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SEVENTH INTERNATIONAL CONFERENCE ON BIOTHERAPY JUNE 21 - 24, 2007 The Convention Hall, The KimKoo Museum SEOUL, KOREA 1 Organized by International Biotherapy Society CAM, Pochon CHA University SEVENTH INTERNATIONAL CONFERENCE ON BIOTHERAPY JUNE 21 ~ 24, 2007 The Convention Hall, The KimKoo Museum SEOUL, KOREA ORGANIZATION International Biotherapy Society Complementary & Alternative Medicine, Pochon CHA University SPONSOR Korean Oriental Medical Practitioners Association World Foundation of Apitherapy (Apimeds, Inc.) 2 CONTENTS Page 1 2 3 6 12 13 78 112 115 IBS, Organizing Committee Welcome General Information Scientific Program Poster Presentation Abstracts – Speakers Abstracts – Contributing Papers Notes Subway Map 3 INTERNATIONAL BIOTHERAPY SOCIETY President: Kosta Y. Mumcuoglu, Ph.D. (Israel) Vice-President: Terence J. Ryan, M.D. (UK) Secretary: Andrew Jarvis, M.D. (UK) Treasurer: John C. T. Church, M.D. (UK) Executive Directors: Theodore Chebuliez, M.D. (USA) Wim Fleischmann, M.D. (Germany) Olga S. Gileva, D.D.S. (Russia) Carita Hansson, M.D. (Sweden) Christopher MH Kim, M.D. (Korea) Theo Rufli, M.D. (Switzerland) Kathryn Vowden, R.N. (UK) Home Page ☞ http://biotherapy.md.huji.ac.il LOCAL ORGANIZING COMMITTEE Honorary President: Sae I. Chun, M.D. President: Christopher MoonHo Kim, M.D. Members: Bank S. Choi, O.M.D. Duck H. Kim, V.M.D. Hyun S. Kim, O.M.D. Kyung H. Kim, O.M.D. Young J. Lee, M.D. Dae W. Shin, M.D. 4 WELCOME On behalf of the International Biotherapy Society (IBS) and the Organizing Committee, we would like to welcome and the Seventh International Conference on Biotherapy. Our warm and sincere greeting goes out to every participant, particularly oversea guests and to those who are visiting Korea for the first time. We hope this Conference will give you a good opportunity to renew existing links with friends and colleagues around the world, and to fulfill new collaborations and friendships. The program of conference is going to take three days and comprise about 17 keynote lectures, 15 contributed papers, and 12 posters will be presented. We hope you enjoy the scientific program to your full extent. The Organizing Committee will assist you in any way during the Conference. We hope you have a pleasant time in Seoul. The Conference Organizing Committee 5 GENERAL INFORMATION The Convention Hall, The KimKoo Museum 255 Hyochang-dong, Yongsan-ku, Seoul, Korea Phone: xxx822-719-1311 Fax: xxx-822-718-1311 ☞ www.kimkoomuseum.org June 21-24, 2007 The official language of the Conference will be English. The weather in Seoul in June is mainly sunny with temperatures ranging from 15ºC/60ºF to 28ºC/82ºF. Clothing is informal for all occasions. All participants, accompanying persons and exhibitors are kindly requested to wear their badges throughout the meeting in order to be admitted to the lecture halls and other scheduled activities. The local currency is the Korean Won (1 US Dollar = 940-960 Korean Won). Cash and traveler's checks can be exchanged at the airport, hotels, or at local banks. Visa, Mastercard, American Express, and other credit cards 6 are accepted at most restaurants, department stores, and for travel. Certificate of attendance will be available for all participants at the registration desk from June 23, 2007. Upon request, the Conference will send a personal invitation to participate. This invitation is not a commitment on the part of the organizers to provide any financial support. Applications for invitation letters must be received by May 30, 2007. Please check with your travel agent if you require an entry visa to Korea. It is the responsibility of the participant to obtain a visa, if required. The IBS and ICB do not accept responsibility for individual, medical, travel or personal insurance. Participants are recommended to take out their own travel policies. - The registration fee will be $350 for regular participants, $175 for accompanying persons and students, and $150 for one day registration. - The breakfast is included in the hotel, and the lunch will be provided all participants at the conference. - On Friday (June 22) for a fee of $80, the participants will have the opportunity to participate to one full day tour to the Korea's most famous tourist sides of the Capital city of Seoul and Korean Folk Village. The price included lunch and dinner. 