2. LAL-Lab PowerPoint Biomedical Use of Horseshoe Crabs and Importance to Human Health

3. Long ago people realized that microbes caused certain diseases. Vaccines were then created to prevent some of these diseases. However, sometimes the vaccines themselves made people sick. This was known as “injection fever”. A bit of history on the subject

4. After the invention of the microscope, scientists were able to distinguish two distinct types of bacteria. Some years later, the cause of “Injection Fever” was discovered. “Injection Fever” was realized to be an immune response to a component of one of these two types of bacteria. Eventually they found that bacteria could cause illness.

5. These two kinds of bacteria came to be distinguished by a simple procedure: This procedure known as the: GRAM STAIN test (after Hans Christian Gram who developed it) works as follows: Two stains are added in turn to the bacteria sample If the bacteria stains blue from the primary stain GRAM-POSITIVE If the bacteria does not take the primary stain, but counterstains red GRAM-NEGATIVE

6. These staining tendencies are determined by the differences in composition of the bacterial cell wall. In gram-positive bacteria the cell wall is thin... allowing stain to penetrate and turn the cell blue

7. In gram-negative bacteria, keeping the stain from penetrating, so it won’t turn blue the cell wall has an extra layer

8. Gram- positive Comparing the two-types of bacteria: Gram- negative: > thin cell wall [made of protein peptideoglycan] > stains blue during Gram-stain procedure > features mainly terrestrial bacteria > stains pink during Gram-stain procedure > features mainly aquatic bacteria > thick cell wall [made of lipopolysaccharide (LPS)] > includes bacteria that cause strep throat & staph infections > includes bacteria that cause salmonella, septi- cemia, dysentery, meningitis & other diseases

9. Within the outer Lipopolysaccharide (LPS) layer of the double-layered cell wall of gram-negative bacteria are pyrogens, or fever-inducing agents, known as: ENDOTOXINS

10. So, what’s the big deal about ENDOTOXINS? ENDOTOXINS are a bacterial toxin found only in gram-negative bacteria. Endotoxins cause fever in mammals. Fever is the immune response to combat foreign invaders. Prolonged intense fever can lead to tissue breakdown, shock, and death. Once endotoxins enter the bloodstream, there are no effective treatments. Antibiotics can kill bacteria, but are ineffective against endotoxins. Bacteria do not have to be viable for endotoxins to produce immune response.

11. Dr. Ronald Berzofsky, Technical Director Cambrex Bio Science, Endotoxin Detection Section ENDOTOXINS are prevalent in the environment and don’t always constitute a human health threat. In fact, endotoxins can be found inside the digestive system of our own bodies.

12. Pharmaceutical companies must perform intensive screening of all medical products to avoid introducing endotoxins into a patient’s bloodstream during surgery or a routine procedure. Because endotoxins in our bodies (outside of the digestive tract) can cause serious health problems: Vaccines, allergy or insulin shots & other injectable medicines IV bags & solutions Implanted pins & plates Pacemakers, heart valves & other surgical implants Numerous other pharmaceutical products This includes:

13. What does this guy have to do with any of this? In the past, rabbits provided the only way of testing products for endotoxins. A sample of the product to be tested was injected into a live rabbit. The rabbit’s temperature was monitored for 3 hours. If the rabbit developed a fever, the sample was considered contaminated and thus rejected.

14. In the 1960’s, scientists Frederik Bang and Jack Levin discovered a more effective endotoxin test: Dr. Bang, while researching horseshoe crabs, noticed massive blood clotting in response to injected gram-negative bacteria. Upon further research, Dr.’s Bang and Levin determined that white cells (amebocytes) in horseshoe crab blood formed a clot in the presence of endotoxin. Bang & Levin sought to isolate and refine this clotting reaction. Later, the potential bio-medical applications were recognized. The procedure they developed is much like the one now in use by pharmaceuticals worldwide to test for endotoxins. HSC Amebocytes

15. Horseshoe crabs possess a simple, yet effective, system to guard against bacterial infections

16. How horseshoe crab blood responds to endotoxins  Blood clots at the site of the wound in response to endotoxin.  The clot encapsulates any entering bacteria.  This prevents bacteria from infecting the entire body cavity.

