The Indian horseshoe crab, Tachypleus gigas (Muller) and its biomedical applications
THESIS SUBMITTED TO GOA UNIVERSITY
FOR THE DEGREE
OF DOCTOR OF PHILOSOPHY
IN
MARINE SCIENCES
BY
MS HUMA ALAM
BIOLOGICAL OCEANOGRAPHY DIVISION NATIONAL INSTITUTE OF OCEANOGRAPHY DONA PAULA -403 004, GOA, INDIA
DECEMBER' 2007 The Indian horseshoe crab, Tachypleus gigas (Milner) and its biomedical applications
THESIS SUBMITTED TO GOA UNIVERSITY
FOR THE DEGREE
OF
DOCTOR OF PHILOSOPHY
IN
MARINE SCIENCES
BY
MS HUMA ALAM
UNDER THE GUIDANCE
OF
Dr. ANIL CHATTERJI SCIENTIST
BIOLOGICAL OCEANOGRAPHY DIVISION NATIONAL INSTITUTE OF OCEANOGRAPHY DONA PAULA -403 004, GOA, INDIA 1§A cyn@r CERTIFICATE
This is to certify that that the thesis entitled "The Indian horseshoe crab, Tachypleus gigas (Mailer) and its biomedical applications" submitted by Ms. Huma Alam for the award of the degree of Doctor of Philosophy in Marine Sciences, Goa University, Goa based on her original studies carried out by her under my supervision..The thesis or any part thereof has not been previously submitted for any other degree or diploma in any university or institution.
(Dr. Anil Ch tterji) Research Guide Scientist, National Institute of Oceanography Dona Paula-403 004, Goa, India.
Place: NIO, Goa Dr. Anil Chatterji SCIINTIST Date No I but it, ite of Oceon OCiN" rAU C.iO41.-44., 3uU4. IN1)1A
Cf).A.A-ec_il 0 A-4
e e r\ DECLARATION
As required under the university ordinance 0.19 .8 ( VI ), I state that the present thesis entitled "The Indian horseshoe crab, Tachypleus gigas (Muller) and its biomedical applications" is my original contribution and that the same has not been submitted elsewhere for award of any degree to any other university on any previous occasion. To the best of my knowledge the present study is the first comprehensive work of its kind from the area mentioned.
The literature related to the problem investigated has been cited. Due acknowledgements have been made wherever facilities and suggestion have been availed of. List of Tables
Table 4.1: A comparison in the sensitivity of amoebocyte lysate produced from both in vitro and in vivo stock List of Figures
Figure 1.1: Attachment of CD 34+ cells to the surface after 22 days of culture
Figure 1.2: Formation of colonies
Figure 1.3: Pro-angiogenic activity of PVF (Number of capillaries tube formation
Figure 1.4: Pro-angiogenic activity of PVF (Number of intersections)
Figure 1.5: Pro-angiogenic activity of PVF (length of capillaries)
Figure 2.1: Expression of Von Willebrand factor
Figure 2.2: Expression of smooth muscle cell actin
Figure 2.3: Migration of human bone marrow endothelial cells
Figure 3.1: FPLC profile of peri-vitelline fluid
Figure 3.2: Assessment of most effective fraction of PVF
Figure 3.3: Assessment effective dose of PVF by the expression of myoglobin
Figure 3.4: Sequence of fraction 8th of PVF
Figure 3.5: Expression of myoglobin by differentiating CD 34 + Cells
Figure 4.1: Experiment to select a suitable culture media to culture gill flaps
Figure 4.2: Amoebocyte yield in males (in vitro production)
Figure 4.3: Amoebocyte yield in females (in vitro production)
Figure 5.1: Oxygen consumption of the trilobite larvae with different salinity
Figure 5.2: Oxygen consumption of the trilobite larvae with different temperatures
Figure 5.3: Oxygen consumption of the trilobite larvae with different pH
Figure 6.1: Regeneration of gill lamellae in females.
Figure 6.2: Regeneration of gill lamellae in males List of Plates
Plate 1.1: Formation of colonies
Plate 1.2a: Capillary network formation-migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 0 & 4 hrs)
Plate 1.2b: Capillary network formation-Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 8 & 12 hrs)
Plate 2.1: Hematopoietic and stromal stem cell differentiation
Plate 2.2: Resource for stem cell research
Plate 2.3: Expression of Von Willebrand factor with PVF
Plate 2.4: Expression of smooth muscle actin with PVF
Plate 2.5a: Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 0 hrs)
Plate 2.5b: Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 3 hrs)
Plate 2.5c: Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 6 hrs)
Plate 2.5d: Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 9 hrs)
Plate 2.5e: Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 24 hrs)
Plate 3.1: Gel profile of the 8 th fraction of PVF
Plate 3.2: Effect of PVF in the differentiation of CD 34 + cells into cardiomyocytes (expression in myocytes)
Plate 3.3: Peroxidase activity in presence of PVF
Plate 4.1: A comparison of the amoebocyte cells produced by gill culture and amoebocytes obtained from wild stock
Plate 6.2 (a) Arrow shows incised gill lamellae (in circle). (b) Arrow shows regenerated gill lamellae (in circle) CONTENTS
Page No
ACKNOWLEDGEMENTS 1
INTRODUCTION 3
CHAPTER —1 Role of peri-vitelline fluid of the fertilized eggs in maintaining the 22 original phenotype of CD 34 + stem cells, enhances colony and capillary formation in vitro
CHAPTER — 2 Role of peri-vitelline fluid of the fertilized eggs of the Indian 43 horseshoe crab in vasculogenesis
CHAPTER — 3 Role of peri-vitelline fluid in myocyte differentiation of bone 74 marrow stem cells in vitro
CHAPTER — 4 A comparison of the sensitivity of amoebocyte lysate produced 96 from In vitro grown amoebocytes and amoebocytes collected from wild stock
CHAPTER — 5 Respiratory metabolism of the trilobite larvae 120
CHAPTER — 6 Regeneration of gill lamellae of the Indian horseshoe crab 137
REFERENCES 156 Acknowledgements
It is indeed a great pleasure to me in expressing my deep sense of gratitude and indebtedness to Dr. Anil Chatterji, the senior scientist of
National Institute of Oceanography, Goa for his constant interest, encouragements, moral support and the guidance during the entire period of my research that has made my work successful on the Indian horseshoe crab. Dr. Chatterji always took care to bolster up my sagging sprits whenever I found myself faced up with frustrating moments.
I take this opportunity to thank Dr. S. Z. Qasim, the former Director of
National Institute of Oceanography, Goa whose abiding involved concern and encouragements for making me see the true light and dimension to carry out this hard task. I also thank Dr. Satish R. Shetye, Director of
Institute of Oceanography, Goa for encouragements and providing laboratory facilities for carrying out this work.
I wish to express my profound thanks to Professor G. N. Nayak,
Head of Department of Marine Sciences, Dr. C. U. Rivonkar, Department of
Marine Science of Goa University, Goa for their encouragements and help during my Ph.D. work. I especially thank Professor Amu Therwath and Dr.
Massoud Mirshahi of INSERM 736, Faculte de Medecine Paris VI, 15, rue de I'Ecole de Medecine, 75006 Paris, France for their supervision and moral support during my research work.
I am grateful to my fellow colleagues, Ms. Janette Soria, Mr. Pejman
Mirshahi, Mr. Alexende Berout, Ms Julia Rodrigiz, Ms Sylvi, Ms Shah Sultan
1 of Faculte de Medecine Paris VI, 15, rue de l'Ecole de Medecine, 75006
Paris, France for their valuable contributions in the project during my stay in
France.
The present work might have not been up to the mark had it not been for the support, encouragements and efforts of my dearest colleagues; Ms
Reena Rodrigues, Dr. Savita Kotnala, Ms Rashmi Vinayak, Sreekumar, P.
K. and Mr. M. K. Tari of the National Institute of Oceanography, Goa.
I cannot ignore the silent contribution of my dearest husband, Samir
but for whose cooperation and inspiration over a long period of time through thick and thin, I would have found it difficult to carry on. I thank my parents,
in laws, sisters and my dear brother in-law Akhil for their encouragements
during the entire period of my research.
I specially thank to embassy of France in India and Professor John
Piere Marie of Hospitel Hotel Dieu, Paris (France) for providing the funds to
enable me to carry out my research work.
Last but not the least I would like to thank the Hospitel Hotel Dieu,
Paris (France) National Institute of Oceanography, Goa and University of
Goa for providing me necessary facilities to carry out my entire research
work.
Huma Alam
2 INTRODUCTION Life is complex process and we have a very little knowledge about its origin. There are two main views on the appearance of the life on earth. The origin of life was either due to external source created supernaturally by mankind or a gradual orderly progression from non-living matter to living organisms (McAlester, 1968). The vast scientific community does not accept the view on the possibility of origin of life by supernatural power. It is generally believed that the life is originated due to long sequences of chemical evolution.
In the modern world, all organisms showed unique chemical and physical attributes broadly on their construction and compositional activities although they differ in many of their basic biogenic activities (McAlester, 1968).
However, their basic biogenic activities like utilization of energy and reproduction show similar pattern.
The first definite trace of life in the form of blue-green algae was found in Canada, which is considered to be at least thousand million years old (Weller, 1969). All marine plants are primitive forms and they have no significant difference from the ancient forms. It is believed that many developments had occurred in the plants after they led the way ashore some 400 million years ago. As compared to 250,000 species of flowering plants on the lands, only less than one hundred species of plants are found in the ocean (Price, 1971).
There are many theories to demonstrate the biologic evolution. It was the ancient Greeks who visualized for the first time the nebulous transition from the inorganic to the organic and further from the plants to animals in a series, attaining more perfection and leading up to man. Later on, Buffen—a
French Zoologist and Linnaeus—a Swedish Botanist, suggested the
3 possibility of some kind of evolution among organisms subsequent to their creation. In the middle of the eighteenth century, Lamarck— a French
Zoologist presented for the first time, a complete integrated theory of biologic evolution. Lamarck's theory postulated that the animals acquired new characters according to their need. As soon as the single-celled animals became multi-celled animals, diversity in their forms and habits occurred rapidly. These characters slowly started diversifying by use or disuse process and they were passed on by inheritance to their descendants. According to Lamarck's theory, modifications in the characters of animal took place from a simple organism. But the theory of Lamarck on inheritance of acquired character was not accepted by the naturalists
(Weller, 1969).
In the middle of the nineteenth century with the declaration of
Darwin's theory, a new era in the understanding of biologic evolution started. Darwin's theory described three main bases for the evolution; 1) the individuals making up a species vary more or less among themselves in form, physiology and behavior; 2) the potential for reproduction of every species far exceeds the necessity to maintain its number, and 3) competition for food and shelter for living resulted in the elimination of the weaker and less well adapted animals. The theory of survival for the fittest and principal of natural selection presented a new idea of biologic evolution.
According to Darwin's theory, many animals that were present in the past geologic era have become extinct because of their weak adaptability
(Weller, 1969).
4 Ocean is a treasure house of many living creatures. About 26 phyla of marine organisms are found in the ocean whereas arthropods contribute four-fifths of all marine animal species with over 35,000 varieties. These arthropods have jointed limbs and an outer shell, which they molt as they grow. Surprisingly, quite a few marine organisms, which were once suspected to be extinct, still flourish as living animals. Horseshoe crab, a chelicerate arthropods belonging to the class merostomata, is one such amazing creature. The horseshoe crab is known as the oldest "living fossil" because the archeal body of this animal has not shown any significant phenotypic change even after a span of 350 million years (Mikkelsen, 1988; Chatterji and Abidi, 1994).
Evolutionary history of the horseshoe crab
The horseshoe crab has descended from mud dwelling primitive arthropods called trilobite, which lived in the Precambrian seas, nearly 600 million years ago (Price, 1971). The horseshoe crab evolved into its present shape, about 350 million years ago and remained unchanged. Although the horseshoe crab has a long history, it left a very little geological record
(Chatterji, 1994). The first fossilized horseshoe crab (Mesolimulus walchi,
Desmerest, 1822) was found from the upper Jurassic layer of famous lithographic lime stone of Bavaria in West Germany, Limulitella decheni,
Mesolimulus syriacus, M. nathrosti, M. woodwardi and Psamalimulus gottingenesis were the other species of the horseshoe crab reported after some time from the Oligocene layer of a brown coal of Teuchern near
Mersberg. In the Cretaceous strata of Lebanon and Jurassic layers of
Australia, fossil record of Austrolimulus fletheri were also found. The
5 Precambrian layers of Kansas (U.S.A) and cretaceous layers of Colorado
(U.S.A) contributed further to the fossil records of Palaeolimulus avitus and
Limulus .coffini, respectively (Mikkelsen, 1988; Chatterji, 1994). Considering how so many animals including dinosaurs and many other late corners to the Mesozoic era have became extinct completely, it is a wonder how the humble horseshoe crab has survived for such a long period. It appears that the animal can tide over all kinds of situations arising from its estuarine and coastal shallow habitats. It can tolerate a wide range of salinity, temperature, desiccation and submergence.
It is generally believed that the ancestors of the horseshoe crab inhabited in brackish or freshwater environments (Weller, 1969). The fossil record showed that the oldest horseshoe crab was similar to the aglaspids with less abdominal segments. The body of the horseshoe crab slowly started shortening from the late Paleozoic period and the abdominal segments were reduced from ten to six. These segments were fused from behind at a later stage and the posterior part of the body developed a rigid shield, similar to pygidium of a trilobite but terminated into a movable telson
(Mikkelsen, 1988). The fossil records showed that these forms were not having well defined appendages. The five pairs of walking legs, discontinuing up to abdomen, were present in the primitive forms. Gradually the first four pairs started developing pinching claws whereas, the last pair terminated in primitive spines. In modern horseshoe crab, these five pairs are highly specialized appendages with broad, flat and overlapping plates.
The external gills of the horseshoe crab were developed partly from these appendages.
6 The horseshoe crab belongs to the benthic community. They prefer calm sea or an estuary with muddy sand bottom (Quammen, 1982; Grant, 1984; Hicklin and Smith, 1984; Kelsey and Hassall, 1989). Most of the biogenic activities of the horseshoe crab occur in the open ocean. They specifically migrate towards the shore for the purpose of breeding. As such, the horseshoe crab is subjected to face a wide range of change in environmental conditions. Although detailed knowledge on the complete life cycle of the animal is not yet known, it is, generally believed that the animal inhabited the littoral zone of the sea, for most part of its life. During their migration for breeding towards inter-tidal zone, they experience lower salinity than the littoral zone (Chatterji, et al., 1994). Similarly, the juveniles, while migrating out towards the sea, also experience the low saline conditions of the inter-tidal zone to comparatively high saline conditions of the littoral zone. The animal can also tolerate a wide range of temperature.
The fluctuating habitats encountered by the amazing horseshoe crab during their different stages of life, reflect their ability to tolerate and adapt to different environmental conditions.
It is rather interesting to know that an animal like the horseshoe crab whose distribution pattern is patchy, has highly specialized methods for detecting its selected habitat for reproduction and dispersal (Shuster and
Botton, 1985). These methods ensure the animal of their safe arrival to their respective places and reduce the chances of mortality during their
migration. Such characteristics could possibly be responsible for providing
an evolutionary advantage to the horseshoe crab for their very long survival.
On the contrary, the uniformly distributed animals are subjected to more
7 competition for their biological needs and get less opportunity to survive for a longer period (Botton et a/., 1988).
The study on the distribution of horseshoe crab is difficult because migration of the animal is totally dependent on two important physical stimuli
- the tide and - lunar periodicities (Botton et al., 1994). There have been a few attempts to study the distribution of the horseshoe crab in the last few decades. The occurrence of a species of the horseshoe crab, Tachypleus hoeveni, was reported for the first time from the coast of Moluccas
(Mikkelsen, 1988; Chatterji, 1994). Two more species of the horseshoe crab, Limulus and Cancer mollucensis were reported from the same region, afterwards (Mikkelsen, 1988). .
The most diversified Paleozoic group is represented only by four species of the horseshoe crab with discernible morphology and three genera confirmed, so far, all over the world. It is rather interesting to note that among these four species, Limulus polyphemus and Tachypleus tridentatus occur in a north-south-north direction whereas, Tachypleus gigas and Carcinoscorpius rotundicauda in east-west-east direction including India
(Mikkelsen, 1988; Chatterji, 1994). Among the Asian species of the horseshoe crab, T. gigas is morphologically more closely related to T. tridentatus than C. rotundicauda (Mikkelsen, 1988). The classification of the easily identifiable features shows high degrees of similarity among all the species of the horseshoe crab (Chatterji, 1994).
8 The horseshoe crab and its uses
The horseshoe crab has been useful to human in different ways for the past several decades (Shuster et al., 2003). It is believed that the early
Indians used the tail spines of the crab as spear tips and the body after grinding as a fertilizer for their fields and ponds (Chatterji, 1994). In India, some of the tribes inhabiting the northeast coast of Orissa still use the tailpiece to get relief from different types of pains by tying it on the arms or pricking it on the forehead (Chatterji and Vijayakumar, 1987). It has been reported that the tail tips are used for healing arthritis or other joint pains and sold by faith healers in West Bengal (personal communication). The tail of the horseshoe crab is also used for making small ornaments in China. It is believed that Indians in early days used to eat the appendages of the horseshoe crab. They also used the hard carapace as a vessel for eating food and drinking water. In China, people use the carapace as a hat or ladle as the size of the carapace of T. tridentatus species is very big.
The eggs of T. gigas are used as a delicacy in Singapore, Malaysia and Borneo (Mikkelsen, 1988). People of southern part of China blend the meat of the appendages with sauce and serve it as food (Shuster et al.,
2003). It is said that in Singapore, pregnant women eat the egg mass of the horseshoe crab to get immunity to their fetus. In India, the dead carapace of the crab is boiled with mustard oil and used for treating rheumatic pain in
West Bengal.
In Korea, the fishermen sell hundreds of dried shells of the horseshoe crab to tourists and to the entrepreneurs involved in the tourism industry, for decorative purposes (Mikkelsen, 1988). But in India, at
9 present, the fishermen are not fully aware of the commercial importance of the horseshoe crab and they throw them back into the sea when they are caught in the trawl net while fishing.
In Balramgari (Orissa) India, the horseshoe crab is found in abundance and known as Rama-Lekhani' (pen of Lord Rama). The tribes inhabiting the nearby area call this valuable crab as `Bombkachchi'
(Chatterji and Singh, 2002). in the present day world no body consumes this crab, as there is hardly any flesh in the body (Mishra, 1991; Vijayakumar, 1992).
The unique physiology of the heart and functioning of the optic nerve of the horseshoe crabs make them even more interesting to the biomedical researchers (Mikkelsen, 1988; Shuster et al., 2003). The crab has altogether nine eyes, which are located under the prosoma. Out of these, two pairs are known as true eyes though the rest of organs are also sensitive to light (Chatterji, 1994). The compound eyes of the horseshoe crab can polarize light and the crystal lining cones concentrate it tenfold.
This is an adaptation for their continuous living in translucent and dark muddy waters. The vision is also sensitive to infrared and ultraviolet lights.
They help the horseshoe crabs to find their way on cloudy days when the
Sun is not visible and a small patch of clear sky is enough for the animal to locate the Sun. This principle has been very useful in developing a compass for navigation in Polar Regions where magnetic compass is not reliable and the celestial navigation is also difficult as the Sun and stars are not visible in the long polar twilight. The design of eye structure of the horseshoe crab has also been successfully adopted in designing solar energy collectors.
10 The optic nerve cells of the crab are cross connected in such a way that when one nerve is stimulated, its partner nerve gets inhibited. These results in an increase in contrast of the images looked at by the horseshoe crab. In 1967, Prof. H. K. Hartline of U.S.A. shared a Nobel Prize for his notable work on the functioning of optic nerves of the horseshoe crab
(Mikkelsen, 1988). General Electrical Company U.S.A developed a video system based on this principle to provide sharper TV images. The principle
has also helped in developing improved radar system
Pharmaceutical uses
The blood or haemolymph of the horseshoe crab is white but it turned
into blue when exposed to air. Hence, it is also known as blue-blooded
animal. The blue colour is attributed to haemocyanin, a copper based
molecule that carried oxygen throughout the circulatory system of the crab
(Mikkelsen, 1988).
The blood cells of T. gigas are known as amoebocyte (Shuster,
1982). These amoebocytes are elliptical with a prominent nucleus. The
cytoplasm in the amoebocyte is densely packed with large refractile
granules that consist of all factors required for blood clotting mechanism.
The amoebocytes aggregate quickly when exposed to minute amount of
endotoxins (Levin and Bang, 1968). In presence of endotoxins the shape of
the amoebocyte changes and simultaneously long processes extending
from one amoebocyte to the other appear in pseudopod like fashion (Levin
and Bang, 1964 a & b). This process is followed by degranulation of the
amoebocyte and release of all the clottable proteins or coagulogen that
11 immobilized and engulfed the endotoxins to form a firm gel or clot
(Jorgensen and Smith, 1973; Chatterji and Singh, 1997 a & b).
There are two types of coagulogen mRNAs present in the haemolymph of the horseshoe crab. A detailed biochemical study of the haemolymph shows that these coagulogens consist of 175 amino acids with
20 amino acids in a pre segment (Srimal and Bachhawat, 1985; Srimal et a/., 1985). Besides this, the haemolymph also consists of an important serum protein, known as LACTIN, which is useful in the diagnosis of gram- positive bacteria (Mikkelsen, 1988).
It has also been found that the intensity of the fragrance of perfumes could be increased by mixing the haemolymph (Chatterji, 1994). The
haemolymph of the horseshoe crab is also important in homoeopathic
practices. Limulus medicine introduced for the first time by Dr. C. Herring in
homoeopathic therapies is prepared from the haemolymph of the horseshoe
crab. There are number of applications of this medicine for the treatment of
mental exhaustion gastro-enteric symptoms, cholera and drowsiness after
sea bathing.
Endotoxin is present in the triple layered cell wall of gram-negative
bacteria, which is composed of polysaccharide lipids and smaller peptides
(lipopolysaccharide or LPS). The amount of endotoxins in a cell is reported
to be 104 g. The release of endotoxins in the surrounding medium is due to
rupture or lysing of the bacterial cell wall. Toxicity of endotoxin is basically
due to the presence of lipid-A fraction with LPS. The endotoxin has an
extraordinary resistance power of tolerating a wide range of temperature
and tolerance to all types of detergents (Young et al., 1990).
• 12 The gastro-intestinal tract of the human being is a house for gram-
negative bacteria. The most common gram-negative bacteria belong to the families of Psudomonadaceae, Entererobacteriaceae (mainly Eschrichia
cob), Bacteriodaceae and Neisseriaceae. An important symbiosis exists
between the gram-negative bacteria and human being for mutual benefits.
A little LPS of gram-negative bacteria is beneficial in producing stimulation
in our immunological surveillance and increased non-specific body
resistance to different pathogens. Endotoxins have also been reported to
induce necrosis and regression of malignant tumors and helpful in providing
protection against the vascular and haemodynamic effects of radiation
(Levine et al., 1988). The prevention of absorbing endotoxins from the
gastro-intestinal tract to blood circulatory system is mainly controlled by the
liver-regulated mechanisms, which involved detoxification of endotoxins.
But if any chance the endotoxins of these gram-negative bacteria even in a
small fraction, enter into the blood stream, it becomes extremely toxic and
badly affects the body especially the hypothalamus of the brain, the blood
cells, the hormonal system, the metabolic system and the immunological
surveillance system (Chatterji, 1994). Dissemination of intravascular
coagulation, hemorrhage, hypertension, circulatory collapse or even deaths
is the effects of endotoxins. The most common example of the effect of
gram-negative bacteria is septicemia where 30% mortality rate has been
reported in spite of taking all the necessary precautions.
The presence of these endotoxins in human beings is generally
assessed with the help of rabbit vaccine test. Apart from the method being
not very sensitive, it results in the death of the animal. In India,
approximately 30,000 to 40,000 rabbits are sacrificed every day, for this
13 purpose. In addition a huge amount of expenditure is also involved in the maintenance of rabbit colony. But the pioneering research of Levin and Bang (Chatterji, 1994) on endotoxin diagnosis with the help of a reagent prepared from the blood of the horseshoe crab has opened a new chapter in the history of diagnosis of endotoxins. This process is popularly known as
Limulus Amoebocyte Lysate or LAL test Dr. R. Nandan, a USA based, an Indian scientist was the first man who got a patent of LAL test in the year
1956.
A clotting enzyme-the lysate has been discovered in the amoebocytes of the horseshoe crab which clots and forms a firm opaque gel, in no time, when exposed to minute amount of endotoxin or bacterial pyrogens (Cohen, 1979; Rudloe, 1979). The rate of gelation of lysate is directly related to the concentration of endotoxins, which in turn is dependent on protein content of lysate. In lysate, the most important factor is the enzymatic activity, which basically controls the rate of gelation at a particular concentration of endotoxin (Chatterji, 1994). The lysate is so sensitive that it can detect one millionth of a billionth gram of endotoxin in just less than an hour (Ross and Bruck, 1982).
The pyrogen test with the help of lysate is simple, sensitive, accurate and easy to perform. The test does not require any special type of equipment. The test sample (0.1 ml) is mixed with lysate (0.1 ml) in a sterile test tube and incubated in a water bath at a constant temperature of 37 ° C for a period of 2 hours. If the solution is clear, waterish and runs down the wall of the test tube, the sample is considered to be free from gram-negative bacteria. But, if the solution turns into a firm opaque gel and sticks to the
14 bottom of the test tube when inverted, the sample is considered to be contaminated with gram-negative bacteria.
The key factor of forming gel by lysate in the presence of endotoxins is yet to be known, though several proteases known as factor B, C and clottable enzymes are identified. These enzymes are present in an inactive form and termed as zymogen (Srimal and Bachhawat, 1985). But in the presence of endotoxin, these inactive enzymes are converted into active enzymes by a series of reaction and called as ecocytosis.
In recent times the biomedical properties of the horseshoe crab have assumed great importance due to unanticipated marketing of Limulus
Amoebocyte Lysate as an endotoxin tester in food, drug and pharmaceutical industries (Cooper, 1979; Mikkelsen, 1988; Chatterji, 1994). The lysate test has been proved to be a better substitute for the rabbit vaccine test because of its efficiency in terms of quick result and economy (Rudloe, 1979).
The pyrogen testing by lysate at present is a promising and sensitive method for the detection of endotoxins (gram-negative bacteria) even up to
100 pg/m1 (Rudloe, 1979; Mikkelsen, 1988). It has become a basis for thee in vitro test for pyrogens associated with parental pharmaceutical and drugs, which are intended for human use. The lysate test is recognized by Drug
Regulatory Authorities and Industry in most of the countries and commonly used as an end-product testing method for endotoxin associated in human, animal injectables and drug products. Endotoxaemia, urinary infection, infection of eye and bacterial meningitis can also be detected efficiently with the help of lysate. Very recently, it has also been discovered that the bacterium responsible for gonorrhea (Neiserria gonorrhea) is a gram-
15 negative bacterium, possessing an endotoxin, which is extremely reactive with lysate. In 1970, the Federal Food and Drug Administration of the U.S.A. permitted lysate test for testing radiopharmaceutical, viral vaccine and various pharmaceutical products. An important protein detecting the vitamin
B12 deficiency in human beings has also been discovered very recently, in the lymph part of the haemolymph of the horseshoe crab.
Enemies of the horseshoe crab
The eggs of the horseshoe crab are reported to be eaten by striped bais, striped killi fish, silver perch, northern king fish, Atlantic silverside and flounders (Shuster et al., 2003). The appendages and gill books of the young limuli are most sought after by the puffer and catfish. Plovers, sand pipers and gulls are also considered to be regular predators of the horseshoe crab eggs and larvae in the beach nest (Shuster et al., 2003).
The other predators causing considerable damage to the horseshoe crab population are tiger sharks, leopard shark, loggerhead turtles, rays and swordfish. The horseshoe crab has great fear of mosquito and it dies if bitten by mosquito (Mikkelsen, 1988).
In India, the local fishermen are not fully aware of the economic importance of the horseshoe crab and discard them with a nuisance value
(Chatterji and Vijyakumar, 1987). This is because they damage their nets when trapped while operating shore seine and cast net during high tides.
Fishermen catch these crabs and throw them on the shore causing a very high mortality of the animals. Besides, a large number of crows also contribute to a great extent to the mortality of the animal during spawning migration to the beach. The crows turn the crabs upside down and then eat
16 the eggs by badly damaging the haemocoel of the crab. The crows also damage the nests and eat the fertilized eggs (Chatterji, 1994).
Apart from these, the indiscriminate exploitation of the horseshoe crab for medicinal and other purposes also threatens its population towards extinction all over the world. In the U.S.A. itself about 50,000 brooder crabs are sacrificed every year for the preparation of Limulus Amoebocyte
(Mikkelsen, 1988; Chatterji, 1994). This activity has resulted in considerable depletion of the population of the horseshoe crab along the coast of the
U.S.A. Japan was the first country to realize the declining trend of the horseshoe crab (T. tridentatus) population and subsequently implemented conservative measures by declaring the horseshoe crab as a national monument. The other possible reason for decrease in the population of the horseshoe crab in several places is the degradation and destruction of the habitat especially the breading ground of the animal.
As the horseshoe crab has assumed great significance in biomedical research, it is essential to protect this valuable creature from extinction along the India coast (Technical Report, 1998). Rearing and breeding of the horseshoe crab under confined environment is quite possible since the animal is hardy and can tolerate a wide range of temperature, salinity and other environmental conditions. As the horseshoe crab migrate regularly towards the shore for breeding purpose, it is rather prudent on our part to protect the breeding beaches from destruction and environmental degradation (Chatterji, 1994; Chatterji and Singh, 2002). The adequate measures should be adopted in protecting the valuable breeding beaches along the coast. Regular plantation of casuarinas trees along the coast to
17 control beach erosion, protection of sand dune vegetation on the beaches, ban on removal of sand gravel from the breeding beaches for construction purpose, enforcement of the technique of removal of blood by entrepreneurs without sacrificing and disturbing the normal life cycle of the horseshoe crab are the other important measures to be strictly followed by the environmentalists.
Genesis for undertaking the present study
Egg of the mysterious horseshoe crab is filled with valuables. The egg of the horseshoe crab is larger than the sperm greenish in colour and surrounded by an extra cellular egg envelop (Chatterji, 1994). The fluid, which is filled between the outer envelope and embryo, is known as peri- vitelline fluid. The peri-vittelline fluid contains important primitive types of proteins. An attempt was made as such to see the uses of peri-vitelline fluid in various purposes. It was observed that the peri-vitelline fluid helped in proliferation of [3-cells (Parab et al., 2004) that may be used in insulin production in human being. The peri-vitelline fluid contains proteins such as hemagglutinins and hemocyanin, which may have an important role during embryogenesis (Sugita and Sekiguchi, 1979; Shishikura and Sekiguchi,
1984 a & b). The embryo in the peri-vitelline fluid goes through four molts before hatching. Shishikura and Sekiguchi (1984 a & b) reported that the hemagglutinating activity in peri-vitelline fluid of the Indo-Pacific horseshoe crab, Tachypleus gigas dramatically increases after the third embryonic molting. Studies on chick developing embryo showed that the peri-vitelline fluid contained some biological active compounds that influenced early vertebrate embryonic development. Chick embryo explants cultured in vitro
18 is used as a model system as several features of chick embryo development are remarkably similar to mammalian development and these embryos are more amenable to in vitro culture and experimentation (Parab et al., 2004). It was observed that peri-vitelline fluid tended to stimulate several aspects of chick embryonic development. Peri-vitelline stimulated the development and differentiation of specific organs such as brain and heart. This clearly indicates that peri-vitelline fluid contains molecules that stimulate growth and differentiation of specific organs. It has also been observed that peri-vitelline fluid of the developing embryo of horseshoe crab showed the presence of both anti- and pro-angiogenic activities in an in vitro study. This is an important observation, since inhibition of angiogenesis is a way to "suffocate" developing tumors by depriving them of vascular irrigation and oxygen supply. Isolation of such a factor and its antagonist present in the peri-vitelline fluid of the developing embryo of horseshoe crab is of biomedical and economic importance (Mirshahi personal communication).
Organogenesis and cell replacement therapy is going to be one of the more popular methods to tackle several dreaded medical complications including diabetes and degenerative diseases related to heart (Mirshahi et al unpublished data). In vitro organ development and stem cell biology are two of the approaches that will underline the success of such a strategy.
However, in order to achieve ideal organ growth, differentiation and/or regeneration, it would be essential to identify, purify and employ specific molecules, either individually or in various combinations. The same is true for successful applications of stem cell biology where in one needs to provide a defined milieu to naive stem cell so as to coax them to take one or the other developmental pathway, at the choice of the researcher. The
19 possible source for such novel molecules is marine animals included amazing horseshoe crab.
The PVF of the Indian horseshoe crab contains peptide(s) capable of positively influencing differentiations of specific organs. Such peptides are likely to be present in minute quantities and may be as proteins in addition to the four major proteins reported in the Japanese horseshoe crab (Nagai et al., 1999). The use of peri- vitelline fluid for maintaining the original phenotype of CD34+ stem cells, enhancements of colony and capillaries formation in vitro, vasculogenesis process and myocyte differentiation were discussed in detail in Chapters 1-3.
The biomedical properties of horseshoe crab depend on the ecological conditions as these parameters play an important role on the physiological aspect of the animals. Hence in Chapter 4 the culture of amoebocytes in vitro considering the importance and requirements of amoebocyte lysate in biomedical science for rapid detection of pyrogens was demonstrated in detail. The main aim of the present study was to produce amoebocyte lysate without disturbing the natural population of the animals. Additionally, production of amoebocyte lysate through in vitro technique would also ensure the uniform sensitivity of the product under different seasons. Similarly in Chapter 5 the culture condition of the horseshoe crab was described in relation to different environmental conditions. This attempt was made to standardise the best culture condition to keep the donors for bleeding the horseshoe crab. The entrepreneurs interested in using horseshoe crab for biomedical purposes would have to follow these culture conditions to maintain the horseshoe crab population under simulated condition.
20 The re-generating properties of the gill lamellae were discussed in Chapter-6. Basically this study will help in finding out this important factor in near future that can be used to develop effective drug for regeneration of degenerated cells like
Islets of langerhan's cells. So all these chapters are interrelated justifying the biomedical properties under the umbrella of marine biological approaches.
21 Chapter-1
Role of peri-vitelline fluid of the fertilized eggs in maintaining the original phenotype of CD 3e stem cells, enhances colony and capillary formation in vitro Diverse processes such as embryonic development, tumor growth and tissue repair are linked by the absolute requirement for a vascular bed to deliver nutrients and oxygen to metabolically active cells. The clinical importance of controlled vascular growth is illustrated by disease states resulting from imbalances in blood vessel formation, such as Diabetes mellitus. Vasculopathies associated with diabetes include excessive blood vessel formation (eg, retinopathy, glomerular nephropathy) and accelerated atherosclerosis leading to coronary artery disease, peripheral vascular disease and cerebrovascular disease (Martin et al., 2003). A number of growth factors essential for wound healing, including VEGF, b-FGF-2 and platelet-derived growth factor (PDGF)-B, have also been found to be reduced in experimental diabetic wounds (Beer et al., 1997).
Vascular endothelial growth factor (VEGF)-A, referred subsequently as simply VEGF and b-FGF members of a family of growth factors with essential roles in vascular and lymphatic growth and patterning, has been shown to be deficient in experimental and clinical diabetic wounds (Frank et al., 1995). VEGF acts through at least two receptors, expressed primarily on endothelial cells, along with other vascular cytokines to induce and maintain the vasculature (Carmeliet, 2000). VEGF and b-FGF are the prototype members of this family and has been shown to be absolutely essential for vascular development (Carmeliet et al., 1996). VEGF and b-FGF facilitate tissue repair by increasing vascular permeability, allowing the efflux of inflammatory cells into the site of injury and increasing the migration and proliferation of pre-existing endothelial cells. However, exogenous administration of VEGF and b-FGF result in leaky malformed vessels, which
22 has raised a great concern regarding its therapeutic usefulness (Drake and
Little, 1995).
Recent studies have established that angiogenesis is not the sole mechanism by which new vessels are formed. It is now apparent that bone marrow-derived cells, including endothelial progenitor cells (EPCs) are mobilized in response to trauma or ischemia and are able to contribute to tissue repair and new blood vessel formation (Takahashi et al., 1999; Gill et al., 2001). The development of blood vessels from blood-borne endothelial precursors, termed as vasculogenesis, was previously thought to be restricted to embryonic development, but is now accepted to play a role in postnatal processes including tissue repair (Crosby et al., 2000).
During early developmental stages of the embryo of horseshoe crab, as the inner egg membrane is being freshly formed, the space between the inner egg membrane and embryo (peri-vitelline space) is filled with peri- vitelline fluid (Sekiguchi, 1988). Since it was previously shown that peri- vitelline fluid (PVF) added to early chick embryo induces an increase of cardiac vasculogenesis (Parab et al., 2003), we were interested to analyze the action of PVF in human stem cell-induced vasculogenesis.
It is now generally accepted that bone marrow stem cells differentiate into endothelial and hematopoietic cell progenitors. The stem cells appear to have antigenic determinants, including CD 34k , ACC133 and VEGFR2, although their phenotype may be plastic (Peichev et al., 2000). These cells respond to growth factors, prominent among them is Vascular Endothelial
Growth Factor (VEGF) that induced CD 34 + to differentiate into endothelial
23 cells (Hill et al., 2003). A number of studies indicate that these cells or their
progeny may be a significant source of endothelial cells in adult pathologic and non-pathologic vascularization and may participate in vascular repair
(Peichev et al., 2000). Treatment with exogenous bone marrow-derived cells can promote vascularization, accelerating restoration of blood flow to
ischemic tissues and improving cardiac function after ischemia. Hence, certain amount of hope can justifiably be entertained that either alone or in combination with other angiogenic factors, these cells can be used to treat
ischemic and vascular diseases in the near future.
Formation of capillaries tubes in vivo is an early event in the establishment of fine blood carrying capillary network during embryogenesis and later in adult during wound healing as well as growth and dissemination
of tumors. Whereas, colony formation is a process of cell organization and
indicate the inherent potential of these cells to become organized
progressively leading to the genesis of organs. The present study
particularly relates to an interesting study on the peri-vitelline fluid obtained from the fertilized eggs of the Indian horseshoe crab (Tachypleus gigas,
Muller) on the maintenance of the original phenotype of CD 34 + stem cells,
enhancement of the formation of capillary tubes as well as large colony formation in vitro.
24 Materials and Methods
The fertilized eggs of the horseshoe crab were collected from the nests located on the sandy beach at Balramgari (Orissa). The eggs were incubated at a constant temperature (37±1 ° C) in artificial incubators till the eggs became transparent and showed the movement of trilobite larvae as described (Chatterji, 1994). The peri-vitelline fluid (PVF) was collected using a sterilized 22-gauge needle under highly aseptic condition immediately after the eggs became transparent and showed the movement of trilobite larvae. The peri-vitelline fluid was aliquoted and stored at -20 ° C and freeze- dried using a tabletop freeze dryer (Edward: Micromodulo). When required, the freeze-dried sample of PVF was dissolved (1 mg/ml) in Endothelial
Basal Medium (EBM MV2). From this stock solution of PVF, 100 pl sample was re-suspended in 1 ml of EBM MV2 medium (to make the final concentration 0.1 mg/ ml) containing IGF 20 pg/L, Gentamycine 50 mg/L,
Arachnoid acid 1 mg/L, EGF 20 [tg/L, Amphotericin 5000 pg/ L and 5% Fcetal Calf Serum.
The human bone marrow from healthy volunteers (donors for bone marrow transplantation) was collected from the Haematology Division of the
Hospital Hotel Dieu, Paris (France). The mononuclear cells from the cells were then immediately separated by Ficoll centrifugation technique and then bone marrow was diluted in 40 ml of L- Glutamin enriched with RPMI 1640 medium. The mononuclear cell ring was collected and separation of CD 34 + cells was carried out using immune-magnetic beads as per the protocol of
Easy Sep Stem Cell Technologies Inc U.S.A.
25 Isolated CD 34+ cells were cultured in 5 different culture flasks
(adjusted at 2x106 cells/flask containing 5 ml EBM MV2 medium enriched with 5% Fetal Calf Serum) and in 5 labteks (50 x 10 3 cells/well containing 0.5 ml of EBM MV2 medium enriched with 5% Fetal Calf Serum). Before transferring the cells, culture flasks were treated with gelatine (1X). Five sets of small culture flasks were prepared in different combinations; i.e. Set A: control cells, Set B: addition of VEGF (10 pl, 0.25 pg/500 pl), Set C: addition of b-FGF (10 pl, 0.25 pg/500p1), Set D: addition of VEGF and b-
FGF, Set E: addition of PVF (0.1 mg/ml). All these sets were incubated for
22 days at 37° C with 5% CO2 and 95% humidity. The number of CD 34+ attached to the surface was counted in each flask.
Colony formation assay: The assay was performed as described by Hill et al. (2003). The isolated CD 34+ cells were re-suspended at 2.5 x 10 6 cells/ml in culture medium in two different sets i.e. Set A: Medium EBM MV2
+ cell suspension + 5 % Fcetal Calf Serum + VEGF and b-FGF ((10 pl, 0.25 pg/500 pl); Set B: EBM MV2 medium + cell suspension + PVF (0.1 mg/ml)
+5 % Fcetal Calf Serum. This was followed by plating the cells at 2 ml/well in fibronectin-coated 6-well plates (Becton Dickinson, UK) and incubated for two days at 37° C with 5% CO2 and 95% humidity. After two days, the non- adherent cells were removed, counted and re-suspended at 0.5 to 1 x 10 6
cells/ml in fresh medium in two different sets as described above.
The colonies in each well were then identified and counted. A typical colony was defined as a central core of «round» cells surrounded by elongated «sprouting» cells at the periphery and was classified as Colony
26 Forming Unit Endothelial Progenitor Cell (CFU-EPC). Colonies of endothelial progenitor cells isolated in this fashion exhibited many endothelial cell characteristics, including expression of CD31 and VEGFR-2
(Asahara et al., 1997).
Capillary formation assay: In vitro, angiogenesis was tested using human bone marrow endothelial cells (HBMEC) kindly provided by Prof. Pienta of
University of Michigan, Comprehensive Cancer Center Ann Arbor, Michigan,
USA. The technique employed for the study of in vitro angiogenesis is same as described by Kern et al. (2003) and modified as given below. HBMEC were prior incubated with endothelial basal medium (EBM) with 5% Fcetal
Calf Serum and antibiotics at 37° with 5%CO2 to obtain the 90% confluence in the presence of PVF (0.1 mg/ml) and in its absence in two different culture flasks. Matrigel depleted in growth factor was used to study capillary tube formation. For the assay, 96-well plates pre-coated with matrigel
(Becton-Dickinson, Le Pont de Claix, France) were employed. The matrigel was allowed to polymerize for 1-2 hours at 37° C after which the previously detached (with accutase) HBMEC were seeded with 3.5 x 10 4 cells / well onto the matrigel-coated wells. With different incubation times at 37° C, capillary tube formation was assessed using an inverted microscope fitted with a digital camera (Nikon-Diafot). The images were acquired and processed using the micro vision computer (Microvision Instruments, Evry,
France). The capillary network was quantitiated by recording the number of connections between three or more capillary-like structures, number of capillaries tubes and the length of the capillaries after 4, 8 and 12 hours.
27 Results
Viability of CD 3e cells in PVF
The results of the present investigation clearly showed that in PVF, a significant increase in the number of adherent cells was observed. The cells remained viable for a relatively longer duration, as compared to cells cultured with VEGF or b-FGF or both (Figure 1.1). The addition of VEGF induced an increase in the number of adherent CD 34 + cells after 22 days in culture as compared to control cultures. The effect of b-FGF was slightly higher than that of VEGF. A combination of VEGF and b-FGF showed a synergetic effect. Interestingly, PVF alone induced a remarkable increase of adherent CD 34+ cells attached to the surface. However, there was no synergetic effect of PVF when combined with VEGF and b-FGF (Figure
1.1).
Colony formation
Similarly, the number of colonies formed in culture wells on days 4, 8 and 12 was ascertained under light microscope (Figure 1.2). In the control experiments (without PVF), the number of colonies was maximal on day 8
(Figure 1.2) whereas in presence of PVF, the number of colonies increased considerably by —55% as compared to the control group where no PVF was used. This increase was continued through days 8 and 12 (Figure 1.2). In addition, in PVF treated cultures, the colonies appeared bigger with a larger number of cells per colony as compared to control group (Plate 1.1).
28 Capillary network formation
The formation of capillaries network is clearly demonstrated in
Plates 1.2 a-d during 1 hours of experiment. The formation of capillaries was relatively much higher when the cells were treated with PVF. The numbers of intersections after 4 hrs were -70% higher as compared to control group (Figure 1.3). The number of intersections started decreasing after 8 hrs and at 12 hrs where no intersection was visible in both the groups (Figure 1.3). Similarly the number of capillary tubes after 4 hrs of experiment was higher (-63%) when the cells were cultured in presence of
PVF (Figure 1.4). To demonstrate the pro-angiogenic effect of PVF, the length of capillaries was also measured and it was found that in presence of
PVF the length of capillaries increased -70% more than in the group where no PVF was used (Figure 1.5).
29 Discussion
The ability of CD 34+ cells to differentiate into endothelial cells can be determined in vitro by plating endothelial cells onto an extracellular matrix, such as collagen, fibronectin and/or gelatin. Adherent colonies of ECs can subsequently be detected by their morphology and immune-reactivity to EC markers (Rafii et al., 1995; Shi et al., 1998; Rafii, 2000). Further analysis of endothelial progenitor cell homing and incorporation into sites of neo- vascularization (e.g. following bone marrow transplant into recipient mice) can be performed using antibodies specific for human endothelial cell markers (Kalka et al., 2000).
A number of growth factors for the in vitro expansion of endothelial precursor cells are available in the market. One such combination comprising VEGF, b-FGF, IGF and EGF resulted in 80 to 90-fold expansion of cells expressing EC antigens (Kalka et al., 2000). Other growth factor combinations described in the literature include VEGF, b-FGF and IGF
(Suhonen et al., 1996) and b-FGF together with VEGF (Peichev et al.,
2000). The identification and isolation of endothelial progenitor cells is difficult due to the absence of specific endothelial markers and functional assays to distinguish migrating endothelial progenitor cells from sloughed mature ECs (Rafii et al., 1995). ECs and matured wall-derived ECs express common endothelial-specific markers including VEGFR-2 (KDR, Flk-1)
(Eichmann et al., 1997), Tie-2 and Tie-1 (Sato et al., 1995, Suri et al., 1996),
VE-Cadherin (Johnstone et al., 1998), CD34, PECAM (CD31) and E-
Selectin (Peichev et al., 2000). Other endothelial markers include von
30 Willebrand factor (vWF), P1H12 (CD146), thrombomodulin, CD36, Endoglin and Integrin a V113. Identification of ECs is further complicated by the fact that hematopoietic stem cells and their progeny (particularly monocytes) express markers similar to those expressed by endothelial cells, such as
VEGFR-1 (Flt-1), CD34, PECAM-1, Tie-1, Tie-2 and Von Willebrand's factor
(Peichev et al., 2000). The antibody P1H12 (CD146) has been used successfully for the enrichment of circulating ECs from peripheral blood prior to analysis (Lin et al., 2000).
The peri-vitelline fluid of the horseshoe crab has been reported to contain proteins such as hemagglutinins and hemocyanin that could play an important role during embryogenesis (Sugita and Sekiguchi, 1979;
Shishikura and Sekiguchi, 1984 a & b). The hemagglutinating activity in peri-vitelline fluid of the Indo-Pacific horseshoe crab Tachypleus gigas increases dramatically after the third embryonic molting (Shishikura and
Sekiguchi, 1984 a & b). A multimeric lectin of 450 kDa composed of a 40- kDa sub-unit was purified from the peri-vitelline fluid of T. gigas, using affinity chromatography (Sugita and Sekiguchi, 1979). Also insulin producing beta cell differentiating factor has been identified and characterized in the peri-vitelline fluid of the fertilized eggs of the Indian horseshoe crab (Parab et al., 2004). The agglutin binding substances from the peri-vitelline fluid of Tachypleus gigas embryo has been purified and characterized (Shishikura and Sekiguchi, 1984 a & b). The expression of
VEGFR-2 and AC133 by circulating human CD 34+ cells has been identified in a population of functional endothelial precursors (Peichev et al., 2000).
31 From the present results, it appears that the number of CD34 + cells attached to the surface of flask is much higher in the presence of PVF than in the presence of VEGF and/or b-FGF. Since vascular development requires many cell adhesion interactions, including cell-matrix interactions, both with basement membranes and with surrounding extra cellular matrices, it is suggested that the contribution of PVF in vascular development could be due to increased in cell attachment. In fact there was a significant increase (2.8 times) of attached CD 34 + cells when exposed to
PVF alone as compared to culture cells exposed to VEGF alone. Similarly, the difference was 1.94 times more in PVF exposure as compared to b-FGF exposure. When both VEGF and b-FGF were used concomitantly, an additive effect on the number of CD 34 + attached cells was observed. This was 2.15 times more as compared to cells exposed to VEGF and 1.49 times more as compared to b-FGF. This clearly indicated that combined presence of VEGF and b-FGF has a cumulative effect in enhancing the number of attached CD 34+ cells. The addition of a third component, namely, PVF further enhanced the cumulative effect. But surprisingly the use of PVF alone surpasses the cumulative effect shown by all the three components combined. Hence the present investigation shows that PVF largely surpasses the beneficial effect rendered by VEGF or b-FGF or even both together in increasing the number of attached CD 34 + cells.
In addition it was shown that PVF added to human endothelial cells was responsible for an increase in capillary tube formation. This process is
a prerequisite in vasculogenesis. The most prevalent in vitro assay to
determine clinical vasculogenesis is to evaluate the colony-forming unit of
endothelial progenitor cells exhibiting expression of CD31 and vascular
32 endothelial growth factor receptor 2. With this in aim, the present investigation was undertaken to show the ability of CD34+ cells to expand the clonogenic-derived cells ex-vivo. The effect of PVF, as well as its combination with VEGF and b-FGF was tested. Concerning the colony formation, PVF induces an increase in the number of colony forming units and the size of the colony formed in the presence of PVF was much bigger.
The data of the present study clearly show the role of PVF in differentiation of stem cells CD34 + into endothelial progenitor cells which form colony forming units (CFUs). They contain more cells per colony than colonies formed in its absence. The action of PVF is much stronger than that of VEGF and b-FGF. This could explain the cardiac promoting effect of
PVF on early embryo of horseshoe crab (Parab et al., 2004). The results of the present study suggest that PVF could improve stem cell therapy for vasculogenesis in patients with severe myocardial ischemia. This is a highly interesting result which can be translated into development of new CD 34 +
+ cells culture medium and procurement of much higher yield of the CD 34
cells in near future for the purpose of transplantation or for exploiting their plasticity to be guided towards differentiation into different sub-lineages.
33 Summary
In the peri-vitelline fluid (PVF) of the fertilized eggs of Indian horseshoe crab, Tachypleus gigas (Muller), a significant increase in the number of adherent cells was observed. The cells remained viable for a relatively longer duration, as compared to cells cultured with VEGF or b-
FGF or both. In the control experiments (without. PVF), the number of colonies was maximal on day 8 whereas in presence of PVF, the number of colonies increased considerably by -55% as compared to the control group.
This increase was continued through days 8 and 12. In addition, in PVF treated cultures the colonies appeared bigger with a larger number of cells per colony as compared to control. The formation of capillaries was relatively much higher when the cells were treated with PVF. The numbers of intersections after 4 hrs were -70% higher as compared to control group.
The number of intersections started decreasing after 8 hrs and at 12 hrs where no intersection was visible in both the groups. Similarly the number of capillary tubes after 4 hrs of experiment was higher (-63%) when the cells were cultured in presence of PVF. To demonstrate the pro-angiogenic effect of PVF, the length of capillaries was also measured and it was found that in presence of PVF the length of capillaries increased by -70% more than the group where no PVF was used.
34
Attachment of CD34+ cells to the surface after 22 days of culture
160 0 140 -secn Vas co 120-
0 a^ 100 L '6 80 - ■ Mean g a 60 - c 40- cm 20- a)L 0 ct> Control VEGF bFGF VEGF+bFGF PVF
Figure 1.1: Attachment of CD 34+ cells after 22 days
35 Number of colonies/well 4 Formation ofcolonies Figure 1.2:Formationofcolonies Number ofdays 8 12 36 F ■ ❑ With PVF Control (WithoutPVF)! Control PVF treated
Plate 1.1: Colony Formation
37 Control Treated with PVF
A: Capillary network formation Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 0 hrs)
Capi lary Junction
B: Capillary network formation-migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 4 hrs)
Plate 1.2a: Capillary network formation: migration of human bone marrow endothelial cells
38 Control Treated with PVF
C: Capillary network formation-migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 8 hrs)
D: Capillary network formation-Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 12 hrs) Plate 1.2b: Capillary network formation Migration of human bone marrow endothelial cells
39
Pro-angiogenic activity (0.1mg/m1 PVF)
o With PVF ■ Control (Without PVF)j
4 hours 8 hours 12 hours Hours
Figure 1.3: Pro-angiogenic activity (capillary tubes formation)
40 Figure 1.4:Pro-angiogenicactivity(Numberofintersections) Num ber of intersections/well 20 - 25 10 - 15- 5 - Pro-angiogenic activity(0.1mg/m1PVF) 1 Hours 2 41 3 ❑ ■ With PVF Control (WithoutPVF) • •
Pro-angiogenic activity (0.1mg/m1 PVF)
25000 c to 20000 -
- ++ = e 15000 - ❑ ful- E With PVF 0 2 ■ 45 .0 10000 - • Control (Without PVF) .c E ts) c 5000 - o _1 0 4 hours 8 hours 12 hours Hours
Figure 1.5: Pro-angiogenic activity (Length of capillaries)
42 Chapter-2
Role of peri-vitelline fluid of the fertilized eggs of the Indian horseshoe crab in vasculogenesis Vasculogenesis is a process of differentiation of stem cell endothelial progenitor cells (EPC) into mature endothelial cells. It contributes to new vessel formation (Rumpold, et al, 2004). Vasculogenesis is closely resembles the embryological process in which 'haemangioblasts' differentiate in primitive vessels. The ability to isolate stem cells recently has opened up exciting vistas to help the re-vascularization of ischemic tissues.
The existence of EPC has been convincingly shown in both animals and humans (Rafii Avecilla, et al 2003, Rafii and Lyden, 2003, Zwaginga and
Doevendans, 2003, lshikawa and Asahara, 2004). Cellular progenitor population for the generation of EC may serve as cellular source for therapeutic vasculogenesis. It could represent a novel approach to treat patients with ischemic disease that are not curable with conventional treatments. Therapeutic angiogenesis / vasculogenesis and ex vivo engineered endothelial progenitor cells have already been successfully used for the treatment of peripheral limb ischemia (Jackson et al., 2001;
Alessandri et al. 2004). Cell based regeneration therapy has been widely touted as a novel means of repairing of damaged heart after myocardial infarction (MI), chemotherapy-induced damages or other injuries leading to heart failure (Yeh et al., 2003).
A key role of stem cell research is to understand how differentiation is controlled and then to know how to direct the cellular development.
Research on both embryonic and adult stem cells will make a complementary contribution to these objectives. Studies of human embryonic stem cells may yield information about the complex events that occur during human development (Malouf et al., 2001). A primary goal of
43 such type of work has been to identify how undifferentiated stem cells become differentiated (Yeh et al., 2003). Scientists know that turning genes on and off is central to this process. Some of the most serious medical conditions, such as cancer and birth defects are due to abnormal cell division and differentiation. A better understanding of the genetic and molecular controls of these processes may yield information about how such diseases arise and suggest new strategies for therapy. A significant hurdle to this use and most uses of stem cells is that scientists do not yet fully understand the signals that turn specific genes on and off to influence the differentiation of the stem cell. Human stem cells could also be used to test new drugs (Malouf et al., 2001).
Stem cells are unspecialized and one of the fundamental properties of a stem cell of not having any tissue-specific structures that allow it to
perform specialized functions. A stem cell cannot work with its neighbours to pump blood through the body (like a heart muscle cell). It cannot carry
molecules of oxygen through the bloodstream (like a red blood cell) and it cannot fire electrochemical signals to other cells that allow the body to move
or speak (like a nerve cell). However, unspecialized stem cells can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells
(Terada et al., 2002). Stem cells are capable of dividing and renewing themselves for long periods. Unlike muscle cells, blood cells, or nerve cell which does not normally replicate themselves, stem cells may replicate
many times. When cells replicate themselves many times over it is called
proliferation. A starting population of stem cells that proliferates for many
months in the laboratory can yield millions of cells. If the resulting cells
44 continue to be unspecialized, like the parent stem cells, the cells are said to be capable of long-term self-renewal (Wang et al., 2003).
The specific factors and conditions that allow stem cells to remain unspecialized are of great interest to scientists. It has taken scientists many years of trial and error to learn to grow stem cells in the laboratory without them spontaneously differentiating into specific cell types. For example, it took 20 years to learn how to grow human embryonic stem cells in the laboratory following the development of conditions for growing mouse stem cells. Therefore, an important area of research is to understand the signals in a mature organism that cause a stem cell population to proliferate and remain unspecialized until the cells are needed for repair of a specific tissue. Such information is critical for scientists to be able to grow large numbers of unspecialized stem cells in the laboratory for further experimentation (Malouf et al., 2001).
Adult stem cells typically generate the cell types of the tissue in which they reside. In a living animal, adult stem cells can divide for a longer period. They can give rise to mature cell types that have characteristic shapes, specialized structures and functions of a particular tissue. The differentiation pathways of adult stem cells are descibed in detail in Plate
2.1 (Stem cell Information, The National Institute of Health Resource for stem cell research).
A blood-forming adult stem cell in the bone marrow, for example, normally gives rise to the many types of blood cells such as red blood cells,
45 white blood cells and platelets (Malouf et al., 2001). Until recently, it was thought that a blood-forming cell in the bone marrow is haematopoietic stem cell that might not give rise to the cells of a very different tissue such as nerve cells in the brain. However, a number of experiments over the last several years have raised the possibility that stem cells from one tissue may be able to give rise to cell types of a completely different tissue, a phenomenon known as plasticity (Plate 2.1). Examples of such plasticity include blood cells becoming neurons, liver cells that can be made to produce insulin and haematopoietic stem cells that can develop into heart muscle. Therefore, exploring the possibility of using adult stem cells for cell- based therapies has become a very active area of investigation by researchers.
An adult stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ can renew itself. They can differentiate to yield the major specialized cell types of the tissue or organ.
The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in which they live. Some scientists now use the term somatic stem cell instead of adult stem cell. Unlike embryonic stem cells that are defined by their origin (the inner cell mass of the blastocyst), the origin of adult stem cells in mature tissues is unknown.
Cellular progenitor cell population for the generation of EC may serve as cellular source for therapeutic vasculogenesis or tumour targeting therefore, representing a new approach to treat patients with ischemic disease that are not curable with conventional treatments. This may also be
46 used in targeting drugs for cancers in which the promotion of neovascular formation is an important matter. Therapeutic angiogenesis / vasculogenesis and ex vivo engineered endothelial progenitor cells have
already been used for the treatment of peripheral limb ischemia (Alessandri
et al. 2004). Various cell populations such as embryonic stem cells, cord
blood cells, mesenchymal cells and bone marrow stem cells have been
suggested as a source for replacement therapy. It will be useful to identify a
readily available cell source that does not require significant manipulation.
47 N More ldlot OKI col tioutrophil Bone Betcaht• tyrtoAold progotiled
7 toilTiOrila
SUM Ca
c 40Gtra-41'40°019s MutCpcoomlel 141yotoo 'Ion coil Foclenher , toti• 4174 col
Piolaiou
Rod blood cols
komolopototle appailvtarnrr4
Immo! rivn coil it •
Pi441140oltg1
Skolalal mock eon colt
illopotocjso gin car?
Plate 2.1. Hematopoietic and stromal stem cell differentiation (Source: Stem cell Information, The National Institute of Health Resource for stem cell research)
48 Liver
Brain
f CNS stem calls
Skeletal Bone muscle marrow
Blood coil
Blood vessel Epithelial cell Fat cell
Cardiac muscle
Plate 2.2 Rresource for stem cell research (Source: Stem cell Information, The National Institute of Health Resource for stem cell research)
49 In a preliminary attempt to examine the presence of differentiating factors on chick developing embryo it was observed that there are some
important compounds present in the peri-vitelline fluid of the early developing embryos of the horseshoe crab that help in organanogenesis
process when the study was carried out on chick model. In a preliminary
study made at the INSERM-Universite Paris 6, it has been found that when
chick embryo is incubated with peri-vitelline fluid it shows the enlargement
of heart tissue. On the basis of these observations it has been concluded
that there can be two possibilities for the enlargement of heart, either it was
due to vasculogenesis or pro-angiogenesis. We know that adult bone
marrow stem cells have been shown to differentiate into different cell
typyes. Hence on the basis of preliminary studies an attempt has been
made and demonstrated that the peri-vitelline fluid stimulated the adult
human bone marrow stem cells to transdifferentiate into cardiomyocytes,
mature endothelial cells or smooth muscle cells using specific markers.
50 Material and Methods
The fertilized eggs of the horseshoe crab were collected from the nests located on the sandy beach at Balramgari (Orissa). The eggs were incubated at a constant temperature (37+1 ° C) in artificial incubators till the eggs became transparent and showed the movement of trilobite larvae. The peri-vitelline fluid was collected using a sterilized 22-gauze needle under highly aseptic condition immediately after the eggs became transparent and showed the movement of trilobite larvae. The peri-vitelline fluid was aliquoted and stored at -20° C.
The peri-vitelline fluid was freeze dried using a table top freeze dryer
(Edward:Micromodulo). The freeze-dried sample of PVF was re-suspended
(1 mg/m1) in Endothelial Basal Medium (EBM MV2) purchased from Promo
Cell (Cat no C-22 221). Bone marrow was collected from the Haematology
Division of the Hospital Hotel dieu Paris 4. Ficoll Centrifugation Technique then immediately separated the adherent cells from the bone marrow. In this technique, at first 1 ml of bone marrow was diluted in 40 ml RPM! 1640 —L-
Glutamin Medium purchased from PAA Laboratories (Cat no E-15-039). The
diluted samples (10 ml each) were then transferred slowly into conical tubes
containing 4 ml of Lymphocyte Separation Medium (Lymphozyten
Separations Medium: Cat No: J 11-004). The four conical tubes were then
centrifuged at 1650 rpm for 30 minutes. The supernatant from all the tube
was collected whereas all pellets were discarded. The supernatant was
diluted to 50 ml with PBS (1X) (Dulbecco's Phosphate Buffered Saline 10X
by PAA Laboratories, Austria: Cat No H15-01) and centrifuged again at
1200 rpm for 10 minutes. The supernatant was discarded and pellets were
51 diluted in to 20 ml RPMI-1640 with 10% Foetal Calf Serum (Promo Cell: Cat
No C 37310). This was followed by centrifugation of the samples again at
1200 rpm for 10 minutes. The supernatant was discarded and pellets were used for separation of CD 34 + cells. 100 pl from the stock solution of PVF was re-suspended in 1 ml of EBM MV2 medium containing IGF 20 p.g/L, Gentamycine 50 mg/L, AA 1mg/L, EGF 20 1.1g/L, Amphotericin 5000 p.g/L, 5% fcetal calf serum (Promocell) to make the final concentration 0.1 mg/ ml.
The separation of CD 34+ cells from the mixed population of the cells was done using Immunomagnetic Beads following the protocol as described
by Easy Sep Stem Cell Technologies Inc U.S.A. The separated CD 34 + cells
were counted by haemocytometer and adjusted at 2x10 6 in 5 culture flasks
containing 5 ml EBM MV2 medium for FACS study and 50x 10 3 cells/well in
2 labteks, each well containing 400 pl of EBM MV2 medium for immuno
fluorescence study. The surface of the culture flasks and labteks was
treated with gelatine (1X) before transferring the medium. The following
scheme was followed for experimentation:
A: For FACS study Set A: Medium EBM MV2 + cell suspension + 5 % Fcetal Calf Serum.
Set B: EBM MV2 medium + cell suspension + VEGF (10 pl, 0.25 pg/500 pl) + 5% Fcetal Calf Serum.
Set C: EBM MV2 medium + cell suspension + bFGF (10 pl, 0.25 pg/500p1) +5% Fcetal Calf Serum.
Set D: EBM MV2 medium + cell suspension + VEGF + b-FGF (10 pl, 0.25 pg/500 pl) +10% serum.
52 Set E: EBM MV2 medium + cell suspension + PVF (0.1 mg/ml) +5 % Fcetal Calf Serum.
B: For immuno fluorescence study Set A: Medium EBM MV2 + cell suspension + 5 °A) Fcetal Calf Serum Set B: EBM MV2 medium + cell suspension + PVF (0.1 mg/ml) +5 % Fceta Calf Serum
All the culture flasks were taken out from the incubator and then the medium was removed. The samples were washed thoroughly with PBS (1X) solution for three times. The cells from each flask were detached by
Accutase (PAA Laboratories GmbH Cat No. L11-007) and transferred into
15 ml centrifuge tubes. The samples were centrifuged at 1100 rpm for 10
minutes to remove Accutase and then re-suspended in 1 ml PBS (1X). The
cells from each centrifuge tube were divided in to 4 different FACS tubes
(100,000 cells / tube) and again centrifuged at 1100 rpm for 10 minutes.
PFA solution (4%) was then added for five minutes at room temperature into
each FACS tube and sample was vortexed. Each tube was again washed
with PBS (1X) and then permeabilized with Triton (1%) for 20 minutes. To
avoid unspecific adsorption in the further steps, PBS: BSA (1%) was added
for 30 min. The samples were washed again with PBS (1X) three times. To
detect the antigens present in the cells, 2 incubations of 1 hr at room
temperature were performed. The first one after adding primary antibodies
diluted 1/100 in PBS:Alb [rabbit Ig anti human von Willebrand Factor (Dako
Cytomation Polycional Rabbit Anti Human von Wiilibran Factor - Cat No A
0082)] and mouse Ig Anti Smooth Muscle actin (Zymed Laboratories Inc;
Cat No 18-0106) in different sets of test tubes. After three times washing of
the samples with PBS (1X), the 2nd incubation was performed by adding
53 the secondary antibodies previously diluted at 1/100 in PBS containing 1% albumin (biotinylated anti rabbit Ig and biotinylated anti mouse Ig by
Amersham). The samples were washed with PBS (1X) and then streptavidine fluorescence (Amersham Cat No RPN 1232 V1) was used for staining. Finally the samples were washed three times with PBS (1X). For negative controls, primary antibodies were not added to the tubes. All the samples were analysed by FACS analysis using a Flow-cytometer (Model: Beckton & Dickenson).
All the labteks were removed from the incubator and washed thoroughly with PBS (1X) solution for three times. 4% para - formaldehyde
(PFA) was added into each well of labtek and kept at 4° C for five minutes for fixation. Each well of labtek was again washed with PBS (1X) and then permeabilized with Triton (0.1%) for 20 minutes. To avoid unspecific adsorption in the further steps, PBS: BSA (1%) was added in the labteks for
30 minutes. The samples were washed again with PBS (1X) three times.
Then, in all the wells of a labtek except negative controls, primary antibodies diluted 1/100 in PBS containing 1% Alb [rabbit Ig anti human von Willebrand
Factor (Dako Cytomation Polyclonal Rabbit Anti Human von Wiilibran
Factor; Cat No A 0082)] and mouse Ig Anti Smooth Muscle actin (Zymed
Laboratories Inc; Cat No 18-0106) was added. After an incubation period of 1 hr at room temperature, the wells of the labteks were washed three times washing with PBS (1X). This was followed by addition of secondary antibodies (Streptavidine conjugated anti rabbit Ig Amersham and RPE conjugated anti mouse Ig; Amersham Cat No RPN 1232 V1) diluted 1/100 in
PBS:BSA for double staining. Both the antibodies were used at the dilution of 1/100 in PBS containing 1% albumin. The labteks were washed three
54 times with PBS (1X). The slides from the labteks were detached and permanent slides were prepared with fluroprep. The slides were analysed using both a microscope for fluorescence and a confocal microscope.
For each test, while incubating the samples, only the 2nd labelled antibody was added and these sets were treated as negative controls. By
immunocytochemistry, it was confirmed (as already found by flux cytometry)
that PVF induced an important differentiation of CD 34+ cells in endothelial
cells (Von Willebrand) and in smooth muscle cells (S-M actin).
The effect of PVF on cell migration was also studied for 24 hrs. For
the analysis of cell migration and for that 5 x 104 HBMEC (human bone
marrow endothelial cells) were cultured in 24 well plates in the presence of
M131 medium containing 5% FCS and 5% MVGF (Microvascular Growth
Factors from Cascade Biologics) until 80% confluency. This was followed by
scratching a line through the layer artificially creating an open scar or
wound. Then, M131 medium containing only 2% FCS (to avoid cell
proliferation) in the presence or absence of PVF was used at a
concentration of 100 µg/ml in each well. The open gap was then inspected microscopically over time as the cells move in and fill the damaged area.
Cell migration was evaluated by measuring the quantum of cells migrating
to the wounded edge after 0, 3, 6, 9 and 24 hrs of intervals with the help of
image analysing system
55 Results
At first a comparison was done on the differentiation of CD 34 + stem cells into endothelial cells using different growth factors such as VEGF, b-
FGF, the mixture VEGF and b-FGF and PVF that was expressed by Von
Willebrand factor as an endothelial cell marker. The FACS analysis showed that the effect of PVF on CD 34+ stem cell differentiation into endothelial cells was found to be stronger than that observed with VEGF, b-FGF and
mixture of VEGF and b-FGF (Figure 2.1). The mean fluorescence intensity was 28 for VEGF, b-FGF and mixture of VEGF and b-FGF whereas it was
38 when the cells were treated with PVF. This showed that in PVF the differentiation of CD 34 + cells into endothelial cells were greater than 73.7%
as compared to other known growth factors. The results were further
confirmed by immuno staining studies where the expression of Von
Willebrand factor in presence of PVF was significantly much more higher as
compared to control group where PVF was not used (Plate 2.3).
The effect of PVF in differentiating CD 34 + stem cells into smooth
muscle cell actin was also studied. The expression of smooth muscle cell was increased approximately by two times as compared to cells cultured in
both VEGF and b-FGF (Figure 2.2). The results of immuno-fluorescence
carried out on slides showed that CD 34 + cells when incubated with PVF
were differentiated into vascular smooth muscle cells expressing vascular
smooth muscle cell actin (Plate 2.4).
56 The effect of PVF on human bone marrow endothelial cells showed that in presence of PVF a significant migration of cells was observed. About 50% cells migrated towards each other and filled the wound created artificially (Plate 2.5a-e). The wound was completely filled with cells within 24 hrs showing a very high activity of PVF (Plate 2.5a-e). The migration of human bone marrow endothelial cells after 6, 9 and 24 hrs of experiment was 5.8, 19.2 and 24.3% respectively that was higher as compared to control groups (Figure 2.3).
57 Discussion
The isolation of human embryonic endothelial cells has potential therapeutic implications, including cell transplantation for repair of ischemic tissues and tissue engineering of vascular grafts. Recently, several studies demonstrated the use of adult endothelial progenitor cells for such applications (Kawamoto et al., 2001). It would be important to examine the efficiency of embryonic stem cells in such applications, because ES cells can be expanded without apparent limit (Amit-et al., 2000). ES cell-derived cells could be created in virtually unlimited amounts and available for potential clinical use. Another area in which embryonic endothelial cells can potentially be beneficial is the engineering of complex tissues where vascularization of the regenerating tissue is essential. Several approaches are now being developed to vascularize engineered tissue in vitro before transplantation (Black et al., 1998). Vascularization in vitro is important to enable cell viability during tissue growth, induce structural organization and promote integration upon implantation. The formation of the first capillaries takes place mostly during early stages of embryogenesis when endothelial cells are generated from precursor cells (Flamme et al., 1997). Isolated
human embryonic endothelial cells or progenitor cells can be critical for such applications. Future studies should include further analysis of the
molecular mechanism underlying endothelial differentiation and vasculogenesis during human embryoid body's differentiation and
investigation of growth factor involvement in this process. In addition, efforts to identify and isolate early embryonic progenitors are important to provide tools for elucidating regulatory elements in vasculogenesis and to potentially
58 shed light on vasculogenic and angiogenic mechanisms involved in pathological situations affecting the vascular system.
Research on adult stem cells has recently generated a great deal of excitement. Scientists have found adult stem cells in many more tissues than they once thought possible. This finding has led scientists to ask whether adult stem cells could be used for transplants? In fact, adult blood forming stem cells from bone marrow have been used in transplants for 30 year (Yeh et al., 2003). Certain kinds of adult stem cells seem to have the ability to differentiate into a number of different cell types provided if they are given right conditions. If this differentiation of adult stem cells can be controlled in the laboratory, these cells may become the basis of therapies for many serious common diseases.
Stem cells can give rise to specialized cells and when unspecialized stem cells give rise to specialized cells, the process is called differentiation
(Orlic et al., 2001, Toma et al., 2002). Scientists are just beginning to understand the signals or factors inside and outside the cells that trigger stem cell differentiation. The internal signals are controlled by a cell's genes, which are interspersed across long strands of DNA and carry coded instructions for all the structures and functions of a cell (Badorff et al., 2003).
The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighbouring cells and certain molecules in the microenvironment. However, there are still many questions pertaining to stem cell differentiation remained unanswered. We still do no know whether the internal and external signals for cell differentiation are similar for all
59 kinds of stem cells or not. Similarly, the information of specific sets of signals that promote differentiation into specific cell types is also lacking. Addressing these questions is critical because the answers may lead scientists to find new ways of controlling stem cell differentiation in the laboratory, thereby growing cells or tissues that can be used for specific purposes including cell-based therapies (Vassilopulos et a/., 2003).
Attempts have also been made to differentiate the stem cells into different cell types and various compounds stimulated differentiation (Parab
et al., 2003). In all these investigations, cells from an adult human bone
marrow were used to demonstrate the vasculogenesis. All recent studies
have focused on the use of embryonic stem cells and bone marrow derived
cells, including adult hematopoietic stem cells, mesenchymal stem cells and
bone marrow side population cells (Orlic et al., 2001). CD34+ cells from the
bone marrow and peripheral blood have been successfully used for
regeneration of the blood vessels after experimental myocardial infection
(Kocher et al., 2001).
In previous attempts, it was observed that peri-vitelline fluid (PVF)
added to early chick embryo induced an increase of cardiac vasculogenesis
(Parab et al., 2003). This interesting observation encouraged seeing the
action of PVF on human stem cell-induced vasculogenesis. In my present
investigation, I demonstrated that the peri-vitelline fluid of Indian horseshoe
crab promoted the differentiation of CD 34+ cells into different cell types in
vitro (endothelial cells and smooth muscle cells) using specific cell markers.
The frequency of differentiation of adult bone marrow stem cell into
60 endothelial and smooth muscle cell is very high. Such differentiation of stem cells into vascular cells is a rare event and an inductor of this differentiation process would certainly be useful in therapeutic applications.
Recent data indicate that the adult stem cell is able to produce differentiated cell types of other tissues or organs. The adult human
peripheral blood cells have been shown to differentiate into mature cells of
non-hematopoietic tissues, such as hepatocytes and epithelial cells of the
skin and gastrointestinal track. These cells were also reported to transdifferentiate into human cardiomyocytes, mature endothelial cells and
smooth muscle cells in vivo (Yeh et al., 2003). Using human HLA as a
marker for the donor cells Yeh et al. (2003) demonstrated transdifferentiation of CD 34+ cells into cardiomyocytes in vivo. However, in
their studies the frequency of the event has been observed to be low in un-
injured hearts. The cells derived from transplanted CD 34 + cells
demonstrate mature cardiomyocyte morphology and seem to be integrated
into the myocardium of the peri-infarct area. The fact that no
transdifferentiated cardiomyocytes were found inside the infarct zone is in
agreement with in vitro observation between the donor and host cells are
necessary for transdifferentiation. CD 34+ cells from the bone marrow and
peripheral blood have long been recognized to contain endothelial
progenitors (Yeh et al., 2003). Consistent with these findings, double
staining of the blood vessels with anti-HLA and anti—VE-cadherin
documented the transdifferentiation of peripheral blood CD 34 + cells into
vascular endothelial cells. It has also been reported that CD 34 + cells in
human blood contain a population of smooth muscle progenitor cells. Yeh et
61 al. (2003) demonstrated the potential of these cells to differentiate into vascular smooth muscle cells in vivo.
Terada et al. (2002) demonstrated in vitro that bone marrow stem cells were able to fuse with embryonic stem cells and to adopt their phenotype. Recently, Vassilopoulos et al. (2003) and Wang et al. (2003) demonstrated that cell fusion was the major mechanism in regeneration of the damaged hepatocytes of mice. Because no chromosome analysis was
performed in my study, I could not exclude the possibility that cell fusion is
partly responsible for the phenotype conversion of the injected CD 34 + cells, especially in myocytes where fusion of myotube is part of the differentiation
process. However, the phenotypic conversion of the injected CD 34 + cells
into endothelial cells or smooth muscle cells may occur predominantly through transdifferentiation.
The mechanism resulting in transdifferentiation is not yet understood
and may be influenced by epigenetic or genetic modifications caused by
various compounds including PVF. The identification of signaling molecules
that promote differentiation of specific subtypes will help in understanding
these processes in adult stem cells. Thus, by continuing basic human adult
stem cell research using the egg extract of Indian horseshoe crab and
comparing gene expression patterns and phenotypes, the future application
of human stem cells (ES, adult stem and progenitor cells) in clinical settings
may become more viable. Therefore, the scope of PVF as an inductor of cell
differentiation for cell therapy is high in near future.
62 Summary
The differentiation of CD 34+ stem cells into endothelial cells using different growth factors such as VEGF, b-FGF, the mixture VEGF and b- FGF and PVF was compared using Von Willebrand factor as an endothelial cell marker. The effect of PVF on CD 34 + stem cell differentiation into endothelial cells was found to be stronger than VEGF, b-FGF and mixture of
VEGF and b-FGF. With PVF, the differentiation of CD 34 + cells into endothelial cells was greater than 73.7% as compared to other known growth factors. The immuno staining studies showed much higher expression of Von Willebrand factor with PVF as compared to other growth factors. Similarly, the effect of PVF in differentiating CD 34 + stem cells into smooth muscle cell actin was increased approximately by two times as compared to cells cultured in both VEGF and b-FGF. The results of
immuno-fluorescence carried out on slides showed that CD 34 + cells when
incubated with PVF were differentiated into vascular smooth muscle cells
expressing vascular smooth muscle cell actin. The effect of PVF on human
bone marrow endothelial cells showed that in presence of PVF a significant
migration of cells was observed helping in healing the wound.
63 Expression of Von Willebrand factor
ity 50
ns 45
te 40
In 35 30 25 20 15
Fluorescence 10 5
Mean 0 Control VEGF b-FGF VEGF+b- PVF FGF Different growth factors
Figure 2.1: Expression of Von Willebrand Factor
64
•
Expression of smooth muscel cell actin
40 • 35 — 30; a) •C.) 25 cu g 20 o 15 -■ z c.) 10 5 a) Q. 0 Control VEGF b-FGF VEGF+b- PVF FGF Different growth factors
Figure 2.2: Expression of smooth muscle cell actin
65
Migration of human bone marrow endothelial cells (0.1mg/m1 PVF) c 400 350 "gc 300 - ❑ With PVF .g 250 - — 200 ■ Control (Without PVF) 7) 150 - raco 100 -
61 50 < 0 1 0 hour 3 hours 6 hours 9 hours 24 hours Hours
Figure 2.3: Migration of human bone marrow endothelial cells
66 Set A: Control Set B: Cell expressing VWF
Plate 2.3 Expression of Von Willebrand factor with PVF
67 Set A: Control Set B: Cell expressing muscle actin
Plate 2.4 Expression of smooth muscle actin with PVF
68 Control Treated with PVF
Plate 2.5a: Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 0 hrs) (Arrows showing artificially created wound)
69 Control Treated with PVF
Plate 2.5b: Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 3 hrs)
70 Control Treated with PVF
Plate 2.5c: Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 6 hrs)
71 Control Treated with PVF
Plate 2.5d: Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 9 hrs) (Arrows showing 50% wound healing)
72 Control Treated with PVF
Plate 2.5e: Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF at 24 hrs) (Arrows showing 100% wound healing whereas circles show less healing)
73 Chapter-3 Role of peri-vitelline fluid in myocyte differentiation of bone marrow stem cells in vitro Cardiac disorders and congestive heart failure has been remaining among the Nation's most prominent health challenges despite many breakthroughs in cardiovascular medicine. In fact, despite successful approaches to prevent or limit cardiovascular disease, the restoration of function to the damaged heart remains a formidable challenge. For those suffering from common, but deadly, heart diseases, stem cell biology represents a new medical frontier. Researchers are working toward using stem cells to replace damaged heart cells and literally restore cardiac function. The destruction of heart muscle cells, known as cardiomyocytes, can be the result of hypertension, chronic insufficiency in the blood supply to the heart muscle caused by coronary artery disease or a heart attack, the sudden closing of a blood vessel supplying oxygen to the heart. Despite advances in surgical procedures, mechanical assistance devices, drug, therapy and organ transplantation, more than half of patients with congestive heart failure die within five years of initial diagnosis. Researches have shown that therapies such as clot-busting medications can re-establish blood flow to the damaged regions of the heart and limit the death of cardio- myocytes. Scientist have been exploring ways to save additional lives by using replacement cells for dead or impaired cells so that the weakened heart muscle can regain its pumping power.
Recently, Orlic et al. (2001 a & b) reported on an experimental application of hematopoietic stem cells for the regeneration of the tissues in the heart. In their study, a heart attack was induced in mice by tying off a major blood vessel, the left main coronary artery. Through the identification of unique cellular surface markers, the investigators then isolated a selected group of adult primitive bone marrow cells with a high capacity to develop
74 into cells of multiple types. Jackson et al. (2001) demonstrated that cardiac tissue can be regenerated in the mouse heart attack model through the introduction of adult stem cells from mouse bone marrow.
New advances in cardiomyocyte regeneration are being made in human embryonic stem cell research and because of their ability to differentiate into any cell type in the adult body, embryonic stem cells are another possible source for cardiac repair cells. The first step in this application was taken by Itskovitz-Eldor et al. (2000) who demonstrated that human embryonic stem cells can reproducibly differentiate in culture into embryoid bodies made up of cell types from the body's three embryonic germ layers. Among the various cell types noted cells that had the physical appearance of cardiomyocytes, showed cellular markers consistent with heart cells and demonstrated contractile activity similar to cardiomyocytes when observed under the microscope. There has however still been an unsolved controversy about the role of endogenous progenitor cells in cardiac homeostasis and myocardial regeneration (Anversa et al., 2007).
Several reports suggest that bone marrow (BM)—derived hematopoietic stem cells (Orlic et al., 2001 a & b), mesenchymal stem cells
(Shake et al., 2002; Barbash et al., 2003) and mononuclear cells (Strauer et al., 2002; Perin et al., 2003) exhibit a high degree of differentiation plasticity and thus could potentially be used to regenerate infarcted myocardium in humans. These reports have however been challenged vigorously and the concept of transdifferentiation of adult hematopoietic stem cells has recently been called into question (Wagers et al., 2002;
Wagers and Weissman, 2004). Furthermore, the identity of the specific BM-
75 derived cellular subset(s) capable of regenerating cardiac tissue remains elusive.
Parab et al. (2004) showed PVF of the Indian horseshoe crab contains peptide(s) capable of positively influencing differentiations of specific organs. Such peptides are likely to be present in minute quantities and may be as proteins in addition to the four major proteins reported in the
Japanese horseshoe crab (Nagai et al., 1999).
In this work, the fertilized eggs of the horseshoe crab were used for their pharmacologic potential. In this study it has been demonstrated that
PVF either alone or in combination with pro-angiogenic factors, improve differentiation of CD34 + stem cells into myocytes. Therefore, it is suggested that PVF contains an active component enhancing the differentiation of stem cells in myocytes, which may have therapeutic application in ischemic cardiopathy and vascular diseases
76 Material and Methods
The fertilized eggs of the horseshoe crab were collected from the nests located on the sandy beach at Balramgari (Orissa) in India. The eggs were incubated at a constant temperature (37+1 ° C) in artificial incubators and the peri-vitelline fluid (PVF) was collected using a sterilized 22-gauze needle under highly aseptic condition immediately after the eggs became transparent and showed the movement of trilobite larvae. The peri-vitelline fluid was aliquoted and stored at -20 ° C for further use. Peri-vitelline fluid was also freezing dried on a tabletop freeze dryer. The freeze-dried sample of peri-vitelline fluid was re-suspended in sterilized distilled water for further purification.
FPLC and SDS-PAGE analaysis: The peri-vitelline fluid was sub- fractionated using a Fast Pressure Liquid Chromatography (Amersham
Bioscience) using Superose 12 Hr 10/30 column at room temperature. The fractions were eluted in phosphate buffered saline (pH 7.4) with 1.45 mM of
NaCI and 0.5 ml fractions were collected and stored at -20 ° C until further use. A comparison was also performed to asses the most effective fraction of PVF. The SDS-PAGE analysis was done using 15% gel with Coomassie
Brilliant Blue (Laemmli, 1970). The amino acid sequencing of the purified fraction was done at Applied Bioscience, Mass Spectrometry Laboratory,
New Delhi for identification of the protein.
77 Assessment of myocyte differentiation activity using purified fraction
Bone marrow CD 34+ cells preparation: At first, bone marrow was collected from the Haematology Division of the Hospital Hotel Dieu Paris 4 in France and Ficoll Centrifugation Technique was immediately used to separate the mononuclear cells from the bone marrow. The cell layer present in the supernatant was collected whereas all pellets were discarded. The cells were washed with PBS (1X) (Dulbecco's Phosphate Buffered
Saline 10X by PAA Laboratories, Austria: Cat No H15-01) and centrifuged again at 1200 rpm for 10 minutes. The cell pellets were suspended in RPMI-
1640 containing 10% Foetal Calf Serum [FCS] (Promo Cell). This was followed by one more centrifugation of the material at 1200 rpm for 10 minutes for the separation of CD 34 + cells. The separation of CD 34+ cells from the mixed population of the cells was done using Immunomagnetic
Beads following the protocol as described by Easy Sep Stem Cell
Technologies Inc U.S.A.
Cell culture: The isolated CD 34 + cells were suspended in EBM-MV2 and their number adjusted at 4x10 5 cells/ml. Then 5 ml of cell suspension was transferred into a culture flask that was previously coated with gelatin (1X).
Nine flasks were used for this study. To analyze the biological activity, in enhancement of colonization of a fraction of the peri-vitelline fluid of the fertilized eggs of horseshoe crab, cells were suspended in EBM MV2
Medium containing 10% FCS and enriched with different growth factors
(VEGF, b-FGF and 8 th fractions of PVF) according to the requirement of different experiments. All culture flasks were kept for three weeks in an
78 incubator at 37°C injected with 5% CO2 to obtain the 90% confluence of the • cells.
Colony formation assay: The assay was performed as described previously in Chapter-1
Peroxidase Staining: Two labteks were prepared to observe peroxidase staining
Set A: Medium EBM MV2 + cell suspension + 5 % Foetal Calf Serum
Set B: EBM MV2 medium + cell suspension + 8th fraction of PVF (0.1 mg/ml) + 5 % Foetal Calf Serum.
Both the labtecks were kept for three weeks in an incubator at 37° C
injected with 5% CO 2 to obtain the 90% confluence of the cells. For the
identification peroxidase activity of cells from colonies, the cells cultured in
the wells of labteks were fixed by 4% paraformaldehyde during 5 min at 4°C
and then permeabilized with Triton (0.1%) for 20 min. Then the cells were
washed with PBS 1X and the peroxidase was detected by incubating cells
for 20 min at room temperature with freshly prepared 100 IA of DAB (0.5
mg/ml + H202 0.1% final concentration). After an incubation period of 20 min
at room temperature, the wells of the labteks were washed three times
washing with PBS (1X). The slides from the labteks were detached and
permanent slides were prepared with fluroprep. The slides were analysed
using light microscope and photographs were taken of both the labteks.
79 Immuno-fluorescence staining: The following two sets were prepared for immuno-fluorescence staining: Set A: Medium EBM MV2 + cell suspension + 5 % Fcetal Calf Serum
Set B: EBM MV2 medium + cell suspension + 8th fraction of PVF (0.1 mg/ml) + 5% Fcetal Calf Serum
The fixation and the permeabilisation of the cells were performed as described above. To avoid unspecific adsorption in the further steps,
PBS:BSA (1%) was added in the labteks for 30 min. After washing, the wells were incubated for 30 min at 4° C in presence of rabbit anti-human myoglobin (Sigma Cat No M 8648) fluorescein-labelled anti-rabbit Ig
(Amersham Cat no 1034). The two antibodies used were diluted by 1/100 in
PBS containing 1% albumin. The labteks were washed three times with
PBS (1X). The slides were analysed using both fluorescence microscope and a confocal microscope. The number of positive cells was counted. For each test, incubating the samples only with fluorescein-labelled anti-rabbit Ig antibody without adding the rabbit anti-human myoglobin antibody performed negative controls experiments.
FACS analysis: Assessment of effective dose of PVF by FAGS analysis for myoglobin expression
Four different flask were prepared for dose assessment activity
1. Medium EBM MV2 + cell suspension + 5 % Fcetal Calf Serum
2. EBM MV2 medium + cell suspension + PVF, 8th fraction of PVF (0.1 mg/ml) +5 % Fcetal Calf Serum
80 3. EBM MV2 medium + cell suspension + 8 th fraction of PVF (0.01 mg/mI)+5 % Fcetal Calf Serum.
4. EBM MV2 medium + cell suspension + 8 th fraction of PVF (0.05 mg/ml) + 5
% Fcetal Calf Serum
81 FACS analysis for myoglobin expression in presence of different growth factors The following protocol was followed for myoglobin expression: Set A: Medium EBM MV2 + cell suspension + 5 % Fcetal Calf Serum
Set B: EBM MV2 medium + cell suspension + VEGF (10 pl, 0.25 pg/500 p1) + 5 % Fcetal Calf Serum.
Set C: EBM MV2 medium + cell suspension + b-FGF (10 pl, 0.25 pg/500p1) + 5 % Fcetal Calf Serum.
Set D: EBM MV2 medium + cell suspension + VEGF + b-FGF (10 pl, 0.25 pg/500 pl) +10% serum.
Set E: EBM MV2 medium + cell suspension + 8 th fraction of PVF (0.1 mg/ml) +5 % Fcetal Calf Serum.
The same method as described in Chapter-2 was used for immunocytochemical study by FACS analysis using a Flow-cytometer
(Model: Beckton & Dickenson) and rabbit anti-human myoglobin antibody
(Sigma Cat No M 8648) was used to observe the expression of myoglobin
82 Results
A total of 10 peaks were detected in the peri-vitelline fluid of the horseshoe crab on FPLC profile (Figure 3.1). Initially all fractions collected by this methods were analysed for the presence of myocyte differentiation activity to select the most effective fraction (Figure 3.2). Before initiating the experiment, the most effective dose of the fraction 8 th was also assessed by
FACS analysis for myosin expression. It was found that 0.1 mg/ml showed the maximum expression (Figure 3.3). Since the 8th fraction was showing highest myocyte differentiation activity, it was further analysed for its purity on SDS-PAGE (15% gel). A single band having 29-30 kDa molecular weights was obtained showing the purity of the fraction (Plate 3.1). The protein sequencing of the fraction 8 th showed 221 amino acids (Figure 3.4).
Fraction 8th of PVF induced the differentiation of bone marrow stem cell to myocyte
In order to identify the myocytes present in the colonies formed after long-termed culture of bone marrow stem cells, cells expressing myosin were identified both by immunohistochemical analysis and FACS analysis.
In immunohistochemical analysis PVF was found to induce a great expression of myosin in some adherents cells incubated with PVF as compared to control cells cultured in the presence of VEGF or b-FGF (Plate
3.2). Similarly, the mean fluorescence intensity of cells cultured in presence of PVF was also much greater as compared to control cells or cells cultured in the presence of VEGF or b-FGF (Figure 3.5).
83 Peroxidase activity was also performed to identify myoglobin of adherent cells (Plate 3.3). It appeared from the results that the number of cells having a peroxidase activity in colonies obtained in the presence of
PVF was greater than that of control experiment. This result was in high correlation with those showing myoglobin detection. The results of the present studies thus showed that PVF helped in improving the differentiation of bone marrow stem cell to myocyte.
84 Discussion
Several molecules isolated from various marine organisms such as microorganisms, algae, fungi, invertebrates and vertebrates; are currently under study at an advanced stage of clinical trials. They are either directly or in the form of analogues deduced from structure-activity relationships. Some of them have already been marketed as drugs.
In this chapter an attempt was made to demonstrate that the purified fraction having molecular weight of 23-26 kDa polypeptide from peri-vitelline fluid of the fertilized eggs from the Indian horseshoe crab improved the differentiation of bone marrow stem cells into myocytes. The present study also indicated that the PVF fraction not only induced stem cell differentiation but also helped in colony formation and promoted conversion of bone marrow progenitor cells to myocytes. In another study Parab et al. (2004) demonstrated that peri-vitelline fluid stimulated cardiovascular regeneration in experimental model. These authors have also discussed the possibility of shunting of bone marrow progenitor cells towards cardiomyocyte
differentiation .
Several colony-stimulating factors already reported from marine origin (Misra et al., 1994). In this study, it has been clearly shown the effect of PVF in the development of myocyte from bone marrow stem cells CD34 + .
However, multilineage developmental. capacity of the CD34+ cells, especially into myocytes and smooth muscle cells is still controversial.
Asahara et al. (2006) conducted a series of experiments to prove the hypothesis that vasculogenesis and cardiomyogenesis after myocardial
85 infarction may be dose-dependently enhanced after CD34 + cell transplantation. The present study also confirms the improvement in the differentiation of CD34 + cell to myocyte in presence of PVF.
Marine natural products have been a source of new leads for the treatment of many deadly diseases such as cancer (Cragg et al., 2006, GuI and Hamann, 2005) and acquired immuno-deficiency syndrome (Tziveleka et al., 2003). The pharmacology of marine compounds with anthelminthic, anti-bacterial, anti-coagulant, anti-diabetic, anti-fungal, anti-inflammatory, anti-malarial, anti-platelet, anti-protozoal, anti-tuberculosis and anti-viral activities affecting the cardiovascular and nervous systems have also been reviewed elsewhere (Mayer et al., 2004). Number of marine peptides has been isolated in recent years, which exhibit potent biological activities and many of the compounds showed promising anticancer activity. Didemnin was the first marine peptide that entered in human clinical trials in USA for the treatment of cancer, and other anticancer peptides such as kahalalide F, hemiasterlin, dolastatins, cemadotin, soblidotin, didemnins and aplidine have entered in the clinical trials (Rawat et al., 2006).
On the basis of the present results it can be concluded that PVF improved the differentiation of the bone marrow stem cells into myocyte. It is also evidenced that b-FGF and VEGF can also be involved but at a lesser extent than PVF in differentiation of bone marrow stem cells to myocytes.
This present study thus opens up entirely new possibilities using PVF for medical application to improve stem cell differentiation into cardiomyocytes on severe ischemic diseases, which can not be improved by classic therapy.
86 Summary
The FPLC profile of peri-vitelline fluid showed 10 peaks where fraction 8th showed highest activity. The most effective dose of the fraction
8th was also assessed by FACS analysis for myosin expression and a dose of 0.1 mg/ml showed the maximum expression. Since the 8 th fraction was showing highest myocyte differentiation activity it was further analysed for its purity on SDS-PAGE where a single band having 23-26 kDa molecular weight was obtained. The protein sequencing of the fraction 8 th showed 221 amino acids. In order to identify the myocytes present in the colonies formed after long-termed culture of bone marrow stem cells, cells expressing myosin were identified both by immunohistochemical and FRCS analysis. In immunohistochemical analysis, PVF was found to induce a great expression of myosin in some adherent's cells incubated with PVF as compared to control cells cultured in the presence of VEGF or b-FGF.
Similarly, the mean fluorescence intensity of cells cultured in presence of
PVF was also much greater as compared to control cells or cells cultured in the presence of VEGF or b-FGF. It appeared from the results that the number of cells having a peroxidase activity in colonies obtained in the presence of PVF was greater than that of control experiment. These results showed that PVF helped in improving the differentiation of bone marrow stem cell to myocyte.
87 112.16 100
80 -
60 - =
E 52.00 40 - 1.28
44.54 75.85 20- 121.46 82 .34 128.70 65.21 7.68 1 0- 50 100 150 ml
Figure 3.1: FPLC profile of peri-vitelline fluid (Buffer: 7.4; sample load 500 pg; Column: Superose 12.2 column; AUFS: 0.1; Fraction size: 0.5 ml
88 4(.
Mean fluoresce nce 20 25 30 10 i 15 Figure 3.2:AssessmentofmosteffectivefractionPVF 5 Assessment ofeffectivefractionPVFbythe expression ofmyoglobin(FACSanalysis) 1 2 Different fractionsofPVF 3 89 4 5 6 7 8
Assessment of effective dose of PVF (8th Fraction) by the expression of myoglobin (FACS analysis) z, 30
25
2 0 Control (Without PVF) PVF 0.1mg/m1 PVF 0.01mg/m1 PVF 0.05mg/m1 Different concentrations of PVF (8th Fraction)
Figure 3.3: Assessment effective dose of PVF(8 t Fraction) by the expression of myoglobin
90 97 .4 KDa 66 KDa
KDa
4-29 KDa
4---20.1 KDa
14.3 KDa
Plate 3.1: Gel profile of the 8 th fraction of PVF
91 VQWHQ1PGKLIVIHITATPHFLWGVNSNQQIYLCRQPCYDGQ WTQISGSLKQVDADDHEVWGVNRNDDIYKRPVDGSGSWV RVSGKLKHVSASGYGYIWGVNSNDQIYKCPKPCNGAWTQ VNGRLKQIDGGQSMVYGVNSANAIYRRPVDGSGSVVQQIS GSLKHITGSGLSEVFGVNSNDQIYRCTKPCSGQWSLIDG LKQCDATGNTIVGVNSVDNIYRSG
Figure 3.4: Sequence of fraction 8 th of PVF
92 Expression of myoglbin by differentiating CD34+ cells (FACS analysis)
30
• 20
15
O 10 0 vs• 5 0 0 Control (Without PVF) PVF (8th Fr) VEGF bFGF
Figure 3.5: Expression of myoglobin by differentiating CD 34 + Cells
93
Effect of PVF in the Differentiation of CD34+ cells into cardiomyocytes (Expression of myoglobin )
Control
Expression of myoglobin With PVF
Plate 3.2: Effect of PVF (8 th Fr) in the differentiation of CD 34 + cells into cardiomyocytes (expression in myocytes)
94 Peroxidase Staining expressed by CD34+ cells in
presence of PVF
Control With PVF (8th fraction)
Plate 3.3: Peroxidase activity in presence of PVF
95 Chapter-4
A comparison of the sensitivity of amoebocyte lysate produced from in vitro grown amoebocytes and amoebocytes collected from wild stock Water and life are closely linked to each other. This has been
recognized throughout history by civilizations and religions and it is still a
matter to study for scientists. Liquid water is required for life to start and for
life to continue. No enzymes work in the absence of water molecules. No other liquid can replace water. Living organisms maintain a constant water
balance in their body in order to survive.
It is believed that the life began in a watery medium i.e. the sea.
Therefore, a significant relationship exists between the general composition
of seawater and body fluids of the aquatic animals (Macallum, 1926).
Animals in all environments (aquatic and terrestrial) must maintain the right
concentration of solutes and amount of water in their body fluids. This
involves excretion i.e. getting rid of metabolic wastes and other substances
such as hormones which would be toxic if allowed to accumulate in the
blood via organs such as the skin and the kidneys. Keeping the water and
dissolved solutes in balance is referred to as osmoregulation.
Many marine invertebrates (such as jellyfish, scallops, crabs, oysters,
etc.) are osmo-conformers and have body fluids that are similar in
composition to seawater. This condition is described as either isosmotic or
hyperosmotic, relative to seawater. Beyond that, they either conform to
environmental conditions, or they regulate. Some invertebrates, such as
brine shrimp (sold in the back of comics as "sea monkeys") and mosquito
larvae are capable of tolerating extremely high salinities (as great as 10
times sea water).
96 Marine arthropods exhibit all possible patterns of osmoregulation (Prosser, 1973) and their extra cellular fluids are in osmotic equilibrium with the surrounding seawater (Mantle and Farmer, 1983). Osmoregulation in marine arthropods is basically an effect of salinity on the osmotic and ionic properties of the haemolymph (Gilles and Pequeux, 1983). Salinity variations during different seasons, significantly, influence the volume of the haemolymph and body weight of the marine arthropods (Shaw, 1958;
Prosser, 1965 and Robertson, 1970). The responses by haemolymph are related to hypo-regulation at low salinity when the volume of haemolymph increases considerably during monsoon period and hyper-regulation or conformity at higher salinities when the volume of the haemolymph decreases during summer and post monsoon periods.
Invertebrates have a characteristic host defensive mechanism, which differs of those of mammalian immune systems (Brehelin, 1986). In the horseshoe crab this defence system is carried out by the haemolymph, which contains a type of cell called amoebocyte or hemocyte (Levin and
Bang, 1964 a & b). The hemocytes are extremely useful to bacterial endotoxins, lipopolysaccharides which are a major outer membrane component of gram-negative bacteria. The amoebocytes aggregate quickly when exposed to minute amount of any endotoxin (Mikkelsen, 1988). The shape of amoebocytes changes and simultaneously long processes extending from one amoebocyte to the other appear in a pseudopod like fashion. When amoebocytes come in contact with gram-negative bacteria or lipopolysaccharide, they begin to degranulate and the resulting granular
97 components initiate haemolymph coagulation (Levin and Bang, 1964 a & b;
Ornberg and Reese, 1979; Iwanaga et al., 1992).
Arthropods have developed a unique immune system without the acquired immunoglobulin-dependent immunity found in vertebrates. Therefore, innate immunity, the pre-extinguished immediate ability to prevent and limit invading microbes and pathogens, is likely to be a major host defence system in arthropods. The hemolymph of horseshoe crabs contains three abundant proteins, hemocyanin, C-reactive protein, and a 2-macroglobulin. One type of granular cells are accounting for 99% of the total hemocytes (Armstrong,
1991; Toh et al., 1991).
The granular cells are extremely sensitive to bacterial endotoxin, i.e. lipopolysaccharides (LPS). The cells release granular components in response to LPS stimulation (Toh et al., 1991; Iwanaga, 1993; Kawabata et al., 1996; Muta and Iwanaga, 1996). This response is thought to be important for host defence in engulfing and killing invading microbes, in addition to preventing the leakage of hemolymph. The hemocytes contain large and small granules that selectively store proteins and defence molecules, including serine protease zymogens, a clottable protein that participates in the coagulation cascade, protease inhibitors, antibacterial peptides, lectins, and others (Kawabata et al., 1996). Lectins are a group of sugar-binding proteins that recognize specific carbohydrate structures and agglutinate a variety of animal cells by binding to cell-surface glycoproteins and glycolipids.
98 It recent times, the biomedical properties of the horseshoe crab a living fossil, has attained unanticipated importance due to marketing of
Limulus Amoebocyte Lysate as a rapid bacterial tester in food, drug and pharmaceutical industries. The horseshoe crab is a hardy animal and can thrive well in diluted or saturated seawater by maintaining steady state.
Salinity, has been reported to significantly influenced the of the hemolymph and body weight of the marine during different months (Chatterji et al.,
1992). Considering seasonal fluctuation in the sensitivity of amoebocyte lysate an attempt has also been made to culture amoebocyte producing tissue for the purpose of generating amoebocyte lysate of uniform sensitivity
(Joshi et al., 2002). In the present investigation, an attempt has been made to compare the sensitivity of amoebocyte lysate produce by in vitro and in vivo techniques.
99 Materials and methods
Preparation of glassware:
All glassware used for the preparation of amoebocyte lysate were kept in liquid soap for 24 hours and then washed thoroughly twice with tap water. After removing the traces of tap water, the glassware was washed three times with pyrogen free double distilled water. All glassware was kept for drying for 24 hours at 20° C in an oven. These glassware were removed and plugged with cotton and cotton gauge. They were autoclaved for 15 minutes at 14 lbs pressure and kept safely for further use.
Preparation of N-ethylmaleimide anti-coagulant: a. 1 N HCI: 8 ml Conc HCI was taken in 100 ml of sterilized double distilled
water. b. 0.1 M TRIS: 1.21 g of Tris was dissolved in 50 ml of double distilled water
and mixed properly. The pH of the solution was adjusted to 7.4 with 1 N
HCI. The volume of the solution was finally made to 100 ml with double
distilled water. c. 3 g NaCI and 0.125 g N-ethylmaleimide (NEM) were taken in 50 ml of
double distilled water. To this 50 ml of 0.1 M IRIS was added. The pH of
the resultant solution was adjusted to 7.4 with 0.1 N HCI. Now the
strength of the TRIS was 0.05M. The solution was then filter sterilized by
using 22-micron glass filter and kept safely at 4 ° C for further use.
100 Reconstitution of endotoxin
The commercially available endotoxin standard prepared from E. coli (0.55:B% lipopolysaccharide) was obtained from Sigma Chemicals (Cat
210-SE). The endotoxin standard was reconstituted in pyrogen free double distilled water (5.9 ml) before performing the endotoxin assay. The solution was vortex to mix the material properly. This gave 4000 EU/ml of endotoxin and treated as stock solution.
Preparation of serial dilutions of endotoxin
In three test tubes, marked A, B and C, 9 ml of pyrogen free double distilled water was taken. Set 1: 1 ml of stock solution was added to test tube marked A, giving 400 EU/ml.
Ser 2: 1 ml of solution from test tube marked A was added to test tube marked B, giving 40 EU/ml.
Set 3: 1 ml of solution from test tube marked B was added to test tube marked C, giving 4 EU/mI.
Preparation of 5X antibiotics
Penicillin (0.5 ml) and streptomycin (0.5 ml) were dissolved in 100 ml of sterilized double distilled water and the solution was kept for further use.
Preparation of Tween 80 solution
Tween 80 (0.5 ml) was mixed in 0.5 ml of sterilized seawater (salinity
-30 ppt). The solution was filter sterilized with 22-micron swiney filters.
101 The present investigation was based on the specimens collected during their migration towards the intertidal zone for spawning at Balramgari in Orissa (Lat 19 ° 16' N Long 84 ° 53' E) during pre-monsoon, monsoon and post-monsoon periods. Monthly observation from June' 2004 to May'
2005 were carried out on 250 specimens (130 females and 120 males) from a marked 200 m transect during highest high tides (Indian Tide Table,
2004). After determining the sex of each individual, specimens were blotted lightly with filter paper and then weighed to nearest 1 g on a single pan electronic balance. A sterile disposable syringe fitted with pyrogen free collected Haemolymph of each of the crab 14-gauge: 3.8 cm hypodermic needle.
Before the collection of haemolymph, the base of the last pair of thoracic appendage of the horseshoe crab was cleaned with a gauze piece moistened with absolute alcohol. Haemolymph (20 ml) was collected from each crab and transferred to a pyrogen free polypropylene centrifuge tube containing 3 ml of N-ethylmaleimyde anticoagulant. The amoebocytes were separated by centrifugation (850 rpm) at a temperature of 4 ° C. The supernatant solution was discarded whereas white packed amoebocytes were washed with pyrogen free 3% sodium chloride solution. The amoebocytes were then kept at 4° C for lysing for 24 hours in pyrogen free double distilled water. The cellular debris was finally removed by centrifugation at 1500 rpm at 4° C and the lysate decanted in sterile pyrogen free vials. Supernatant clear solution was then removed and kept at -20 ° C for pre-freezing. The amoebocyte lysate samples were then freeze-dried using a table top lyophilizer (Edward: Micromodulo) and finally all samples
102 were vacuum sealed with the help of mechanically operated closing device of lyophilizer.
The preparation of lysate required absolute precaution for pyrogen contamination at all processing steps. All apparatus and reagents were pyrogen free as described by Chatterji et al. (1992).
Amoebocyte culture in vitro
The book gills of both male and female specimens of horseshoe crab were sterilized with 70% alcohol. The gill flaps about (100 leaf like structures) were removed with the help of a pair of sharp and sterilized scissors. The gill flaps were dipped for 2-3 min in concentrated Betadine solution and then washed with 70% alcohol for 15 min. The gill flaps were transferred into sterilized seawater of 720 mM molarity. This was followed by addition of 1% antibiotics, 0.5 ml penicillin and 0.5 ml streptomycin for 2 hrs. The gill flaps were again removed and dipped in 10% Betadine solution for 15 min. The flaps were washed again with sterilized seawater. These gill flaps were again immersed in seawater with 5X antibiotics for 10 min. The flaps were washed with seawater and 1X antibiotics. The flaps after 10 min were removed and transferred into seawater with 25 pg/ml of fungizone and kept for 10 min. The flaps were washed again with sterilized seawater. The gill flaps were then transferred in 6-well plate containing 3 ml seawater with
1 X antibiotics and 0.25 pg/ml of fungizone. The solution was incubated at room temperature on a rocker platform for 48 hrs.
103 Selection of a suitable culture medium An attempt was made to select a suitable culture medium for the
cultivation of gills and for that media namely; Grace's insect medium, M M
medium, 2X L-15 and RPMI-1640 were used in the present study (Figure
4.1). The most suitable medium for in vitro maintenance of gill flaps was 2X
L-15 (Figure 4.1).
After 48 hrs of incubation the flaps were transferred into 2x L-15
medium containing 0.5% Tween 80. The flaps were again incubated on a
rocker platform for 24 hrs. This was followed by stimulation of the flaps with
10% sterilized horseshoe crab serum for releasing of amoebocytes after 24
hrs. This resulted in release of amoebocytes within and outside the gill flaps.
The release of amoebocytes was monitored every week for a period of 1
month and at every harvest the cell count was recorded.
Long-term maintenance of amoebocytes in vitro
The amoebocytes produced from the gill flaps were obtained by
centrifugation. These cells were seeded in tissue culture dishes along with
2X L-15 medium and 10% horseshoe crab serum. The cultures were fed at
an interval of 5-10 days with fresh medium and serum. The amoebocytes were maintained by this method showed no change in morphology and viability for more than 3 months.
Harvesting of amoebocytes
The amoebocytes along with medium were collected after their
release within and outside the gill flaps. Release of amoebocytes in males
104 and females was observed every week for a period of one month and at each harvest the number of cells was counted and recorded.
The amoebocytes collected by in vitro cultivation and amoebocytes obtained from the wild population were cultured on sterile pyrogens free cover slips in 2X L-15 medium. The attachment of cells was observed within
48 hrs. These cover slips cultures were processed further for porpidium iodide (PI) staining to study the morphology and size of the amoebocytes cells under confocal laser microscope.
The wells of the 6 well plates were properly washed so that loosely attached cells were removed completely. The content of each well was transferred into a sterile 50 ml tube and centrifuged at 2000 rpm for 15 min at room temperature to obtain amoebocyte pellets. The amoebocytes were washed with sterilized seawater and transferred to eppendorf tube with distilled water. The tube was kept at 4° C for over night for lysing of the amoebocytes and then centrifuged at 3000 rpm for 15 min. The clear supernatant was removed and kept at -20 ° C for pre-freezing. After 6 hrs, the amoebocyte lysate was freeze dried and vacuum-sealed using a tabletop freeze dryer.
Using endotoxin standard assessed the sensitivity of amoebocyte lysate. 0.2 ml sample of amoebocyte lysate with 0.2 ml of solutions either from A or B or C were taken into four sterilized endotoxin test tubes with a control without amoebocyte lysate. All the test tubes were vortexed for 5 minutes and then incubated at 37±1 ° C in a water bath for 2 hours.
105 Formation of gel with different intensity indicated the presence of quantity of endotoxin.
106 Results
The amoebocytes obtained from in vitro culture of gill flaps were in the size range of 4 to 4.5 micron. However, the size of amoebocytes obtained from the wild population was in the range of 12-15 micron (Plate
4.1). The amoebocytes though were maintained for 4 weeks, but no further growth was recorded in the size of amoebocytes cultured by in vitro technique.
No significant difference in the number of yield, cells counts and cell morphology was observed between the amoebocytes obtained from males and females (Figures 4.2 & 4.3). Maximum numbers of cells were obtained in the first week of culture (males-18,00000 and females-22,00000). But during the successive weeks, the numbers of cells were reduced. During second to fourth weeks, the numbers of cells were in the range of 10,00000 to 12,00000 in males and 15,0000 to 18,00000 in females (Figures 4.2 &
4.3).
The sensitivity of amoebocytes was assessed with different seasons and with amoebocyte lysate prepared by in vitro technique. It was found that the sensitivity of amoebocyte lysate varied during different seasons (Table
4.1). Maximum sensitivity of amoebocyte lysate prepared from the wild population was recorded during pre-monsoon period whereas the lowest during monsoon season (Table 4.1). The amoebocyte lysate prepared during pre-monsoon period showed ability to detect endotoxin up to 4 EU/mI whereas a very low sensitivity of amoebocyte lysate was observed in
107 monsoon (4000 EU/mI). The sensitivity was also very low (4000 EU/mI) in the amoebocyte lysate produced by in vitro technique (Table 4.1).
108 Discussion
The horseshoe crab is mainly confined to the polyhaline region of the estuaries but immature specimens have also been found to migrate into mesohaline waters (Magnum et al., 1976). In fact, all stages of the horseshoe crab are able to tolerate a wide fluctuation in the salinity of the environment. Limulus has been found occur in waters of salinity 6-8 x 10 3 showing a lower salinity tolerance when the animal faced a gradual acclimatization (Robertson, 1970). The horseshoe crab shows a perfect mechanism of osmoregulation, which enables the animal to ensure a proper intracellular water balance in particular environment. The animal successfully, maintains more or less hypo-osmotic state in dilute media whereas, in more concentrated media, the haemolymph osmolarity strictly follows the osmolarity of the external environment.
Mature specimens of Tachypleus gigas migrate towards shore and lay their eggs in nests on sandy beaches, round year. The metamorphosis of the larvae takes place in waters where the salinity does not vary greatly.
But in the intertidal zone, the animal is subjected to great osmotic stresses due to high and rapid fluctuations in salinity besides desiccation during the low tide. Adult specimens of the Limulus have also been reported migrating, shoreward during spring and summer for spawning purpose
(Shuster, 1982) and thereby face salinity variations ranging from 6-32 x 10 -3.
It has been reported that during monsoon season when the salinity decreased considerably, due to land run off, the volume of haemolymph increased by 26.0% in females (Chatterji et al., 1992). This is basically due
109 to hypo-osmotic condition when water from the environment passes inside the body of the crab to maintain equilibrium between internal and external osmotic state. Decrease in salinity of the environment results in diffusive movement of water into the animal body across permeable surface. The permeability of water is largely through highly chitinized gills of the horseshoe crab.
The most important factor to minimize the diffusive movement of water is to reduce the concentration and osmotic gradients between the haemolymph and the external conditions. Similarly, during summer and post monsoon periods when the salinity of the environment is relatively high and the hyper-osmotic condition prevails, water passes outside the body of the horseshoe crab resulting in decrease in the values of haemolymph-the maximum of 19.8% in females (Chatterji et a/., 1992). During the monsoon period, the haemolymph volume increased by 15.7% as against 12.9% during post monsoon period in males (Chatterji et at., 1992). However, in normal seawater, Limulus has been found to be iso-osmotic and showed no significant difference in volume of haemolymph of the horseshoe crab and the salinity of the surrounding water are supposed to be in iso-osmotic condition. The animal tissues act adequately in regulating the haemolymph concentrations over this wide range of salinity changes ((Mantel and
Farmer, 1983). It has also been observed that the ionic composition of haemolymph varies from the medium regardless of the osmotic relationships. At high salinities, the haemolymph of the crab has been found hypo-ionic and hyper-osmotic to the medium (Robertson, 1949; 1953;
Robertson and Waterman, 1960). The haemolymph collected from 6
110 samples of Limulus over a period of 3 summers showed hypo-osmotic state
(Cole, 1940), whereas, McManus (1969) reported an iso-osmotic state of haemolymph on L. polyphemus when kept in tanks of running seawater of
25 x 1 0-3 salinity.
At high salinities, the body weight of the horseshoe crab has been reported to decrease considerably, as against an increase at lower salinities
(Robertson, 1970). In shore crab, Carcinus maenas, the body weight of the animal increased when the animal moved into diluted seawater of 50% to the normal seawater (Prosser, 1965). In a similar way, during an investigation the body weight of T. gigas was reported to increase by approximately 9.8% in female (221-240 mm size) and by 3.09% in male
(141-160 mm size) (Chatterji et al., 1992). During post monsoon period, the body weight decreased by 18.2% in females and 6.1% in, males. These fluctuations in body weight are more pronounced in females rather than in males of T. gigas. In study (Robertson, 1970), it has been reported that the body size has no relation with osmotic steady state even though it is related to salinity.
Though salinity plays an important role in the fluctuating the volume of haemolymph in different seasons, it is still not ascertained whether the variation in the salinity has any significant effect on the sensitivity of amoebocyte lysate or not. Jorgenson and Smith (1973) reported that in
June/July through October maximum lysate preparations could be possible.
The variability in the haemolymph is related to eco-biological changes rather than any other factor. The possibility of causing the variability is supported
111 by the fact that the activity of amoebocyte lysate increases when the horseshoe crab gets acclimated to dilute seawater (Robertson, 1970). In
Uca pugilator the activity of certain enzymes have been reported higher when the animal acclimated in low salinity medium (Mantel and Landesman,
1977). Sullivan et al. (1974) reported that in dilute seawater, the concentration of copper based hemocynin increased considerably. At present, the problem of amoebocyte lysate variability with season is of great concern, as a causative factor for this variability has still not been elucidated.
In the attempt made under this investigation, the technique of culture condition for the long-term maintenance of the gill flaps in vitro were optimized for 30 days. Perhaps this is the first investigation carried out on a long-term culture of gill lamellae in the Indian species of the horseshoe crab.
Studies carried out previously described the process of disinfecting the gill flaps with Alcide Expor and modified Grace's insect medium (Friber et al.,
1992). It has been always a problem to insert a pyrogen free glass needle into the blood channel at the edge of the gill and slide it between two layers to open the gill lamellae for purging (Hilly and Gibson, 1989). In the present study the process has been simplified to maintain the intact of gill as organ culture without opening them and thus retaining their tissue architecture, possibly because of the simulation of the in vivo conditions by the use of the rocker platform. In this study instead of Alcide Expor, Betadine was used for disinfecting the flaps. The 2X L-15 medium helped in maintaining the osmolarity similar to that of seawater (720 mOsm) and pH 7.4. This resulted in the successful release of amoebocyte cells in vitro for a longer duration
112 without affecting the cell number till the 4 th harvest. The presence of amoebocytes in large number at every harvest indicated the production and release of amoebocytes in organ-cultured of gill flaps, continuously.
The confocal laser microscopy studies showed significant difference in the size of amoebocyte cells released in vitro process as compared with those obtained form the wild population. There might be immaturity of in vitro-released amoebocytes that required further investigation. This aspect deserves investigations in detail. Future studies on this particular aspect may elucidate methods whereby sensitivity of amoebocyte lysate can be assessed by the measurement of amoebocyte lysate composition in the haemolymph of the horseshoe crab in Indian waters during different seasons.