7 East Side No. 4B, West Side No. 11A → Koreana Hotel → Seoul Plaza Hotel → Lotte Hotel Get OFF! - President Hotel : 1 minutes on foot → KAL Building The conference will provide it. SEVENTH INTERNATIONAL CONFERENCE ON BIOTHERAPY 8 ____________________________________________________________ SCIENTIFIC PROGRAM JUNE 21, 2007 THURSDAY 07:30 - 17:00 Registration 09:00 - 10:00 Opening session, welcome and overview Byung S. Kim, M.D. (Korea), Chairman, College of Medicine, Pochun Cha University, Korea Christopher MoonHo Kim, M.D. (Korea), President of the Local Organizing Committee Kosta Mumcuoglu, Ph.D. (Israel), President of the International Biotherapy Society 09:40 - 12:30 SESSION: MAGGOT THERAPY (Part 1) Keynote Lectures Chair: John Church, M.D. (U.K.) and Ronald Sherman, M.D. (USA) 09:40 - 10:25 Clinical aspects of modern maggot therapy Wim Fleischmann, M.D. (Germany) 10:25 - 11:00 Coffee Break 11:00 - 11:45 Antibacterial substances in maggots of Lucilia sericata Kosta Y. Mumcuoglu, Ph.D. (Israel) 11:45 - 12:30 Integral Medicine & Biotherapy in the 21st Century Sae I. Chun, M.D. (Korea) 12:30 - 14:00 Poster Presentation 12:30 - 14:00 Lunch 14:00 - 15:30 14:00 - 14:45 SESSION: MAGGOT THERAPY (Part 2) Keynote Lectures Chair: Wim Fleischmann, M.D. (Germany) and Andrew Jarvis, M.D. (U.K.) Maggot Therapy - reflections from the past, foretelling the 9 14:45 - 15:30 15:30 - 16:00 16:00 - 17:45 16:00 - 16:15 16:15 - 16:30 16:30 - 16:45 16:45 - 17:00 17:00 - 17:15 17:15 - 17:30 17:30 - 17:45 17:45 - 18:00 18:00 - 18:30 future. Ronald Sherman, M.D. (USA) Maggot therapy in clinical practice and mode of actions Kristin Hasenoehrl, M.D. (Germany) Coffee Break Contributed Papers Aletha W. Tippett, M.D. (USA) Maggots to the Rescue: Using maggot therapy for wounds in hospice patients Sergey Chernysh (Russia) Maggot immunity and drug development Peter H. Nibberin, Ph.D. (The Netherlands) Maggot Excretions/Secretions Inhibit Multiple Neutrophil Pro-Inflammatory Responses Kang-Jun Yoon, M.D. (Korea) Effectiveness of Maggot Therapy in Chronic Infected Wound with MRSA Peter Takac, Ph.D. (Slovakia) Maggot Debridement Therapy (MDT) in Slovakia Shouyu Wang, M.D. (China) The healing and antibacterial function research of the maggot secretion on the ulcer area M.J.A. van der Plas (The Netherlands) Maggot Excretions/Secretions are effective against biofilms of Staphylococcus aureus and Pseudomonas aeruginosa Anders Anderson (Denmark) Maggot debridement therapy (MDT) in diabetic foot wounds – Quorum sensing dependent bacterial niches may promote infection or MDT failure Round-table Discussions (Keynote Speakers) JUNE 22, 2007 FRIDAY City Tour 10 JUNE 23, 2007 SATURDAY 08:30 - 17:00 Registration 09:00 - 12:30 SESSION: HIDUROTHERAPY (Part 1) Keynote Lectures Chair: Kosta Mumcuoglu, Ph.D. (Israel) and Vladmir Savino, M.D. (Russia) 09:00 - 09:45 Hidurotherapy in modern medicine Olga S. Gileva, D.D.S. (Russia) 09:45 - 10:30 Veterinary hidurotherapy Sagiv Ben-Yakir, D.V.M. (Israel) 10:30 - 11:00 Coffee Break 11:00 - 12:30 SESSION: HIDUROTHERAPY (Part 2) Keynote Lectures Chair: Olga S. Gileva, D.D.S. (Russia) and Sagiv BenYakir, D.V.M. (Israel) 11:00 - 11:45 Symbiotic bacterium from medicinal leech as the acting agent of hirudoautohaemotherapy, autoblood from medicinal leech as a means of active nonspecific immunotherapy in oncology. Vladimir Savino, M.D. (Russia) 11:45 - 12:30 1) The use of medical leeches in plastic surgery 2) Elimination of symbiotic bacteria from the gastro-intestinal track of the medicinal leech, Hirudo medicinalis. Kosta Y. Mumcuoglu, Ph.D. (Israel) 12:00 - 14:00 Poster Presentation 12:30 - 14:00 Lunch 14:00 - 16:00 SESSION: HELMINTHOTHERAPY, ICHTIOTHERAPY AND BIODIAGNOSTICS Chair: David Pritchard, Ph.D. (U.K.) and Martin Grassberger, M.D. (Austria), Edwin Cooper, Ph.D. (USA) 14:00 - 14:50 1. Understanding Mechanism of Maggot & Clinical Datas 2. Hookworms in clinical trials for the treatment of seasonal rhinitis and Crohn's disease David Pritchard, Ph.D. (U.K.) 14:50 - 15:20 Ichtiotherapy for patients with psoriasis 11 15:20 - 16:00 16:00 - 16:30 16:30 - 16:45 16:45 - 17:45 16:45 - 17:00 17:00 - 17:15 17:15 - 17:30 17:30 - 18:00 Martin Grassberger, M.D. (Austria) Evidence Based Complementary and Alternative Medicine Edwin Cooper, Ph.D. (USA) The evolution of biotherapy John C. T. Church, M.D. (U.K.) Coffee Break Contributed Papers Galina Korobeinikova (Russia) The Use of Medicinal Leeches and Medicinal Preparation from them in the Complex Treatment of Skin and Mucosal Diseases in UST Health Resort 17:00 - 17:15 Yavuz Dinler, Ph.D. (Turkey) New development about medical leech therapy Jiangning Wang, M.D. (China) Application of maggot therapeutics for repairing serious infective wound Round-table Discussions (Keynote Speakers) JUNE 24, 2007 SUNDAY 08:30 - 12:00 Registration 09:00 - 12:30 SESSION: APITHERAPY (Part 1) Keynote Lectures Chair: Christopher M-H Kim, M.D. (Korea) and Anatoly A. Gribkov, M.D. (Russia) 09:00 - 09:45 Honeybee Venom Clinical and Discovery Research in The United States and Canada in the 20th Century Robert Brooks, Ph.D. (USA) 09:45 - 10:30 Use of Apitherapy in Clinical Medicine Stefan Stangaciu, M.D. (Romania) 10:30 - 11:00 Coffee Break 11:00 - 12:30 SESSION: APITHERAPY (Part 2) Keynote Lectures Chair: Robert Brooks, Ph.D. (USA) and Stefan Stangaciu, M.D. (Romania) 12 11:00 - 11:45 11:45 - 12:30 12:30 - 14:00 14:00 - 15:30 14:00 - 14:15 14:15 - 14:30 14:30 - 14:45 14:45 – 15:00 15:00 – 15:15 15:15 - 15:30 15:30 - 15:45 15:45 - 16:15 16:15 - 17:15 1. Prerequisites and methods for intervertebral hernia treatment by live bee stings and other bees' products 2. Video presentation Anatoly A. Gribkov, M.D. (Russia) Bee Venom Therapy in the modern medicine (1985-2005) C. MoonHo. Kim, M.D. (Korea) Lunch Contributed Papers Duck-Hwan Kim, V.M.D. (Korea) Treatment by Injection-Acupuncture with Bee-Venom (Apitoxin) and Apitoxin Combined by Chinese Herbal Medicine in Patients with Canine Hind Limb Paralysis Duck-Hwan Kim, VMD (Korea) Bee venom (Apitoxin) therapy on canine facial nerve paralysis Byung-Soo Koo, O.M.D. (Korea) Effects of Bee Venom on Glioma Cells Ai-Young Lee, M.D. (Korea) Effects of bee venom on human melanocyte proliferation, melanogenesis and dendricity Nasser M. Shahri, Ph.D. (Iran) A new in vivomodel for testing effects of topically biotherapy wound healing Edwin L. Cooper, Ph.D. (USA) Hepatoprotective potential of earthworm extract (Lampito mauritii, Kinberg) against paracetamol induced liver damage in rats Edwin L. Cooper, Ph.D. (USA) Therapeutic Potential of Tunicates Coffee Break Panel Discussion: Biotherapy Moderator : C.T. Church, M.D. Panel : Wim Fleischmann, M.D. Olga S. Gileva, D.D.S. C. MoonHo. Kim, M.D. Kosta Y. Mumcuoglu, Ph.D. 13 Ronald Sherman, M.D. Stefan Stangaciu, M.D. 17:15 Closing 14 POSTER PRESENTATIONS Anders Andersen (Denmark) Maggot debridement therapy (MDT) in a diabetic foot wound patient suffering from severe gout Takuya Kawabata, Ph.D. (Japan) Induction of antibacterial activity in medicinal maggots by infected environment Myung-Sang Kwon, Ph.D. (Korea) Water extracted propolis inhibits TNF- α release following LPS injection in the rat Myung-Sang Kwon, Ph.D. (Korea) Water extracted propolis attenuates kainite-induced neurotoxicity via adenosine A1 receptor in the rat Hideya Mitsui, M.D. (Japan) Maggot Therapy for severe diabetic foot ulcer Alicia Fonseca Muñoz (Mexico) Efficiency Maggot Debridement Therapy in Fournier’s Gangrene Alicia Fonseca Muñoz (Mexico) Study of Maggot Debridement Therapy in necrotic pressure ulcers, Case Series Nasser M. Shahri, Ph.D. (Iran) Effect of a natural polymer "rabbit vitreous" to wounds therapy on an experimental model Nasser M. Shahri, Ph.D. (Iran) Macroscopic and microscopic surve of the effectiveness degree of some of the biostimulators used in traditional medicine in the process of healing of sheep skin wounds Zohrabyan A. Sureni (Armenia) Maggot therapy in special wound treatment Hitoshi Takase M.D. (Japan) The optimal conditions for medicinal maggots before and during shipment ABSTRACTS 15 for Keynote Speakers Veterinary Hirudotherapy Dr. Sagiv Ben-Yakir BSc(in Biology), DVM(in honor), MRCVS, CVA(IVAS), CVHomotox’(Baden-Baden,Germany) “ORSHINA” – The Israeli Veterinary Institute for Holistic Medicine Medicinal leeches were used for bloodletting, and have been applied to congested or inflamed parts of the animal body for over 