17. A note about Blue Blood … Human blood appears red due to hemoglobin, an oxygen-carrying molecule containing iron. When exposed to air it turns red, just like iron rusting. Horseshoe crab blood inside it’s body is actually straw-colored, but it’s oxygen-carrying molecule, hemocyanin, is copper-based. So it turns blue when oxidized. Please note: The color of HSC blood has nothing to do with its endotoxin-detecting properties!

18. HSC’s are bled in the lab under sterile conditions A needle is injected through the hinge muscle and into the heart to start the blood flowing Bleeding Horseshoe Crabs for Biomedical Use The blood flow will stop due to clotting after about 30% of the blood is collected

19. Effects of bleeding on horseshoe crabs Spawning continues without ill effects. HSC’s are returned to the water after the bleeding procedure. There is about a 10% mortality rate due to handling. Research continues on how to minimize harm to the crabs. Blood cell count returns to normal in about 2-3 months.

20. This clip shows the first step in processing HSC blood for biomedical use centrifuging to separate amebocytes (white blood cells) from the plasma. Processing horseshoe crab blood for biomedical use

21. Final steps in processing of the blood product for biomedical use Distilled, endotoxin-free water is added to the isolated blood cells. As water enters the cells, the cells expand and eventually rupture, or lyse. As the cell bursts, the clotting granules or coagulogens contained within the blood cell are released into the solution. Coagulogens are then isolated from the solution and freeze-dried to an easily-packaged powder. This powdered product is called ‘LAL’.

22. L IMULUS (generic name for our Horseshoe Crab) A MEBOCYTE (blood cells that it’s made from) L YSATE (refers to ‘lyse’ or rupture of cells) or LAL for short How ‘LAL’ gets its name

23. Packaged Product Bottling Lysate Photo courtesy of Associates of Cape Cod Packaging and use of LAL Use of LAL for testing biomedical products gained FDA approval in the 1980’s. It has replaced the rabbit test as the preferred means of endotoxin testing. LAL manufacture is a multimillion dollar industry. It is used globally.

24. Performing the LAL Test > add sample to be tested to LAL powder > incubate for 60 minutes at 37º Celsius > invert the test tube > check for clot reaction > if sample remains liquid: endotoxin-free > in lab testing, controls of known amounts of endotoxin for comparison are used to quantify amounts of endotoxin present > if sample forms a clot: endotoxin present

25. What Are the Advantages of LAL? Faster results : 1 hour (compared to 3 hours for the rabbit test) More accurate results: LAL is more sensitive than rabbit test (can detect lower levels of endotoxin) Lower maintenance: HSC’s are returned with little ill effects; Rabbits must be caged, kept and cared for

26. So what does all this mean to us as humans? The risk of infection from all injected medicines and implanted medical devices is greatly minimized. The risk of patients getting septicemia (blood poisoning from endotoxins), a disease that kills tens of thousands of people a year, is also reduced. The speed at which bacterial meningitis can be diagnosed and treated is much improved, also saving many human lives. AS YOU CAN SEE, HORSESHOE CRABS PLAY A VITAL YET LITTLE KNOWN ROLE IN PROTECTING HUMAN HEALTH!

27. HOORAY FOR HORSESHOE CRABS! Isn’ t it amazing that an animal that’s been around for millions of years is so essential to protecting human health today? Don’t you think we should protect them as well?

28. Developed by: Tricia Cosbey, Melissa Pierce and Gary Kreamer Delaware Aquatic Resources Education (ARE) Center Technical support from: Trina Cale-Rosario of Delaware ARE and Susan Raymond, Teacher, Millsboro, DE Special thanks to: Dr. Ronald Berzofsky of Cambrex Bio Science for sharing his ideas, images, expertise and wonderful way of making complex concepts comprehensible! Video clips and images supplied by: Michael Oates of Anew, Inc. LAL PowerPoint Credits Inspired by: Glenn Gauvry, Ecological Research Development Group