Substances & Homeopatic Remedies

Limulus cyclops

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How does the horseshoe crab protect the public health?

The horseshoe crab plays a vital, if little-known, role in the life of anyone who has received an injectable medication. An extract of the horseshoe crab's blood is used by the pharmaceutical and medical device industries to ensure that their products, e.g., intravenous drugs, vaccines, and medical devices, are free of bacterial contamination. No other test works as easily or reliably for this purpose. Read below for more detail.

Why are we concerned about bacterial contamination of pharmaceutical products?

Bacteria are everywhere-from our intestinal tract, to soils, rivers, and oceans. For the most part, bacteria are beneficial, acting to degrade organic waste and recycle nutrients back into the food chain. Sometimes, however, bacteria cause disease. We are all familiar with many specific bacterial diseases such as Salmonella food poisoning or more serious ones such as Cholera and Tetanus.

Bacteria that cause these diseases are referred to as pathogens and usually require an animal host for multiplication or transmission even though they may persist in a soil or aquatic environment for long periods of time. Other bacteria, generally considered non-pathogenic, can cause disease if they enter parts of our body that are usually bacteria-free, such as the bloodstream. In this case, even the ordinarily benign gut bacterium E. coli can cause sepsis and death. Therefore, the pharmaceutical industry takes great care in producing drugs, vaccines, and medical devices (items that deliver drugs or are implanted) that are sterile-free of living microorganisms. Unfortunately, certain bacterial components can, in and of themselves, be toxic. Thus, pharmaceutical manufacturers not only need to be sure their products are sterile but also non-toxic, i.e., contain no bacterial components left from pre-sterilization bacterial contamination.



Illustration credit Charles River Endosafe, SC
 



The bacterial toxin of greatest concern is termed endotoxin, and it is able to withstand steam sterilization. Endotoxin occurs as part of the cell structure of a large class of bacteria that includes both pathogens and non-pathogens. This class of bacteria is known as Gram-negative, for their characteristic of being easily decolorized during the Gram staining procedure. Surprisingly, it is the non-pathogenic members of the Gram-negative group, those that love aquatic environments, which cause the most problems for the pharmaceutical industry.

Over fifty years ago it was recognized that some sterile solutions, when injected into humans or rabbits, caused a fever or pyrogenic response. Scientists soon learned that these so-called "injection fevers" were caused by endotoxin (a potent pyrogen) left over from bacterial components that remained in the injected solutions after sterilization. Fortunately, it was also found that solutions could be screened for pyrogens by injecting small amounts of the batch into rabbits. If the rabbit exhibited a fever, the solution was deemed pyrogenic and was rejected. The rabbit or pyrogen test, along with a sterility test, became the two most important tools of the pharmaceutical industry. The Pyrogen Test employing rabbits is still in limited use, although as you will see below, an endotoxin test using an extract from the blood cells of the horseshoe crab is the predominant pyrogen test today.

How was the horseshoe crab test discovered?

In the 1960's, Dr. Frederik Bang, a Johns Hopkins researcher working at the Marine Biological Laboratory in Woods Hole, Massachusetts, found that when common marine bacteria were injected into the bloodstream of the North American horseshoe crab, Limulus polyphemus, massive clotting occurred. Later, with the collaboration of Dr. Jack Levin, the MBL team showed that the clotting was due to endotoxin, a component of the marine bacteria originally used by Dr. Bang. In addition, these investigators were able to localize the clotting phenomenon to the blood cells, amebocytes, of the horseshoe crab, and, more importantly, to demonstrate the clotting reaction in a test tube. The cell-free reagent that resulted was named Limulus amebocyte lysate, or LAL. The name LAL is extremely descriptive: Limulus is the generic name of the horseshoe crab, amebocyte is the blood cell that contains the active components of the reagent, and lysate describes the original process used by Levin and Bang to obtain these components. In Levin and Bang's process, amebocytes, after being separated from the blue-colored plasma (hemolymph), were suspended in distilled water where they lysed (ruptured) due to the high concentration of salt contained in the amobocytes versus the absence of salt in the distilled water. Surprisingly, this same process with some minor modifications is still used today to produce LAL.