3,500 years. They are used for engorged blood vessels as in acute equine laminitis, canine aural hematoma, swollen testicles, laryngitis, canine prolapsed rectum or bovine prolapsed uterus and inflamed vulva. Leeches are used also in reconstructive surgery on swollen faces, limbs and digits following successful arterial re-vascularization but limited venous repair. Leeches are also used in the treatment of such disorders as inflammation and peripheral venous and arterial diseases as in cat’s saddle thrombus. We can use leeches to treat inflammatory and traumatic processes such as keratitis, chorioretinitis, periorbital hematoma, subretinal hemorrhage, glaucoma and cataract. Hirudotherapy is being used to treat inflammatory diseases of the ear, nose and throat such as acute and chronic otitis, sinusitis, aural hematoma, and laryngitis, and to treat clinical forms of dermatitis and dermatosis (e.g eczema, paronychia, acral lick dermatitis etc). Leeches can be used to treat acute and chronic inflammatory immune--associated oral mucosa and peridontium lesions as in Feline Immunodeficiency Virus(F.I.V) positive cats with severe stomatitis and gingivitis, diseases of the salivary glands as sialadenitis and sialoadenosis. Furthermore, leeches are used to treat inflammatory conditions of the urogenital tract as endometritis, dysfunction and injuries of the genital organs in postoperative rehabilitation etc. 16 Leeches are used to rehabilitate animals with postpartum pyo-septic complications. Hirudotherapy was also found to be effective in healing intractable and non-granulating wound or ulcers, especially in elderly and immunocompromized animals. Leeching has been used as well for treating cats with polycythemia vera, or to reduce scrotal edema post-castration procedure in adult dogs. In osteoarthritis leeches do lessen the pain and restore lost mobility as in canine hip dysplasia (H.D) cases or knee osteoarthrits. Keep in mind that osteoarthritis is more than a disease of joint’s wear and tear, it is also a vascular disorder. Small clots can occur in the blood vessels supplying bone & joint, and starve the tissue for oxygen. Chemicals that thin the blood and break clots apart could ease animal’s misery as well as assisting in regeneration. Before applying the leeches the area to be treated should be cleansed with clean non-chlorinated water. Remove any traces of povidone-iodine (Betadine) or any other skin preps, as well as flea spray, dip or trans dermal preparations. These traces of solutions on the skin may discourage the leech from attaching. Leeches should be applied in adequate numbers to the general area of maximal congestion or other treated area/tissue. One or two leeches may be sufficient to treat an area of 5 square cm. A larger area may require 3-4 leeches depending on initial clinical response. The History of Honeybee venom in North America Robert Brooks, Ph.D. President, PVA Pharmaceutical Consultant Honeybee venom in North America emerged as a topic of discussion and interest in 1935 in New York City with the publishing of the book Bee Venom Therapy by a Hungarian/American physician Dr. Bodog Beck. Dr. Beck maintained an active bee hive in his office to provide a supply of live bees for his patients. In 1941 Dr. Joseph Hollander, then a young Rheumatologist at the University of Pennsylvania reported the disappointing results of his well designed double blind clinical trial. Dr. 17 Hollander’s trial drug did not utilize stings or the whole venom but rather the filtrate of ground up whole bee parts containing little if any whole bee venom. From 1938 through 1964 several attempts were made to demonstrate the effectiveness of bee venom in the treatment of arthritis. In 1966 a group of scientists gathered at Walter Reed Army Institute of Research including Biochemist William Shipman from San Francisco, California. Shipman began to use a then new technique to separate the components of bee venom, column chromatograph. Using G75-120 and G25-80 sephadex columns Shipman was able to separate the major components of bee venom. Shipman named two components mellitin and apamine identified by their molecular weights and concentrations in the whole venom. Quickly following him other scientists in England and Bulgaria published confirmation of his findings with technical additions and observations. Because of the available technology Shipman was unable to move beyond the 6-7 fractions he was able to separate but he always thought that their were a number more whose molecular weights were closely aligned. He would be surprised to learn how many more fractions were actually found in the whole venom. Shipman also characterized the stability of not only the whole venom but all of the fractions he identified. Working in the pharmacology laboratory at Walter Reed Army Institute of Research Dr. James Vick and I defined the LD50s for the whole venom in mice, dogs and monkeys and published our first work on bee venom. We confirmed the results of Artemov’s work in Russia. Artemov was the first to publish his in vivo results of ascorbic acid depletion from which he theorized indirectly the significant increase in endogenous circulating plasma cortisol. Artemov speculated that this amount of endogenous cortisol had a therapeutic effect similar to the injection of cortisone without the unwanted side effects that caused the abandonment of cortisone as a standard treatment. Dr. Vick and I confirmed this theory through a new blood study that measured circulating plasma cortisol directly in the blood. We studied and evaluated this in a number of ways including a dramatic study showing injected whole bee venom increased cage activity in older dogs. We interpreted our data as a demonstration of the therapeutic effectiveness of whole bee venom in arthritic conditions. We similarly studied the fractions of bee venom with special attention to their cardiovascular effect. We believed that the 18 therapeutic activity of bee venom was much more complicated than the production of endogenous cortisol but venom fractionation produced only small amounts of the fractions which limited out ability to explore our ideas and theory. From our beginning studies which I will provide in detail in this presentation many hundreds of papers have followed with increasing depth of knowledge that the improving technology has allowed. I can specifically offer you some interesting anecdotal details of the beginnings of a few of these works. As our knowledge of medicine has also evolved so have the medical specialties of our time like geriatric medicine and more recently genomic medicine. Only recently have physicians questioned the need of patients to be free of pain and the development of pain management as a medical specialty has led to a better use of whole bee venom for what it has been known for centuries, not the treatment of arthritis, but the often dramatic and sustained relief of pain. The Evolution of Biotherapy. Leeches, Maggots, Worms, Rats & Dogs; parasites or colleagues? John C T Church, M.D. In the evolution of scientific discovery, and its exploitation, over the past two centuries, physics and chemistry can be said to have dominated the scene. By contrast, the twenty-first century is already being identified as the Biological Century (1). The scientific emphasis throughout has been reductionist, but with no end in sight as the complexities of quantum physics and the genome unfold. By contrast again, natural biological evolution is holistic. A multiplicity of unicellular organisms interact, giving rise to a staggering diversity of multi-cellular organisms, all in their turn altering the environment and creating ecosystems which are ‘fuzzy’. ‘Biotherapy’ can be defined as ‘the use of living organisms in medicine’. Most of the natural biological ‘research and development’ in this arena has occurred over formidably long periods. Thus, for instance insects and 19 mammals have been interacting for over 200 million years. Host/parasite interactions, whether symbiotic or hostile, are highly sophisticated and complex, perhaps nowhere more so than in the interplay of their respective immune systems. Dogs and men have lived together since the earliest days of their respective evolution. The relationships that can develop between sentient individuals of difference species, such as dog and man, can be quite as complex as those between individuals of the same species. None of these interactions lend themselves readily to scientific measurement. For the greater part, ