrogen compounds on the skin to potentially beneficial nitrites.

Evolve Biosystems is looking at conditions which are rooted in perturbations in the microbiome of infants. An essential organism nicknamed “Baby Bif” is not present in high numbers in infants as a result of our modern aseptic, antibiotic filled environment. A skewed microbiome in infants may lead to conditions such as asthma/allergies, diabetes and obesity, conditions which could be prevented if friendly bacteria were provided in infant formula and foods. Laurel Lagenaur from Osel, Inc. presented data on lactobacillus products targeted to urinary tract and vaginal infections. Osel’s product, designed to restore a healthy vaginal microbiome, will likely be the first microbiome product to receive drug approval.

We heard about the OpenBiome stool bank which is providing materials for fecal transplants in multiple US centers. Success of the transplant procedures in resolving reoccurring C. difficile infections is fueling enthusiasm for development of pure culture therapies. Janssen Research and Development and Seres Therapeutics reported on projects to develop good gut bacteria as potential remedies for C.difficile, IBD, and Crohn’s disease. Janssen is using a network of collaborators to make progress in this area. Although the gut is the current focus, everyone is thinking beyond to using microbes to re-establish the balance of microbes to influence many different disease states.

Personalized nutrition was the headline from Lihi Segal of Day Two. The company is developing a personalized medicine approach to normalizing blood sugar. Feedback from a glucose monitor along with analysis of the gut microbiome allows Day Two to apply an algorithm suggesting meals to regulate blood sugar. Under such a regime the blood sugar roller coaster has been flattened for trial patients.

It is an exciting time to work in the microbiome arena, and I welcome the opportunity to connect with colleagues, meet up with customers and learn more about what others are doing to advance the study of the human microbiome. At List Labs, we pride ourselves on partnering to deliver live biotherapeutic products that yield results. If you are embarking on a new product or ready to identify a partner to ease into clinical trials with superior research, process development and manufacturing, contact us and find out more about how we collaborate.

Regards,
Karen

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

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

Research Only VS Preclinical/Clinical/Human Use

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

reagent vs gmp products

cGMP = Higher Production Standards

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

See more about our cGMP production capabilities.

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

For example….

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

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

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

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

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

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

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

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

References

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

By: 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://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

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