How does the horseshoe crab protect itself from disease?

One may wonder why the horseshoe crab is sensitive to endotoxin and, furthermore, how does the crab benefit from this phenomenon? As we know, seawater is a virtual "bacterial soup". Typical near-shore areas that form the prime habitat of the horseshoe crab can easily contain over one billion Gram-negative bacteria per milliliter of seawater. Thus, the horseshoe crab is constantly threatened with infection. Unlike mammals, including humans, the horseshoe crab lacks an immune system; it cannot develop antibodies to fight infection. However, the horseshoe crab does contain a number of compounds that will bind to and inactivate bacteria, fungi, and viruses. The components of LAL are part of this primitive "immune" system. The components in LAL, for example, not only bind and inactivate bacterial endotoxin, but the clot formed as a result of activation by endotoxin provides wound control by preventing bleeding and forming a physical barrier against additional bacterial entry and infection. It is one of the marvels of evolution that the horseshoe crab uses endotoxin as a signal for wound occurrence and as an extremely effective defense against infection.

How are the horseshoe crabs collected? Are they harmed?

In shallow water, horseshoe crabs are collected by hand from a small boat using a clam rake, and the animals are not injured during this process. In deeper water, a dredge is used, and in this case, some horseshoe crabs do get injured. Injured crabs are released immediately and most will survive. It is quite common to find crabs with "scars" of old injuries that have healed.

Once the crabs are caught, they are transported to the laboratory from the fishing pier by truck. Sometimes a refrigerated truck is used, but as long as the animals are kept cool and dark during transport, they exhibit no adverse affects. During the bleeding process, up to 30% of the animal's blood is removed. Research has shown that once returned to the water, the horseshoe crab's blood volume rebounds in about a week.

It takes longer for the crab's blood cell count to return to normal, about two to three months. Theoretically, crabs can be bled several times a year, but LAL manufacturers bleed them only once per year.

The Associates of Cape Cod and other LAL manufacturers have studied horseshoe crab mortality following the bleeding procedure and have found it to be quite low, less than 3% when compared to controls handled similarly but not bled. There are no records of a horseshoe crab dying during the bleeding process itself. Other studies conducted by government agencies and universities indicate a mortality of 10-15%. However, the horseshoe crabs in these studies were not handled as carefully as those collected by the LAL industry.

Studies done by the Associates of Cape Cod show that not only do the crabs survive one bleeding, but that they can be captured year after year to donate their life-saving blood-much like human blood donors. In addition, their studies indicate that crabs, which are bled and returned to their spawning area, will continue their breeding activity without any ill effect.

The companies that produce LAL go to great lengths to ensure that the animals used in the making this valuable, life-saving test are handled with care and respect. They recognize that a stable horseshoe crab population is vitally important not only to the biomedical community, but also to the survival of millions of shorebirds, sea turtles, and other marine creatures that have a symbiotic relationship with this remarkable creature. These companies will continue to support sound, scientifically-based conservation measures that will ensure a sustainable population for the future.

Horseshoe Crabs and Vision

It was in 1926 that H. Keffer Hartline began to study electrical impulses from the optic nerve of horseshoe crab eyes. From these studies, some important principles about the function of human eyes were discovered. As a result, Dr. Ragnar Granit of Sweden and Americans H. Keffer Hartline and George Wald were awarded the 1967 Nobel Prize in Medicine.
Horseshoe Crabs and Vision
At the turn on the century, little at all was known about how vision worked. Scientists of course knew that the eyes were responsible but exactly what happened when light entered the eye and how the brain actually received the information was a mystery. - Look here -  
For a more fundamental look at the physics and biochemistry of vision, take a look at the work of former MBL researcher and Nobel-prize winner, George Wald.
The compound eye of Limulus has served as a model for studying vision since the early part of the century and before we describe some of that work it deserves an introduction.