113 Summary
No significant difference in the number of yield, cells counts and cell morphology was observed between the amoebocytes obtained from males and females. However, amoebocytes obtained from in vitro culture of gill flaps were in the size range of 4 to 4.5 micron and from the wild population
12-15 micron. Maximum numbers of cells were obtained in the first week of culture (males-18,00000 and females-22,00000). But during the successive weeks, the numbers of cells were reduced. During second to fourth weeks, the numbers of cells were in the range of 10,00000 to
12,00000 in males and 15,0000 to 18,00000 in females. The sensitivity of amoebocyte lysate varied during different seasons and a maximum sensitivity from the wild population was recorded during pre-monsoon period
(4 EU/ml) whereas the lowest during monsoon season (4000 EU/mI). The sensitivity was also very low (4000 EU/ml) in the amoebocyte lysate produced by in vitro technique.
114 Selection of an appropriate media
50000- 45000 40000 - 35000 30000 Cells/ml 25000 20000 15000 10000 --1 5000 -I - _ - MP /MI INV 7 0-1 ,—. -,- , , 2X L15 GIM RPMI MM Different culture medium
Figure 4.1: Experiment to select a suitable culture media to culture gill flaps
115 Yield of amoebocytes in males
2000000 1800000
l 1600000
/m 1400000
tes 1200000 cy 1000000 bo
e 800000 o 600000
Am 400000 200000 0 1111 2 3 4 Weeks
Figure 4.2: Amoebocyte yield in males (in vitro production)
116 Yield of amoebocytes in females
2500000
2000000 E t 1500000 0 0 • 1000000 0 E 500000
1 2 3 4 Weeks
Figure 4.3 Amoebocyte yield in females (in vitro production)
117 Amoebocytes produced by in vitro gill culture
Amoebocytes obtained directly from the wild population
Plate 4.1: A comparison of the amoebocyte cells produced by gill culture And amoebocytes obtained from wild stock
118 Table 4.1: A comparison in the sensitivity of amoebocyte lysate produced from both in vitro and in vivo stocks
Sensitivity Amoebocyte lysate (endotoxin unit / ml of lysate) Wild population In vitro Pre-monsoon Monsoon Post-monsoon 4000 ++++ + + + + + ++ 400 + + + + +++ 40 + + + + ++ 4 ++ +
[++++ = Highly sensitive] [+ =Less sensitive]
119 Chapter-5 Respiratory metabolism of the trilobite larvae of Indian horseshoe crab Respiration is a process by which an organism exchanges gases with its environment. The term now refers to the overall process by which oxygen is abstracted from air and is transported to the cells for the oxidation of organic molecules while carbon dioxide (CO 2) and water - the products of oxidation, are returned to the environment. In single-celled organisms, gas exchange occurs directly between cell and environment, i.e. at the cell membrane. In plants, gas exchange with the environment occurs in special organs called as the stomata, which are located mostly on the leaves.
Organisms that utilize oxygen obtain energy are aerobic or oxygen- dependent. Some organisms can live in the absence of oxygen and obtain energy from fuel molecules solely by fermentation or glycolysis. However, these anaerobic processes are much less efficient, since the fuel molecules are merely converted to end products such as lactic acid and ethanol with relatively little energy-rich ATP produced during these conversions.
In complex animals, where the cells of internal organs are distant from the external environment, respiratory system facilitates the passage of gases to and from the internal tissues. In such systems, when there is a difference in pressure of a particular gas on opposite sides of a membrane, the gas diffuses from the side of greater pressure to the side of lesser pressure and each gas is transported independently of other gases. For example, in tissues where carbon dioxide concentration is high and oxygen concentration is low as a result of active metabolism, oxygen diffuses into the tissue and carbon dioxide diffuses out.
120 In lower animals, gas diffusion takes place through a moist surface membrane, as in flatworms; through the thin body wall, as in earthworms through air ducts, or tracheae, as in insects or through specialized tracheal gills, as in aquatic insect larvae. In the gills of fish the blood vessels are exposed directly to the external (aquatic) environment. Oxygen-carbon dioxide exchange occurs between the surrounding water and the blood within the vessels and the blood carries gases to and from tissues (Chatterji,
1994).
In other vertebrates, including humans, gas exchange takes place in the lungs. Breathing is the mechanical procedure in which air reaches the lungs. During inhalation muscular action lowers the diaphragm and raises the ribs as such atmospheric pressure forces air into the enlarged chest cavity. In exhalation the muscles relax and the air is expelled. This combined rhythmic action takes place about 12-16 times per minute when the body is at rest. The rate of breathing is controlled mainly by a respiratory center in the brain stem that responds to changes in the level of hydrogen ion and carbon dioxide in the blood, as well as to other factors such as stress, temperature changes and motor activities.
In higher vertebrates, oxygen-poor, carbon dioxide-rich blood from the right side of the heart is pumped into the lungs and flows through the net of capillaries surrounding the alveoli, the cup-shaped air sacs of the lungs.
Oxygen diffuses across the capillary membranes into the blood and carbon dioxide diffuses in the opposite direction. The oxygen combines with the
121 protein hemoglobin in red blood cells as the blood returns to the left side of the heart, is pumped throughout the body and is released into tissue cells.
Carbon dioxide passes in the opposite direction from the cells of the tissues to the red blood cells. In the blood, carbon dioxide exists in three forms as bicarbonate ion, in which form it serves as a buffer, keeping blood acidity fairly constant. The free carbon dioxide gas is available for diffusion from the
blood into the lungs. •
Respiration in marine organism is to obtain sufficient quantity of oxygen and release carbon dioxide. It is also an important phenomenon in
controlling different physiological activities of the animal such as
maintenance of the blood pH, proper oxygen tension of the blood and
normal body temperature. The most frequently used parameter in
crustacean respiratory metabolism is the oxygen consumption by the
animal. Respiratory metabolism of marine organism is dependent on many
environmental factors. Among those salinity, temperature and pH are the
main external factors controlling the respiratory metabolism of the marine
organisms (Wolvekamp and Waterman, 1960). There are some important
intrinsic factors like body size of the animal that also play an important role
on the metabolic rate of the organisms (Bliss, 1983).
Horseshoe crabs have well developed external gills in the form of
appendages, which are known as gill lamellae. Due to the low concentration
of oxygen in water, the gills must be as efficient as possible in order to
extract oxygen. These lamellae are made of extremely thin membranes (1
cell thick). They are the primary sites of gas exchange. Water flows across
122 the gill lamellae and oxygen is removed and passes into the blood by diffusion. The actual uptake of oxygen depends on a number of factors the surface area of the lamellae, the thickness of the gill epithelia across which the oxygen moves and the oxygen tension across the membranes. Since the rate of oxygen diffusion depends on an appreciable gradient existing in any decrease in oxygen tension that occurs will affect the rate of oxygen uptake. As the blood absorbs more oxygen from the water in the gill chamber, the oxygen content of the water decreases and the rate of diffusion decreases as well.
The gill lamellae have well-developed circulating system with blood vessels that helped the animal in respiration (Chatterji, 1994). Gases diffuse from the surrounding water through moist epithelium of the gill and circulate these to the blood vessels. In the present study, an attempt has been made and reported here to determine the influence of salinity, temperature and pH on the respiratory metabolism of the young trilobite larvae of the Indian horseshoe crab, T. gigas.
123 Materials and methods
A 6 volts 900 mA DC current was used to collect eggs and sperms from the adult male and female of horseshoe crab. The fertilization process was completed in a Petri dish as described earlier by Chatterji (1994).
Hatching of the trilobite larvae was occurred between 42 and 45 days after incubation at a constant temperature of 27±1 ° C. The trilobite larvae were collected and divided into several groups immediately after hatching. The larvae were acclimated separately in 10 L filtered seawater (5 pi) of different salinities (range: 10-40 psu), pH (range: 5-9) and temperature (range: 10-
40° C) before the commencement of the experiment.
Seawaters of different salinities were prepared by diluting 40 psu of seawater with sterilized freshwater. A refractometer (Atago, S/Mill, Japan) was used to determine the salinity. The pH of seawater was adjusted from
5-7 by 1 N HCI and 7-9 by 1 N NaOH solutions. Thermostatic titanium heaters were used to control temperatures of the experimental tanks ranging from 20-40° C whereas temperature from 10-20 ° C in refrigerated and heated water bath with a circulator (2160 Bath and Circulator) (range: -
30 to 100° C; tank capacity 36.7 L).
Oxygen consumption in trilobite larvae was studied in specially designed glass respiratory chambers of 4 L capacity. A small PVC tube was fitted at one end of the glass respiratory chamber to insert the oxygen probe. The oxygen concentration was recorded with the help of a pinpoint
124 dissolved oxygen monitor (Make: YSI Hand Operated Oxygen: Temperature
Meter Model 55/12FT).
To study the effect of salinity on the respiratory metabolism, the trilobite larvae ranging in weight from 0.063-0.089 g were initially acclimated for one hour in 10 L seawater of different salinities ranging from 10 to 40 psu (Grashhoff, 1979). A single larva was then transferred from the acclimatization tank to each respective respiratory chamber. Oxygen value was recorded immediately after transferring the larvae into respiratory chambers. This was followed by sealing of the surface of respiratory chamber with liquid paraffin to avoid any diffusion of oxygen from the atmosphere. The respiratory chamber was kept inside the constant temperature water bath with a continuous circulation at a constant temperature of 27±1 ° C. The pH of the water was maintained at 7.0 in each set of experiment.
In the second set of experiment the trilobite larvae were acclimated in
5 acclimatization tanks of 10 L capacity and at salinity of 30 psu. The pH of each tank was ranged from 5 to 9. A portable pH meter (Philips: PP 9046) was used for recording the pH of the seawater after calibrating it with standard pH Buffers of 4.2, 7.0 & 9.2.
In the third set of experiment, the effect of temperature ranging from
10 to 40° C on the respiratory metabolism of trilobite larva was studied.
About 50 L filtered seawater of salinity of 30 psu and pH of 7.0 was aerated for two hours at the beginning of the experiment. A single trilobite larva was
125 then transferred into a respiratory chamber. The respiratory chamber was kept in a temperature-controlled water bath. The temperature of the water bath was adjusted slowly to the desired level to avoid any physical stress to the larva. Before sealing the surface of the respiratory chamber with liquid paraffin, oxygen value was recorded with the help of an oxygen meter.
In all sets of experiment, the value of oxygen was recorded at an interval of 2 hours till 12 hours. The depletion of oxygen (g -1 body weight / hour) was considered as the amount of oxygen used by the trilobite larvae during the experimental period.
126 Results
During the acclimatization and experimental period, no mortality was observed in the trilobite larvae that showed sturdiness of the animal under different stress conditions. The influence of salinity on the respiratory metabolism of trilobite larva is presented graphically in Figure 5.1. The oxygen consumption was more or less uniform at all salinity ranging from
10-40 psu indicating the least influence of salinity on the oxygen consumption of trilobite larvae. The correlation coefficient values showed that the relationship between oxygen consumption and salinity was not significant (P>0.05; r=0.245).
The effect of temperature on the oxygen consumption is presented in
Figure 5.2. Variation in oxygen consumption was not much between temperature 20° and 30° C. At temperature 10 ° C the rate of oxygen consumption was the minimum. A sudden increase in the oxygen consumption was recorded at a temperature 40 ° C. The data were tested statistically by applying analysis of variance where a significant relationship was observed between temperature and oxygen consumption (P<0.05; r=0.97).
A similar trend in oxygen consumption was observed pH 6 and 7 where moderate consumption of oxygen was observed (Figure 5.3).
However, at pH 5, 8 and 9, the rates of oxygen consumption were found to be relatively greater indicating unfavorable conditions (Figure 5.3). The data were tested statistically and it was found that a high degree of correlations existed between pH and oxygen consumption (r=0.98). The analysis of
127 covariance showed a significant relationship between oxygen consumption and pH (P <0.05).
128 Discussion
The metabolic rate varies with change in salinity, temperature and pH. In addition to these parameters, other factors like size of the animal,
light condition and oxygen present in the environment also play a significant
role in controlling the oxygen consumption. Several studies have been
carried out to show that the oxygen consumption varied in juveniles and
post-larvae of the penaeid prawns in relation to changes in salinity (Kutty, et
al., 1971; Gaudy and Sloane, 1981, Janakiraman, et al, 1985 and Diwan, et
al., 1989), temperature (Kutty, et al., 1971; Yagi, et al., 1990; Villarreal and
Rivera, 1993) and body weight (Kurmaly et al., 1989). In the present study,
salinity did not show any significant effect on the oxygen consumption in
trilobite larvae. Similarly, in P. monodon, the influence of salinity on the
oxygen consumption has not been found to be significant (Gaudy and
Sloane, 1981; Lei et al., 1989). However, in P. stylirostris an increase in the
respiration was observed at low salinities (Gaudy and Sloane, 1981).
The rate of oxygen consumption by Amphibola crenata has been
reported to be unaffected by the salinity of the external medium in the range
0-125% seawater (Shumway, 1981). There has been no significant
difference between the rates of oxygen consumption in air and water and
oxygen consumption varied with the 0.45 power of body weight. During
exposure to anaerobic conditions the snails showed an increase in the post-
anaerobic preanaerobic respiratory rate. This ratio reached maximum after
6 hrs of anoxia over the range of 0-125% seawater. Specimens of
Amphibola crenata exposed to declining oxygen tensions at full salinity
show almost no ability to regulate their rate of oxygen consumption. As
129 salinity decreases, the zone of critical pressure decreases, i.e. the snails become more oxygen independent; the maximum decrease in the zone of critical pressure coming in animals exposed to 0 and 25% seawater. When exposed to both salinity and anoxic stress, the animals reach their maximum degree of oxygen independence (Shumway, 1981).
When a marine organism is exposed to water of altered temperature or salinity, it must accommodate in some way to this physiological stress.
Short-term adjustments require additional expenditure of metabolic energy, which can be measured by increased respiration. On the other hand, some organisms respond to sub-optimal conditions by reducing their metabolic rates either by withdrawing into a shell or as a result of lowered enzymatic activity (Gaudy and Sloane, 1981). Marine organisms can respond to changing salinities as osmoregulators or osmoconformers. Regulation is an active process and usually results in an increased metabolic rate.
Conforming usually uses less energy and may result in lowered metabolic rate as enzymes work poorly at sub-optimal conditions.
Temperature also has a profound effect on metabolic rate.
Endothermic animals expend extra energy to maintain body temperature as the environmental temperature increases or decreases (Villarreal and
Rivera, 1993). Exothermic organisms cannot alter their internal temperature and usually show an increased metabolism as temperature rises, within a zone of tolerance. Outside of the tolerance zone, metabolism falls as enzymatic process no longer work properly.
130 Pagurus longicarpus or the mussel Mytilus edulis are common intertidal animals and must be able to cope with fluctuations in temperature and salinity. Hermit crabs respond to stress by withdrawing into their shell.
Placing the organisms into a known volume of water and measuring the oxygen concentration at the start and end of a known time period can measure respiration.
Attempts have also been made by several workers to demonstrate the effect of temperature on the survival of the larvae of marine animals
(Costlow, et al., 1960; 1962, 1966; Choudhury, 1971; Young and Hazlett,
1978; Moreira, et al., 1980). Not much information has been available that show the influence of temperature on the respiratory metabolism. However, in adult penaeid prawns the respiratory metabolism was moderate between temperatures 20° to 30° C (Dail, 1986; Liao and Murai, 1986). The present study demonstrates that the temperature plays a significant role in influencing the respiratory metabolism at higher temperature (<40 ° C). The trilobite larvae were also found to be influence by changes in pH of the seawater where a wide fluctuation in oxygen demand was observed. The normal oxygen consumption was at pH 6 and 7 whereas significant variation was observed at pH 5, 8 and 9.
There has always been a controversy to interrelate the respiratory metabolism with osmotic regulation in marine animals (Gross, 1957;
Panikkar, 1969). However, most of the work carried out on energy spent during osmotic regulation is based on the consumption of oxygen by the animals in a particular environment (Rao, 1968). Hence the respiratory
131 metabolism is considered to be an important biological process especially for the aquatic cultivable animals, which helps in assessing the oxygen requirement of the organism under different environmental conditions. It is also a best indicator for evaluating the energetic expenditure, precisely in the process of the osmotic regulation of any aquatic organism.
The estimation of oxygen consumption during a brief experimental period, however, does not seem to be a reliable index of the respiratory metabolism as compared to the natural conditions which is primarily a culture system for extended periods. However, the oxygen is an important environmental constituent and the scarcity of oxygen in the environment results in various phenotypic and physiological changes in the animal for their survival in a particular environment due to stress occurred on account of the depletion of oxygen.
132 Summary
During the acclimatization and experimental period, no mortality was observed in the trilobite larvae that showed sturdiness of the animal under different stress conditions. The oxygen consumption was more or less uniform at all salinity ranging from 10-40 psu indicating its least influence on the oxygen consumption of trilobite larvae of Indian horseshoe crab. The correlation coefficient values showed that the relationship between oxygen consumption and salinity was not significant (P>0.05; r=0.245). Variation in oxygen consumption was not much between temperature 20 ° and 30° C. At temperature 10 ° C, the rate of oxygen consumption was the minimum. A sudden increase in the oxygen consumption was recorded at a temperature
40° C. The data were tested statistically by applying analysis of variance where a significant relationship was observed between temperature and oxygen consumption (P<0.05; r=0.97). A similar trend in oxygen consumption was observed at pH 6 and 7 where moderate consumption of oxygen was observed. However, at pH 5, 8 and 9, the rates of oxygen consumption were found to be relatively greater indicating unfavorable conditions. The data were tested statistically and it was found that a high degree of correlations existed between pH and oxygen consumption
(r=0.98). The analysis of covariance showed a significant relationship between oxygen consumption and pH (P <0.05).
133 0.12
L A
E .gC
0.07 - C O 0 C 0 0)
0
0.02 10 20 30 40 Salinity (psu)
Figure 5.1. Oxygen consumption of the trilobite larvae with different salinity
134 0.12
E E C O tit." 0.07 0 a) ca>, O
0.02 10 20 30 40 Temperature (oC)
Figure 5.2. Oxygen consumption of the trilobite larvae with different temperatures
135 0.12 -
E Q0
g 0.07 0
a) >, 0
0.02 5 6 7 8 9 pH
Figure 5.3. Oxygen consumption of the trilobite larvae with different pH
136 Ch = pter-6
Regeneration of gill lamellae of the Indian horseshoe crab Regeneration is a form of tissue repair or the restoration of lost or damaged tissues, organs or limbs. Aside from being used to generally describe any number of specific healing processes, regeneration also is a specific method of healing that is noted for its ability to regrow lost limbs, severed nerve connections and other wounds. In other words regeneration is the restoration of body parts which have been removed either by injury or autopsy (self-amputation of body parts). Maintenance of an organism depends upon the continuation of the turnover by which all tissues and organs constantly renew themselves. Regeneration is a fundamental attribute of all living things and yet organisms greatly differ in their ability to regenerate parts. Some grow their new structure on the stump of the old one, unlike others that simply heal over the stump without replacement; a form of regeneration at the tissue level of organisation (Sullivan, 1988).
Scientsists have been fascinated, for ages, with the idea that lost parts of animals can be regenerated. According to Greek legend, one of the twelve "labors" of Hercules was the destruction of the Hydra, a gigantic monster with nine serpents' heads. Finding that as soon as one head was cut off two new ones grew in its place and at last he burned out their roots with firebrands. Regeneration began to be formally studied over
250 years ago in many lower invertebrates (ReAaumur, 1712). The work and research has been continuing ever since and yet a satisfactory molecular mechanistic explanation is still fully unavailable.
It is difficult to understand the actual mechanism of regeneration among living families. How flatworms can regenerate heads and tails from
137 any level of amputation, while other species are only able to regenerate in one direction or sometimes or not at all? We wonder what it is that makes earthworms, a close relative of the leeches, has a great capacity of regenerating whilst the leeches themselves fail to regenerate at all. There are many inconsistencies, which make it hard to explain regeneration as natural selection operating on the principle of efficiency.
The methods by which organisms reproduce have much in common with the regenerative processes. Two examples of such reproductions are - the vegetative reproduction which is the process where a whole new organism is produced from fractions of the parent organism and secondly - the sexual reproduction occurring in all organisms that reproduce sexually. It is a process where a whole organism develops from a single cell known as the zygote. These remarkable events testify to the universality of the regenerative processes.
Regeneration depends upon the ability of un-injured cells to produce the kinds of cells destroyed. Unspecialised cells are perfect for the job, since they are still able to produce any type of required cell. These unspecialised cells are totipotent. Thus regeneration involves re- establishing local tissue differentiation. Some examples require long-range interaction and extensive cell movement (morphallaxis), while others involve short-range interactions and extensive growth (epimorphosis). Morgan
(1898), the Nobel Prize winning geneticist/developmental biologist, began his research career studying regeneration. He coined the terms morphallaxis and epimorphosis to describe the two major types of
138 regenerations. Morphallaxis refers to the type of regeneration that occurs in the absence of cellular proliferation, while epimorphosis refers to regeneration that requires active cellular proliferation as reported by Morgan
(1898).
Morphallaxis is the most efficient way for simple organism to regenerate where the wound is healed and neighbouring tissues reorganised themselves into whatever parts are lost. The process occurs in coelenterates such as the jellyfishes and the hydra. The higher animals usually form a blastema made out of cells that look alike. Despite their different origins, it is produced at the site of amputation, which provides the tissue that will form the regenerated part. The process occurs in higher animals and made its first evolutionary appearance in flatworms. The common earthworm or night crawler of our lawns, gardens and bait cans, has a body made up of a series of 100 or more segments marked off by shallow grooves. If the worm is cut in half, the head end can grow a new tail is formed. If only 15 or 20 segments of the head end are cut off, a new head with five segments replace them.
Regeneration occurs in many, if not in all vertebrate embryos and is present in some adult animals such as salamanders (e.g. the newt and axolotl), hydra, horseshoe crabs and a type of mouse. Mammals exhibit limited regenerative abilities although not as impressive as salamanders.
Examples of mammalian regeneration include antlers, finger tips and holes in ears. Finger tip regeneration has been well characterized and these studies have resulted in the first demonstration of a genetic pathway
139 controlling regeneration in a mammal. Several species of mammals can regenerate ear holes - a phenomenon that has been most studied in the MRL mouse. If the processes behind regeneration are fully understood, it is believed this would lead to better treatment for individuals with nerve injuries
(such as those resulting from a broken back or a polio infection), missing limbs and/or damaged or destroyed organs.
In contrast to all other vertebrates, salamanders can regenerate their limbs throughout their lives and thus they provide a unique window to observe how this can be done. Successful limb regeneration in a salamander is a two-step process consisting of an early phase that begins with wound healing and ends with the formation of a regeneration blastema.
This is followed by the second re-development phase that is a recapitulation of the events that occurred during limb development in the embryo. The unique events of regeneration include the rapid closure of the wound by epidermal cells, interactions between the wound epidermis and underlying stump cells. This stimulates re-differentiation, cell migration and proliferation that give rise to the blastema. De-differentiation of stump cells to give rise to the relatively undifferentiated blastema cells, though not well understood mechanistically, is a necessary step for limb regeneration.
Simply there are two major pathways of regeneration of a lost limb.
The first de-differentiation of adult cells into a stem cell state similar to embryonic cells and the second; development of these cells into new tissue more or less the same way it developed the first time (Bryant et al., 2002).
Some animals like planarians instead keep clusters of non-differentiated
140 cells within their bodies which migrate to the parts of the body that need healing.
All animals have the power of regeneration to a greater or lesser degree. In man and higher animals it is quite limited. We see it most often in the healing of wounds and the mending of bones. A lost fingernail can be replaced but not a lost finger. Lower animals have a much greater ability to replace parts. For instance, the little half-inch flatworm — planarian- that lives under rocks in clean creeks can be cut into as many as 32 pieces. Each fragment is able to rebuild a miniature flatworm complete with head, tail, eyes, mouth and internal organs. Planarian have a supply of unspecialized cells distributed throughout the mesenchyme layer. These small regeneration cells with large nuclei and rich in ribonucleic acid (RNA) are called neoblasts. Neoblasts can differentiate into all of the many different types of specialized planarian cells and are therefore also known as pluripotent stem cells. In a normal worm they appear to be inactive whilst in a wounded worm (or during the course of cell turnover), they become very active. They are signalled to divide and/or differentiate and so replace lost cell types.
Lobsters have a greater ability to regenerate enabling them to replace or repair almost any lost or injured appendage. The new part may however, require several molts to reach normal size. When chelipeds (fourth thoracic segment used for defence better known as chelas, crushers or pincers) snap off, it usually occurs at the preformed breakage plane, which is close to the body. At this plane, the stump is quickly covered by a membrane and then a valve which blocks all remaining small passages. The
141 valve is held in place by blood pressure within the limb base. Thus if the damage occurs at the breakage plane not only does the regeneration occur more readily than anywhere else along the limb, but it also involves little blood loss and minimal tissue damage.
Adult vertebrates, particularly mammals show a very limited capacity for neuronal replacement following trauma or disease. This limitation is one of the main driving forces that underpin the current high level of interest in those small areas of neuronal renewal now known to exist in the mammalian brain. In dramatic contrast to this, both tunicates and echinoderms exhibit a remarkable plasticity as adults and are capable of extensive regeneration of large parts of their bodies following traumatic loss or damage. This potential has been exploited adaptively in both groups of animals as a mechanism for budding. Many adult tunicates produce asexual buds that develop into new and independent adults. Some adult and larval echinoderms can undergo fission and so produce two or more animals from a single original.
The outer shell of horseshoe crab consists of three parts. The carapace is the smooth frontmost part of the crab which contains the eyes, the walking legs, the chelicera (pincers), the mouth, the brain and the heart
(Chatterji, 1994). The abdomen is the middle portion where the gills are attached as well as the genital operculum. The last section is the "telson"
(caudal spine) which is used to flip itself over if stuck upside down The carapace of the horseshoe crab consists of two parts, viz. prosoma and opisthosoma. The opisthosoma is a hexagonal, broad plate connected to the carapace by a flexible joint, allowing it to move up and down. There are
142 broad, flat, thin and double membrane structures attached on the underside of the opisthosoma. As these structures look like pages of a book, they are called book gills or gill lamellae. Horseshoe crabs which have six pairs of them, though the first pair (called the operculum) serves as a cover for the other five. Book gills are flap like appendages that are designed for gas exchange within water and seem to have their origin as modified legs. More than a thousand gill lamellae are present on either side of the opisthosoma.
A well-developed circulating system with blood vessels in the gill books helps the animal in oxygen exchange. These appendages move with rhythmic movements to drive blood in and out of the lamellae and to circulate water over them. Respiration being their main purpose, they can also be used for swimming in young individuals. If they are kept moist, the horseshoe crab can live on land for many hours. As long as the gills are moist, the horseshoe crab can get oxygen from the air while they are on the beaches for breeding. The gill books can be opened and moved easily in such a manner that they help young horseshoe crabs to swim upside down.
Considering the seasonal and batch-to-batch variability in the sensitivity of amoebocyte lysate and threat to the horseshoe crab population, an attempt was made to cultivate amoebocyte-producing tissue in vitro (Joshi et al., 2002). This investigation was undertaken to eliminate the collection of amoebocytes from the wild stock where about 200 gill lamellae were removed for in vitro cultivation for mass production of amoebocytes. During the experiment, it was observed that within 3-4 months, the lost gill lamellae were completely regenerated. Considering this as an important phenomenon, a detailed study was carried out to examine
143 the regeneration rate of the gill lamellae of the Indian horseshoe crab, as
information on the regeneration of lost parts has not been reported so far.
144 Material and methods
Live adult horseshoe crabs both males and females were collected from the breeding beach in Orissa (lat 21°27' N; long 87°04' E) for the
present study. The specimens were transported to the laboratory in Goa in
specially designed containers and maintained in fibreglass tanks (capacity
2000 I). The fibreglass tanks were provided with a sand bed (grain size: 65-
125 pm) and continuous supply of re-circulating filtered seawater (0.5 pm)
of 30%o salinity.
In the present study, about 200 gill lamellae were removed from ten
adult males and female's horseshoe crab for in vitro cultivation of tissue to
harvest amoebocytes. The dissected part of the animal was washed with
antibiotic (5% betadine solution) and 70% alcohol. Specimens both males
and females with lost gill lamellae were tagged and kept separately in the
laboratory in fibreglass tanks. Temperature of the water was maintained at
about 27°C by thermostatically regulated titanium heaters throughout the
period of experiment.
The animals both males and females were examined weekly for a
period of 175 days to evaluate the percentage of regeneration of gill
lamellae. The data obtained were computed to compare the rate of
regeneration in both the sexes.
145 Results
In the present experiment, about a third of the lost gill lamellae in females were regenerated on the 50 th day and half on the 75th day (Figure
6.1). The total regeneration of the gill lamellae in females was observed on day 125th (Plate 6.1- a & b).
The regeneration of gill lamellae in males was observed to be slow as compared to females. About a third of gill lamellae were regenerated on
75th to day and half on the 125 th day. The rest of the gill lamellae were regenerated on the 175 th day (Figure 6.2).
146 Discussion
In marine organisms, regeneration of lost tissue is considered to be a natural phenomenon (Barnes, 1980; McVean, 1982). Most of the work reported on regeneration of lost parts is related to predation. In natural environment it has been observed that the regeneration process is not only species-specific but also it is dependent on seasons. There have been few records showing high regeneration rate in warmer months (Fielman et al.,
1991). A clear picture of the regeneration process has been demonstrated by conducting experiments on brittle stars in the laboratory (Dobson, 1988;
Fielman et al., 1991). The regeneration process in the laboratory has been found to be temperature-dependent and is completed within 100 to 200 days (O'Connor, 1986). Attempts have also been made to study the regeneration process at the molecular level (Sanchez and Newmark, 1999;
Salo and Baguna, 2002). However, due to lack of knowledge of molecular and genetic manipulations of the commonly studied vertebrate models, not much success has been achieved so far. Sanchez and Newmark (1999) made an attempt to study the introduction of double-stranded RNA that abolished the gene function in planarians, which is a classical example of the regeneration process. An extensive study has also been carried out on the genes and molecules involved in the regeneration process in planarians
(Salo and Baguna, 2002).
Regeneration is a process that requires more energy compared to that required for growth of the animal (Sullivan, 1988). Marine animals are capable of differentially allocating nutrients to different functions of body
147 such as maintenance, metabolism, somatic growth, wound repair and reproductive tissue growth. The allocation is a dynamic process depending on the physiological and reproductive states of the organisms (Williams,
1966). It has been a well-known fact that the crustaceans are able to re- grow their limbs and regenerate muscle fibres. Crustaceans often tend to molt and grow throughout life. They therefore never lose the ability to grow back missing appendages. When a leg is lost, a new outgrowth appears even if the animal is not destined to molt for many months. The discovery of the factors responsible for regeneration may provide vital information to determine how to replicate such a process with human tissues. The implications of this for human health are enormous because crustacean muscles seem remarkably similar to humans. Protein structure in human is almost the same and the crustacean molecular switches work the same way as human muscles do.
Science may be one step closer to understanding how a limb can be grown or a spinal cord can be repaired. Scientists at The Forsyth Institute have discovered that some cells have to die for regeneration to occur. This research may provide insight into mechanisms necessary for therapeutic regeneration in humans, potentially addressing tissues that are lost, damaged or non- functional as a result of genetic syndromes, birth defects, cancer, degenerative diseases, accidents, aging and organ failure (Sullivan,
1988). Through studies of the frog (Xenopus) tadpole, the Forsyth team examined the cellular underpinnings of regeneration.
148 Although mammals (including humans) cannot regenerate an entire limb, they do possess some regenerative abilities and we should recognize that regeneration is a basic biological process exhibited in all animals. It is just that salamanders are exceptionally good at regeneration. The most
important difference between salamanders and mammals appears to be in the functioning of fibroblasts. In mammals fibroblasts respond to injury by forming scars. In salamanders they function to control regeneration of the
other cell types in the limb. Thus regenerative failure is a consequence of the failure of mammalian fibroblasts to carry out this function. The challenge
is to identify the signals that fibroblasts use to regulate cellular behaviour
(Fielman et al., 1991).
In fact, the problem of animal regeneration has withstood the probing
of scientific inquiry for over 250 years and still awaits a satisfactory
mechanistic explanation. The idea of regenerating lost body parts has
captured human imagination since the beginning of history. Almost every
phylum possesses one or several species capable of regenerating missing
body parts. In some cases, even entire organisms may regenerate from
parts of their bodies. Solid molecular evidence suggesting a common
ancestral origin for regeneration is presently lacking (Fielman et al., 1991).
The evidence available thus far is correlation and comes primarily from the
study and comparison of vertebrate regenerative events with their better-
understood developmental counterparts. For instance, when molecular
analyses are extended to limb ontogeny and regeneration, it becomes clear
149 that both of these events deploy similar sets of regulatory genes to accomplish their respective parts (Fielman et al., 1991).
The evolutionary conservation of the molecular events leading to limb ontogenesis among different phyla is well known and it is used regularly as an example of a homologous metazoan trait with a functional conservation of key molecular events. Nevertheless, the phylogenic relationships that exist, for example, between insects and vertebrates indicate that none of their common ancestors possessed appendages and thus it is difficult to explain how these appendages may have arisen homogonously in the first place. Obviously, these ideas have to be tempered with molecular experimentation. The diversity of experimental tools available today demands it. As more molecular data are accumulated on the phenomenon of regeneration and life in general, we should not be surprised to find some of these ideas eliminated, others transformed and still others that are altogether new to emerge on this subject.
Considering the association that exists between regeneration and cellular pluripotentiality and given the different strategies used by the metazoa to regulate these properties future research on regeneration must become multiphyletic and integrative in nature. Such an approach as logical as it seems, would require the identification and isolation not only of those sets of genes activated during regeneration but also during injury, wound repair, dedifferentiation and asexual reproduction. This is further complicated by the fact that there may be other unsuspected or even unknown biological processes acting on regeneration whose effects would
150 have to be defined and then characterized at the molecular level. Hence a comprehensive, multiphyletic and integrative approach to the problem of regeneration seems, at first glance, rather difficult to deploy.
151 Summary
The animals both males and females showed regeneration of gill lamellae. About a third of the lost gill lamellae in females were regenerated on the 50th day and half on the 75 th day. The total regeneration of the gill lamellae in females was observed on day 125 th . The regeneration of gill lamellae in males was observed to be slow as compared to females. About a third of gill lamellae were regenerated on 75 th day and half on the 125th
th day. day. The rest of the gill lamellae were regenerated on the 175
152 Figure 6.1. Regeneration of gill lamellae in females.
153 Plate 6.2. a, Arrow shows incised gill lamellae (in circle). b, Arrow shows regenerated gill lamellae (in circle).
154 Regeneration of gills in males
300
e 250 lla 200 me 150 ill la f g 100 o
No 50
0 0 50 100 150 200 Days
Figure 6.2 Regeneration of gill lamellae in males
155 REFRENCES Alessandri, G., Emanueli, C., Madeddu, P. (2004). Genetically engineered stem cell therapy for tissue regeneration. Ann. N. Y. Acad. Sci., 1015: 271-84.
Amit, M., Carpenter, M. K., Inokuma, M. S., Chiu, C. P., Harris, C. P., Waknitz, M. A., Itskovitz-Eldor, J. and Thomson, J. A. (2000). Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol., 227: 271-278.
Anversa, P.,Leri, A., Rota, M., Hosoda, T., Bearzi, C., Urbanek, K., Kajstura, J. and Bolli, R. (2007). Concise review: stem cells, myocardial regeneration, and methodological artifacts. Stem Cells, 25:589-601.
Armstrong, P. B. (1991) In: Immunology of Insects and Other Arthropods (Gupta, A. P. ed.) CRC Press, London, 4-17.
Asahara, T., Murohara, T. and Sullivan, A. (1997) Isolation of putative endothelial progenitor cells for angiogenesis. Science, 275:964-967.
Asahara, T., Iwasaki, H., Kawamoto, A., Ishikawa, M., Oyamada, A., Nakamori, S., Nishimura, H., Sadamoto, K., Horii, M., Matsumoto, T., Murasawa, S., Shibata, T., and Suehiro, S. (2006). Dose-dependent contribution of CD34-positive cell transplantation to concurrent vasculogenesis and cardiomyogenesis for functional regenerative recovery after myocardial infarction. Circulation,113 (10):1275-1277.
Badorff, C., Brandes, R. P, Popp, R., Rupp, S., Urbich, C., Aicher, A., Flemming, I., Busse, R., Zeiher, A. M. and Dimmeler, S. (2003). Transdifferentiation of blood derived human adult endothelial progenitor cells into functionally active cardiomyocytes. Circulation, 107: 1024- 1032.
Barbash, I. M., Chouraqui, P., Baron, J., Feinberg, M. S., Etzion, S., Tessone, A., Miller, L., Guetta, E., Zipori, D., Kedes, L.H., Kloner, R. A., Leor, J. (2003). Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution. Circulation. 108: 863-868.
156 Barnes, R. D. (1980) Invertebrate Zoology, Saunders College, Philadelphia, 4th edn
Beer, H. D., Longaker, M. T., Werner, S. (1997). Reduced expression of PDGF and PDGF receptors during impaired wound healing. J. Invest. Dermatol., 109: 132-138,
Black, A. F., Berthod, F., L'Heureux, N., Germain, L., Auger, F. A. (1998). In vitro reconstruction of a human capillary-like network in a tissue- engineered skin equivalent. FASEB. J., 12 (13):1331-1340.
Blau, H. M., Brazelton, T. R. and Weimann, J. M. (2001). The evolving concept of a stem cell: entity or function. Cell, 105: 829-841.
Bliss, E. D. (1983) The Biology of Crustacea. Internal Anatomy and Physiological Regulation. (Mantel, L. H. ed.). Academic Press, New York, 321-322.
Botton, M. L., Loveland, R. E. and Jacobsen. T. R. (1988). Beach erosion and geochemical factors: influence on spawning success of horseshoe crabs (Limulus polyphemus) in Delaware Bay. Mar. Biol., 99: 325-332.
Botton, M. L., Loveland, R. E. and Jacobsen, T. R. (1994). Site selection by migratory shorebirds in Delaware Bay, and its relationship to beach characteristics and abundance of horseshoe crabs (Limulus polyphemus) eggs. Auk., 111: 605-616.
Brehelin, M. (1986). Immunity in invertebrates (New York: Springer-Verlag), 126 pp
Bryant, N.J., Govers, R. and James, D. E. (2002). Regulated transport of the glucose transporter GLUT4. Nat. Rev. Mol. Cell Biol., 3:267-277.
Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K., Eberhardt, C., Declercq, C., Pawling, J., Moons, L., Collen, D., Risau, W., and Nagy, A. (1996). Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature, 380: 435-439.
157 Carmeliet, P. (2000). Mechanisms of angiogenesis and arteriogenesis. Nature Medicine, 6: 389-395
Chatterji, A. and Vijayakumar, R. (1987). The amazing Horseshoe crab, Science Today, September, 62-64.
Chatterji, A., Vijayakumar, R. and Parulekar, A. H. (1992).Seasonal variation in the volume of the Haemolymph and body weight of the Horseshoe crab, Tachypleus gigas (Muller). Pak. J. Sci. Ind. Res., 35: 495-498.
Chatterji, A. (1994) The Horseshoe Crab-A Living Fossil, A Project Swarajya Publication, 157 pp.
Chatterji, A. and Abdi, S. A. H. (1994) The Indian Horseshoe crab-A Living Fossil. J. Indian Ocean Stud., 1:42-48.
Chatterji, A. Parulekar, A. H. and Qasim, S. Z. (1994). Nesting behaviour of the Indian horseshoe crab, Tachypleus gigas (Xiphosuria). In: India's Economic Zone by S. Z. Qasim and G. S. Roonwal, Eds, Omega Scientific Publishers, New Delhi, 142-155.
Chatterji, A. and Singh, U. (1997a) Bhartiya RajKankra Ka Manav Jeevan Main Mahatvya aim Sangralshan,Rashtriya Vigyan Sangoshthi,Vaigyanic Vishyon Ka Hindi Main Sampereshan, CEERI, Pilani,17
Chatterji, A., and Singh, U. (1997 b). Raj Kankra Ka Ausidhi Vigyan Main Upyog.Vigyan Sabha, Rashtriya Vaigyanic Hindi Sangoshthi ka Lekh Sangrah, 28-29 August,183-185.
Chatterji, A. and Singh, U. (2002). Vichitra Raj Kankra Aur Uska Ausidhi Upyog (in Hindi).Bharat Main Vigyan Ki Uplabdhiyan- Ek Jhalak, Rashtria Antarctic Ayam Samudria Anusandhan Kendar,120-123.
Choudhury, P. (1971). Responses of larval Macrobrachium carcinus (L.) to variation in salinity and diet (Decapoda: Palaemonidae). Crustaceana, 20: 113-120.
158 Cohen, E. (1979) Biomedical Applications of the Horseshoe Crab (Limulidae). Alan. R. Liss. Inc., New york, 668 pp.
Cooper, J. F. (1979) Acceptance of the limulus test as alternative pyrogen test for radio pharmaceuticals and interathecal drugs. Progress Clin. Biol. Res. 29:345.
Cole, W. H. (1940). The composition of fluids and sera of some marine animals and of the sea water in which they live. J. Gen. Physiol., 23: 575-584.
Costlow, J. D., Bookout, C. G. and Monore, R. (1960) The effect of salinity and temperature on the larval development of Sesarma cinererum (Bosc.) reared in the laboratory. Biol. Bull. Mar. Biol., 118: 118-202.
Costlow, J. D., Bookout, C. G., and Monore, R. (1962) Salinity-temperature effects on the larval development of the crab Panopeus herbstii Milne- Edwards, reared in the laboratory. Physiol. Zool., 35: 79-93.
Costlow, J. D., Bookout, C. G. and Monore, R. (1966). Studies on the larval development of the crab Rhithropanopeus harrisii (Gould.) I. The effect of salinity and temperature on the larval development. Physiol. tool., 39: 81-100.
Cragg, G. M., Newman, D. J. and Yang, S. S. (2006). Natural product extracts of plant and marine origin having antileukemia potential. The NCI experience. J. Nat. Prod. 69 (3):488-98.
Crosby, J. R., Kaminski, W. E., Schatteman, G., Martin, P. J., Raines, E. W., Seifert, R. A., Bowen-Pope, D. F. (2000). Endothelial cells of hematopoietic origin make a significant contribution to adult blood vessel formation. Circ. Res., 87:728-730.
DaII, W. (1986). Estimation of routine metabolic rate in a penaeid prawn, P. esculentus (Haswell). J. Exp. Mar. Biol. EcoL, 96: 57-74.
Diwan, A. D., Laxminarayana, A. and Kakati, V. S., (1989). Osmoregulatory ability of P. monodon (Fabricius) in relation to varying salinities. Indian J. Fish., 36(1): 53-57.
159 Dobson, W. E. (1988) Studies of the process,mechanism and limiting factors affecting tissue regeneration after autotomy in ophiuroid echinoderm Macrophiophiolis gracillima. Ph. D. dissertation. University of S. Carolina, Columbia, S. Carolina pp 175.
Drake, C .J. and Little, C. D. (1995). Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization. Proc. Natl. Acad. ScL, USA, 92:7657-7661.
Eichmann, A., Corbel, C., Nataf, V., Vaigot, P., Breant, C. and Nicole Douarin, M. Le (1997). Ligand-dependent development of the endothelial and hemopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2. Proc. Natl. Acad. ScL USA, 94, 5141-5146.
Fielman, K. T., Stancyk, S. E., Dobson, W. E. and Clements, L. A. (1991). Effects of disc and arm loss on regeneration by Microphiopholis gracillima (Echinodermata: Ophiuroidea) in nutrient free seawater. J. Mar. Biol.,111:121-127
Flamme, I., Frolich , T. and Risau, W. ( 1997). Molecular mechanisms of vasculogenesis and embryonic angiogenesis. J. Cell Physiol., 173(2):206-210.
Frank, S., Hubner, G., Breier, G., Longaker, M. T., Greenhalgh, D. G. and Werner, S. (1995). Regulation of vascular endothelial growth factor expression in cultured keratinocytes. Implications for normal and impaired wound healing. J. Biol. Chem., 270,12607-12613.
Friber, J. A., Weathers, P.J., Gibson, D.G. III (1992). Culture of amoebocytes in a nutrient mist bioreactor. In vitro Cell. Dev. Biol., 28A(3): 215-217.
Gaudy, R. and Sloane, L. (1981). Effect of salinity and oxygen consumption in postlarave of the penaeid shrimps, P. monodon and P. stylirostris without and with acclimation. Mar. BioL, 65:2 97-301.
Gibson, G. D. III, Teaticket, Joan, B. Hilly (1992) Production of horseshoe crab amebocytes in vitro United States Patent No 5,082782.
160 Gill, M., Dias, S., Hattori, K., Rivera, M. L., Hicklin, D., Witte, L., Girardi, L., Yurt, R., Himel, H. and Rafii, S. (2001). Vascular trauma induces rapid but transient mobilization of VEGFR2NAC133(4-) endothelial precursor cells. Circulation Research, 88 : 167-174.
Gilles, R. and Pequeux, A. (1983) Environmental Adaptations, (Vernberg, F..J. and Vernberg, W. B. eds.), Academic Press, New York, 8:109- 165.
Grant, J. (1984). Sediment microtopography and shorebird foraging. Mar. Ecol. Prog. Ser., /9:293-296.
Grasshoff, K. (1979) Methods of Seawater Analysis (Verlag, Chemic, New York) 318 pp.
Gross, W. J. (1957). An analysis of response to osmotic stress in selected decapod crustacean. Biol. Bull. Mar. Biol., 112: 43-62.
Gul, W. and Hamann, M. T. (2005). Indole alkaloid marine natural products: an established source of cancer drug leads with considerable promise for the control of parasitic, neurological and other diseases. Life ScL, 78(5):442-453.
Hicklin, P. W., and Smith, P. C. (1984) Selection of foraging sites and invertebrate prey by migrant semipalmated sandpipers, Calidris pusilla (Pallas) in Minas Basin, Bay of Fundy. Candy. J. Zool., 62:2201-2210.
Hill, J. M., Zalos, G., Halcox, J. P., Schenke, W. H., Waclawiw, M. A., Quyyumi, A. A. and Finkel, T. (2003) Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N. Engl. J. Med., 348: 593-600.
Hilly, J. B. and Gibson, D. G. III (1989). Culture of amoebocytes on opened gill lamellae of the horseshoe crab, Limulus polyphems. Am. Zool., 29(4): 112.
Ishikawa, M. and Asahara, T. (2004). Endothelial progenitor cell culture for vascular regeneration. Stem Cells Dev.,13: 344-349.
161 Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, 0., Amit, M., Soreq, H., and Benvenisty, N. (2000). Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. MoL Med., 6: 88-95. lwanaga,S., Miyata, T., Tokunaga, F., and Muta, T. (1992) Molecular mechanism of hemolymph clotting system in Limulus. Thromb. Res., 68:1-32
Iwanaga, S. (1993). The limulus clotting reaction. Curr. Opin. Immunol., 5:74-82.
Jackson, K. A., Majka, S. M., Wang, H., Pocius, J., Hartley, C. J., Majesky, M. W., Entman, M. L., Michael, L. H., Hirschi, K. K. and Goodell, M. A. (2001). Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J. Clin. Invest.,107 (11):1395-1402.
Janakiram, B., Venkateswarlu, Y., Rajarami Reddy, G. and Sasira Babu, K. (1985). Oxygen uptake rhythm in penaeids prawns, P. indicus (H. Milne Edwards) in different salinities. Ind. J. Mar. ScL,14: 222-223.
Johnstone, B., Hering, T. M., Caplan, A. I., Goldberg, V. M., Yoo, J. U. (1998). Stem Cell and gene based therapy — Frontiers as regenerative medicine. Exp. Cell Res., 238:265-272.
Jorgenson, J. H. and Smith, R. F. (1973) Preparation, .sensitivity, and specificity of Limulus lysate for endotoxin assay. Appl. MicrobioL, 26:43-48.
Joshi, B., Chatterji, A. and Bhonde, R. (2002). Long term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Muller). In vitro Cell. Dev. BioL-Anim., 38, 255-257.
Kalka, C., Masuda, H., Takahashi, T. (2000). Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc. Natl. Acad. ScL USA, 97:3422-3427.
162 Kawabata, S., Muta, T. and Iwanaga, S. (1996). In: New Directions in Invertebrate Immunology (So"derha" II, K., Iwanaga, S. and Vasta, G. R., eds.). SOS Publications, Fair Haven, N. J., 255-283.
Kawamoto, A., Gwon, H. C., lwaguro, H., Yamaguchi, J. .1, Uchida, S., Masuda, H, Silver, M., Ma H, Kearney, M., Isner, J. M. and Asahara, T. (2001) Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation, 103:634-637.
Kern, B. E., Balcom, J. H., Antoniu, B. A., Warshaw, A. L. and Fernandez- del Castillo, C. (2003). Troponin I peptide (G1u94-Leu123), a cartilage- derived angiogenesis inhibitor: in vitro and in vivo effects on human endothelial cells and on pancreatic cancer, J. Gastrointest. Surg., 7: 961-968.
Kelsey, M. G. and Hassal, M. (1989). Patch selection by Dunluin on a hetergenous mudflat. Ornis. Scand., 20:250-254.
Kocher, A.A., Schuster, M.D., Szabolcs, M.J., Takuma, S., Burkhoff, D., Wang, J., Homma, S., Edwards, N.M., Itescu, S. (2001). Neovascularization of ischemic myocardium by human bone-marrow- derived angioblasts prevents cardiomyocytes apoptosis reduces remolding and improve cardiac function. Nature Medicine, 7(4):430- 436.
Kurmaly, K., Yule, B. A. and Jones, D. A. (1989). Effect of body size and temperature on the metabolic rate of P. monodon. Mar. Biol., 103: 25- 30.
Kutty, N. M., Murugapoopathy, G. and Krishnan, T. S. (1971). Influence of salinity and temperature on the oxygen consumption in young juveniles of the Indian prawn P. indicus, Mar. Biol.,111:125-131.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227 (5259): 680-685.
163 Lei, C. H., Hsieh, L. Y. and Chen, C. K. (1989). Effect of salinity on the oxygen consumption and ammonia-N excretion of young juveniles of the grass shrimps, P. monodon (Fabricius). Bull. Inst. Zool. Acad. Sinica, 28: 245-256.
Levin, J. and Bang, F. B. (1964a).The role of endotoxin in the extracellular coagulation of Limulus blood. Bull. Johns. Hopkins Hosp.,115: 265- 274.
Levin, J. and Bang, F. B. (1964b). A description of cellular coagulation in the Limulus. Bull. Johns. Hopkins Hosp.,115: 337-345.
Levin, J. and Bang, F. (1968). Clottable protein in Limulus; its localization and kinetics of its coagulation by endotoxin.Thromb. Diat. Haemorrh.,19(1):186-97.
Levine, R. J. C., Chantler, P. D. and Kensler, R. W. (1988). Arrangements of myosin heads on Limulus thick filaments. J. Cell Biol., 107:1739- 1747.
Liao, I. C., Murai, T. (1986). Effects of Dissolved Oxygen temperature and salinity on the oxygen consumption of the grass shrimp P. monodon. In: Maclean J. L., Dizon, L. B., Hosillos, L. V. eds. The First Asian Fisheries Forum. Asian Fisheries Society, Manila, Phillipines, 641-646.
Lin, Y., Weisdorf, D., Solovey, A. (2000). Origins of circulating endothelial cells and endothelial outgrowth from blood. J. Clin. Invest., 105:71-77.
Macallum, A. B. (1926). The palaeochemistry of the body fluids and tissues. Physiol. Rev., 6:316-357.
Magnum, C. P., Booth, C. E., Defur, P. L., Heckel, N. A., Henry, R. P., Oglesby, L. C. and Polities, G. (1976). The ionic environment of haemocynin in Limulus polyphemus. Biol. Bull., 150, 453 pp.
Malouf, N. N., Coleman, W.B., Grisham, J. W., Lininger, R. A., Madden, V. J., Sproul, M. and Anderson, P. A. W. (2001). Adult-derived stem cells from the liver become myocytes in the heart in vivo. Am. J. Pathol., 158(6):1929-1935.
164 Mantel, L.H. and Farmer, L.F. (1983) Internal Anatony and Physiology Regulation (Mantel, L.H. ed.) Academic Press, New York, 5: 54-153.
Mantel, L. H. and Landesman, J. (1977) Osmotic regulation and Na+K- activated ATPase in the green crab, Carcinus maenas, and the spider crab, Libinia emarginata. Biol. Bull.,153: 437-438.
Martin, L., Stricher, F., Misse, D., Sironi, F., Pugniere, M., Barthe, P., Prado-Gotor, R., Freulon, I., Magne, X., Roumestand, C., Menez, A., Lusso, P., Veas, F., Vita, C. (2003). Rational design of a CD4 mimic that inhibits HIV-1 entry and exposes cryptic neutralization epitopes. Nat. BiotechnoL, 21(1):71-6.
Mayer, A. M. S. and Hamann, M. T. (2004). Marine pharmacology in 2000: marine compounds with antibacterial, anticoagulant, antifungal, anti- inflammatory, antimalarial, antiplatelet, antituberculosis, and antiviral activities; affecting the cardiovascular, immune, and nervous systems and other miscellaneous mechanisms of action, Mar Biotechnol., (NY) 6:37-52.
Mayer, A. M. S.and Gustafson K. R. (2006) Marine pharmacology in 2003- 2004: Anti-tumour and cytotoxic compounds, European Journal of Cancer, 42 (14): 2241-2270.
McAlester, A. L. (1968). The History of Life, Printice-Hall, Inc., Englewood Cliffs, New Jersey,152 pp
McManus, J. J. (1969). Osmotic relations in the horseshoe Crab, Limulus polyphemus. Amer. Midland Naturalist, 81 (2): 569-573.
McVean, A. (1982) Autotomy, In The Biology of Crustacea Neural Integration and Behaviour (eds Sandeman, D. C. and Atwood, H. L.), Academic Press, New York, 4:107-132.
Mikkelsen, T. (1988). The Secret in the Blue Blood. Science Press Beijing, China,124 pp.
165 Mishra, J. K. (1991) Embryonic development and variation in the haemolymph of indian horseshoe crab, Tachypleus gigas (Muller). Ph. D. Thesis, Behrampur University, 145 pp.
Misra, S., Ghosh, A. and Varticovski, L. (1994). Naturally occurring ether- linked phosphatidylcholine activates phosphatidylinositol 3-kinase and stimulates cell growth. J. Cell Biochem., 55(1):146-53.
Moreira, G. S., McNamara, J. C., Moreira, P. S. and Weinrich, M. (1980) Temperature and salinity effects on the respiratory metabolism of the first zoeal stage Macrobrachium holthusi Genofre and Loboa (Decaposa: Palaemonidae) J. Exp. Biol. Mar. Ecol., 47: 141-148.
Morgan T. H. (1898) Experimental studies of the regeneration of Planaria maculata. Arch. Entw. Mech. Org., 7:364-397.
Muta, T., and Iwanaga, S. (1996). The role of hemolymph coagulation in innate immunity Curr. Opin. ImmunoL, 8, 41-47.
Nagai,T., Kawabata, S. I., Shishikura, F., Sugita, H. (1999). Purification and characterization of an amino acid sequence of an embryonic lactin in peri-vitelline fluid of the embryo of the horseshoe crab. J. Biol. Chem., 274:37673-37678.
O'Connor, B., Bowmer, T., McGrath, D. and Raine, R. (1986). Energy flow through an Amphiura filiformis (Ophiuroidea: Echinodermata) in Galway Bay, west coast of Ireland: a preliminary investigation. Ophelia, 26:351- 357.
Orlic, D., Kajstura, J., Chimenti; S., Jakoniuk, I., Anderson, S. M., Li, B., Picket, J., McKay, R., Nadal-Ginard, B., Bodine, D.M., Leh, A., Anversa, P. (2001 a). Bone marrow cells regenerate infarcted myocardium. Nature, 41D: 701-705.
Orlic. D., Kajstura, J., Chimenti, S., Limana, F., Jakoniuk, I., Quaini, F., Nadal-Ginard, B., Bodine, D. M., Leri, A., Anversa, P. (2001 b). Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc. Natl. Acad. Sci. ,U S A., 98: 10344-10349.
166 Ornberg ,R. L., and Reese, T. S. (1979) Secretion in Limulus amebocytes is by exocytosis, Prog. Clin. Biol. Res., 29: 125-130.
Panikkar, N. K. (1969), New Perspectives in Estuarine Biology. First All India Symposium in Estuarine Biology, Madras, Dec. 27-30, (Mimeo), 8 pp.
Parab, P. B., Chatterji. A., (2003) Isolation of peri-vitelline fluid of the fertilized eggs of horseshoe crab and use thereof, US patent #20030017212
Parab, P. B., Ghaskadbi, S., Patwardhan, V., Mishra, G. C., Chatterji, A., Parameswaran, P. S. (2004). Cardiac development promoting activity of peri-vitelline fluid of embryos of Indian horseshoe crab, Tachypleus gigas (Muller). Patent Number :0319NF2004.
Perin, E. C., Dohmann, H. F., Borojevic, R., Silva, S. A., Sousa, A. L., Mesquita, C.T., Rossi, M. I., Carvalho, A. C., Dutra, H. S., Dohmann, H. J., Silva, G. V., Belem, L., Vivacqua, R., Rangel, F. 0., Esporcatte, R., Geng, Y. J., Vaughn, W. K., Assad, J. A., Mesquita, E. T., Willerson, J. T. (2003). Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation, 107: 2294-2302.
Peichev, M., Naiyer, A. J., Pereira, D., Zhu, Z., Lane, W. J., Williams, M., Oz MC, Hicklin, D. J., Witte, L., Moore, M. A., Rafii, S. (2000). Expression of VEGFR-2 and AC133 by circulating human CD34 + cells identifies a population of functional endothelial precursors, Blood, 95: 952-958.
Price, J. T. (1971). The Origin and Evolution of Life. The English University press Ltd., London, 113 pp.
Prosser, C. L. (1965) Comparative Animal Physiology, (C.L. Prosser & F.A. Brown (ed.) (Toppan Company Ltd., Tokyo, Japan, pp. 6-47.
Prosser, C. L. (1973) Comparative Animal Physiology, C.L. Prosser (ed) Sauders Philadelphia, 1-11.
167 Quammen, M. L. (1982). Influence of subtle substrate differences on feeding by shorebirds and inter tidal mudflats. Mar. Biol., 71:339-343.
Rafii, S., Mehmet, M. D., Oz, M. D., J., Alex, S., Barbara, F. B. S., Adam, B. A.S., Asch, M. D., Ralph, L., Nachman, M. D., Fred, S.M. D., Eric, A., Rose, M. D., Howard, R. Levin, M. D. 1995. Characterization of hematopoietic cells arising on the textured surface of left ventricular assist devices. Ann. Thorac Sur, 60:1627-1632.
Rafii S. (2000). Circulating endothelial precursors: mystery, reality and promise. Clin. Invest, 105: 17-19.
Rafii, S., Avecilla, S., Shmelkov, S., Shido, K., Tejada, R., Moore, M.A., Heissig, B., Hattori, K. (2003). Angiogenic factors reconstitute hematopoiesis by recruiting stem cells from bone marrow microenvironment. Ann N. Y. Acad. ScL, 996:49-60.
Rafii, S. and Lyden, D. (2003). Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat. Med., 9:702-12.
Ramarao, K. V. and Suryarao, K.V. (1972) Studies on Indian king crab (Arachnid, Xiphosura). Proc. Indian Acad. ScL, 38(B3-4):206-211.
Rao, G. M. P. 1968. Oxygen consumption in rainbow trout (Salmo gairdnen) in relation to activity and salinity. Can. J. Zool., 46: 781-786.
Rawat, D. S., Joshi, M. C., Joshi, P. and Atheaya H. (2006). Marine peptides and related compounds in clinical trial.References Anticancer Agents. Med. Chem., 6(1):33-40.
ReAaumur RAF. Sur les diverses reproductions qui se font dans les Ecrevisse, les Omars, les Crabes, etc. et entr'autres sur celles de leurs Jambes et de leurs Ecailles. Mem. Acad. Roy. ScL, 1712, 223-245.
Robertson, J. D. (1949) Ionic regulation in some marine invertebrates. J. Exp. Biol., 26(2):182-200.
Robertson, J. D. (1953). Further studies on ionic regulation in marine invertebrates. J. Exp. Biol., 30:277 pp.
168 Robertson, J.D. and Waterman, T. H. (1960). Osmotic and Ionic Rregulation. In: T.H. Waterman, Editor, The physiology of Crustacea Vol. I, Academic Press, New York and London, pp. 317-340.
Robertson, J. D. (1970) Osmotic and ionic regulation in the horseshoe crab, Limulus polyphemus (Linnaeus). Biol. Bull., 138:157-183.
Ross, V. C. and Bruck, C. W. (1982). Endotoxin testing of medical devices with LAL:FDA requirements. In:Endotoxin and their detection with the Limulus Amebocyte Lysate Test, Watson, S. W., Levin, J. and Novitsky, T. J. (Eds). Alan R. Liss. Inc., NewYork, 39-48.
Rudloe, A. (1979). Limulus polyphemus. A review of the ecologically significant literature. In : Biomedical application of the Horseshoe crab (Limulidae). E. Cohen ecol. Alan R. Liss. Inc., New York, 27-35.
Rumpold, H., Wolf, D., Koeck, R., Gunsilius, E. (2004). Endothelial progenitor cells: a source for therapeutic vasculogenesis. J. Cell. Mol. Med., 8:509-18.
Salo, E. and Baguna, J. (2002) Regeneration in planarians and other worms: New findings, new tools, and new perspectives. J. Exp. Zool., 292:528-539.
Sanchez Alvarrado, A. and Newmark, P. A. (1999). Proc. Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc. Natl. Acad. ScL U. S. A., 96:5049-54.
Sato, T. N., Tozawa, Y., Deutsch, U., Wolburg-Buchholz, K., Fujiwara, Y., Gendron-Maguire, M., Gridley, T., Wolburg, H., Risau, W., Qin, Y. (1995). Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature, 376:70-74.
Sekiguchi, K. (1988) In: Biology of Horseshoe Crabs (Sekiguchi, K, ed.) Science House Co, Ltd, Tokyo), 139-181.
Shake, J.G., Gruber, P. J., Baumgartner, W. A., Senechal, G., Meyers, J., Redmond, J. M., Pittenger, M. F., Martin, B. J. (2002). Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann. Thorac. Surg., 73: 1919-1925.
169 Shaw, J. (1958). Osmoregulation in the Muscle Fibres of Carcinus Maena. J. Exp. Biol., 35:920-929.
Shi, Q., Rafii, S., Hong-De Wu, M., Errol, Wijelath, S., Cong Yu, Ishida, A., Fujita, Y., Kothari, S., Mohle, R. Lester R. Sauvage, Malcom, A. S., Moore, R., Storb, F. and Hammond, W. P. (1998). Evidence for circulating bone marrow-derived endothelial cells, Blood, 92:362-367.
Shishikura, F., Sekiguchi, K., (1984a). Studies on perivitelline fluid of horseshoe crab embryo. Purification and properties of agglutininfrom the perivitelline fluid of Tachypleus gigas, embryo. J. Biol. Chem., 96 : 621- 628.
Shishikura, F., and Sekiguchi, K. (1984b). Studies on peri-vitelline fluid of horseshoe crab embryo. II. Purification of agglutinin-binding substance from the peri-vitelline fluid of Tachypleus gigas embryo. J. Biol. Chem., 96 (3):629-636.
Shumway, and Sandra, E. (1981) Factors effecting oxygen consumption in marine pulmonate, Amphibola crenata (Gmelin, 1971). Biol. Bul1.,160 (2): 332-347.
Shuster, C. N., Jr. (1982). A pictorial review of the natural history and ecology of horseshoe crabs Limulus polyphemus, with reference to other Limulidae. In physiology and biology of horseshoe crab: Studies on Normal and Environmentally stressed animals, eds, J. Bonaventura, C. Bonaventura and S. Tesh. New York : Alan R. Liss 51 pp.
Shuster, C. N., Jr. and Botton, M. L. (1985). A Contribution to the population biology of horseshoe crabs (Limulus polyphemus). In Delaware Bay. Estuaries, 8: 363-372.
Shuster, C. N., Jr., Barlow, R. B. and Brockmann, H. J. (2003). The American Horseshoe Crab, Harvard University Press, Cambridge, Massachusetts and London, England, 1-421.
Shuster, C. N. Jr. (1982) Physiology and Biology of the Horseshoe crab, J. Bonaventurs and C. Bonaventura (ed.), New York, 1-51.
170 Srimal, S. and Bachhwat, B. K. (1985) Blood Brother-a living fossil helps to save human life. ScL Age, 51-55.
Srimal, S., Miyata, T. Kawabata, S. and Iwanaga, S. (1985). The complete amino acid sequence of coagulogen isolated from southeast Asian horseshoe crab, Carcinoscorpius rotundicauda. J. Biochem. (Tokyo), 98: 305-318.
Sugita, H. and Sekiguchi, K. (1979) Protein components in the peri-vitelline fiuid of the embryo of the horseshoe crab. Dev. Biol., 73: 183-192.
Suhonen, J. 0., Peterson, D. A., Ray, J., Gage, F. H. (1996). Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo. Nature, 383:624-627.
Sullivan, K. M. (1988) Physiological ecology and energies of regeneration in reef rubblebrittle-stars. In Echinoderm Biology (eds Burke, R. D. et al.), A. A. Balkema, Rotterdam, 523-529.
Sullivan, B., Bonaventura, J. and Bonaventura, C. (1974). Functional Differences in the Multiple Hemocyanins of the Horseshoe Crab, Limulus polyphemus. Proc. Natl. Acad. ScL U S A., 71(6): 2558-2562.
Strauer, B. E., Brehm, M., Zeus, T., Kostering, M., Hernandez, A., Sorg, R. V., Kogler, G., Wernet, P. (2002). Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation, 106: 1913-1918.
Suri, C., Pamela, Jones, F., Patan, S., Bartunkova, S., Maisonpierre, P. C., Davis, S., Sato, T.N. and Yancopoulos, G. D. (1996). Requisite Role of Angiopoietin-1, a Ligand for the TIE2 Receptor, during Embryonic Angiogenesis, Cell, 87: 1171-1180.
Technical Report (1998). Department Of Biotechnology, Government of India New Delhi, September 1998.The development of appropriate technology for the conservation controlled breeding and larval rearing of the Indian horseshoe crab along the coast of Orissa, 125 pp.
171 Terada, N., Hamazaki, T., Oka, M., Hoki, M., Mastalerz, D. M., Nakano, Y., Meyer, E. M., Morel, L., Petersen, B. E. and Scott, E.W. (2002) Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature, 416: 542-545.
Takahashi, T., Kalka, C., Masuda, H., Chen, D., Silver, M., Kearney, M., Magner, M., Isner, J. M., Asahara, T. (1999) lschemia- and cytokine- induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat. Med., 5: 434-438.
Toh, Y., Mizutani, A., Tokunaga, F., Muta, T., and Iwanaga, S. (1991). Morphology of the granular hemocytes of the Japanese horseshoe crab, Tachypleus tridentatus and immunocytochemical localization of clotting factors and antimicrobial substances. Cell Tissue Res., 266: 137-147.
Toma, C., Pittenger, M. F., Cahill, K. S., Byrne, B. and Kessler, P. D. (2002). Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation, 105: 93-98.
Tziveleka, L. A., Vagias, C. and Roussis, V. (2003) Natural products with anti-HIV activity from marine organisms. Curr. Top. Med. Chem., 3(13):1512-1535.
Vassilopoulos, G., Wang, P. R. and Russell, D. W. (2003). Transplanted bone marrow regenerates liver by cell fusion. Nature, 422:901-904.
Vijayakumar, R. (1992) Ecology and biology of the horseshoe of the Indian coast. Ph. D. Thesis,University of Karnataka, Dharwad,143pp
Villarreal, H. and Rivera, J. A. (1993). Effect of temperature and salinity on the oxygen consumption of laboratory produced P. californiensis post larvae. Comp. Biochem. Physiol., 106 (1), 103-107.
Wagers, A. J., Sherwood, R. I., Christensen, J. L., Weissman, I. L. (2002). Little evidence for developmental plasticity of adult hematopoietic stem cells. Science, 297: 2256-2259.
Wagers, A. J., Weissman, I. L. (2004) Plasticity of adult stem cells. Cell, 116: 639-648.
172 Wang. X., Willenbring, H., Akkar, Y., Torimaru, Y., Foster,M., Al-Dhalimy, M., Lagasse, E., Finegold, M., Olson, S., and Grompe, M.( 2003). Cell fusion is the principalsource of bone-marrow—derived hepatocytes. Nature, 422:897-901.
Weller, J. M. (1969). The Course of Evolution. McGraw-Hill Book Company New York, 661 pp
Williams, G. C. (1966) Natural Selection, the costs of reproduction and a refinement of Lack's principle Am. Nat., 100:687-690.
Wolvekamp, H. P. and Waterman, T. H., (1960) Respiration in physiology of crustacean (T. H. Waterman Ed.). Academic Press, New York, 100 pp.
Yagi, H., Ceccaldi, H. J. and Gaudy, R. (1990). Combined influence of temperature and salinity on the oxygen consumption of the larvae of the pink shrimp, Palaemon serratus (Pennant) (Crustacea: Decapoda Palaemon). Aquaculture, 86:77-92.
Yeh, E. T. H., Zhang, S., Wu, H. H., Korbling, M., Willerson, J. T. and Estrov, Z. (2003). Transdifferentiation of human peripheral blood CD 34 + enriched cell population into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. Circulation, 108:2070-2073.
Young, A. M. and Hazlett, J. L. (1978). The effect of salinity and temperature on the larval development of Clibanarius vittalus (Bosc.). (Crustacea: Decapoda: Diogenidae). J. Exp. Mar. Biol. Ecol., 34:131- 141.
Young, N. S., Levin, J. and Prendergast, R. A. (1990). An invertebrate coagulation system activated by endotoxin: evidence for enzymatic medication. J. Clin. Invest., 51 : 170-1797.
Zwaginga, J. J., Doevendans, P. (2003). Stem cell-derived angiogenic / vasculogenic cells: possible therapies for tissue repair and tissue engineering. Clin. Exp. Pharmacol. Physiol., 30:900-908.
173 Outcome of the thesis
Publications
1. Regeneration of gill lamellae of the Indian horseshoe crab, Tachypleus gigas (Muller).Current Science, 87 (11), 1511-1512.
Anil Chatterji, Huma Alam, Bhupali K. Joshi and R. R. Bhonde (2004)
2. Effect of salinity on the growth of the adult horseshoe crab, Tachypleus gigas (Muller). J. Expt. Biol. (communicated).
Anil Chatterji and Huma Alam 3. Peri-vitelline fluid of the fertilized eggs of the Indian horseshoe crab (Tachypleus gigas, Muller) helps in maintaining the original phenotype of CD 34 + stem cells and enhances colony formation in vitro, Marine Biotechnology (communicated).
Massoud Mirshahi, Pezhman Mirshahi, Jeannette Soria, Amu Therwath, Huma Alam, P. S. Parameshwaran and Anil Chatterji
4. Perivitelline fluid of the fertilized eggs of the Indian horseshoe crab (Tachypleus gigas, Muller) promotes vasculogensis. J. Oncologie (communicated)
Massoud Mirshahi, Pezhman Mirshahi, Jeannette Soria, Amu Therwath, Huma Alam, Sreekumar, P. K., Sumita Sharma and Anil Chatterji
US Patents
1. The fertilized eggs of the Indian horseshoe crab (Tachypleus gigas, Muller) promotes vasculogenesis.
Massoud Mirshahi, Huma Alam , Pezhman Mirshahi, Jeannette Soria, Amu Therwath, Parameswaran Perunninakulath Subrayan and Anil Chatterji (0221 N F2005, 6/20/2005)
2. The fertilized eggs of the Indian horseshoe crab (Tachypleus gigas, Muller) helps in maintaining the original phenotype of CD 34* stem cells and enhances colony formation in vitro
Massoud Mirshahi, Huma Alam , Pezhman Mirshahi, Jeannette Soria, Amu Therwath, Parameswaran Perunninakulath Subrayan and Anil Chatterji (0222NF2005, 6/20/2005)