Arthropods, like Limulus, possess eyes, but the eyes can vary in complexity. Some are simple, consisting of a few photoreceptors, while others are large and can resolve images. In all of the arthropods, the exoskeloton (the "shell") contributes the lens portion of the eye. As a result, the focus of the eye is fixed as it is part of the outer skeleton of the animal.
Some arthropods possess a type of eye known as a compound eye. The compound eye is made of smaller, simple eye units, called ommatidia. Each ommatidia is composed of a cornea, which as was previously mentioned is formed from the outer exoskeleton. This cornea acts as a lens to focus light into the eye. Following this is an element called the "crystalline cone" which serves as a second lens. It is produced by adjacent cells, usually four in number.
The cone tapers to a receptor unit called a retinula which focuses the light into a translucent cylinder called the rhabdome. The rhabdome is surrounded by light sensitive, or retinular cells. There are generally seven similar retinular cells and one eccentric cell. It is the inward-facing portions of these cells in fact, which form the rhabdome. The rhabdome is the common area where light is transmitted to the reticular cells. Each of these cells is connected to an axon and since each ommatidia consists of seven or eight reticular cells, there are this number of axons which form a bundle from each ommatidia. These axons then form connections with other nerves to create an optic ganglion which passes the visual signal to the brain. Each ommatidia passes information about a single point source of light. The total image formed therefore is a sum of the ommatidia fired. This resultant image can be thought of as a series of dots, just like a computer image is composed of a series of discreet dots (pixels). The more pixels, the better the picture.
Why Limulus
One of the basic tenets of research at the MBL is the use of marine organisms as simple models of more complex biological systems. This is based on an assumption that we can use what we learn from these simple systems in our understanding of the more complex systems we might find in human beings. This has proven to be true and is no different in our understanding of the principles of vision. As H. Keffer Hartline, whom we will learn of more in a moment, used to say to his students when studying vision, "avoid vertebrates because they are too complicated, avoid color vision because it is much too complicated, and avoid the combination because it is impossible."
Limulus provided MBL researchers with a near-perfect model for studying vision. First, it is large, easy to find and easy to handle. As invertebrates go, it is a giant. It possesses both simple and compound eyes. For a marine animal it is also quite hardy and can be safely kept out of water for relatively long periods of time. The compound eyes are relatively large and the optic nerve, which connects the eyes to the brain is not only enormous, up to four inches long, but also lies just below the carapace. For early researchers who wanted to eavesdrop on the signals travelling between eyes and brain one could scarcely design a better animal!

One of the scientists who took advantage of this remarkable animal was H. Keffer Hartline. He was able to isolate and study the activity of single nerve fibers as they relayed signals from single ommatidia to the brain in Limulus. In 1932 he published a paper titled "Nerve Impulses from Single Receptors in the Eye" describing this work. It was his work in clarifying how ommatidia interact with each other, however, that led to understanding the mechanisms of lateral inhibition and won him the Nobel Prize
Lateral inhibition is a process that animals, including humans, use to better distinguish borders. When you look at the ocean horizon the ocean appears darker at the horizon, at the boundary between sea and sky. This apparent difference in light intensity is not actually there but is created by our visual receptors and is known as lateral inhibition. This process increases contrast and results in a sharpening of vision. In fact, computers sharpen images by almost the same process process. (the illustration below illustrates both lateral inhibition and a sharpening filter from a graphics program.) What this means is that the signals coming from the outside are actually altered before being sent to the brain so that what we see isn't necessarily there.
The way this works is as follows. Impulses originate in the eccentric cell when the cell is stimulated by light . This signal is transmitted through the axon then to the optic nerve to the brain.
The ability of an ommatidia to discharge impulses is related to the amount of light that neighboring ommatidia are receiving. Hartline found that if one ommatidia is receiving bright light and a neighbor is receiving dim light, the first ommatidia will inhibit the signal from it's neighbor. The result is that the dimmer signal gets even dimmer and the result is an increased difference between the two which the eye would perceive as an increase in contrast.


Chitin

The chitin from horseshoe crabs is used in the manufacturing of chitin-coated filament for suturing and chitin-coated wound dressing for burn victims (Hall, 1992). Since the mid-1950s, medical researchers have known that chitin-coated suture material reduces healing time by 35 to 50 percent.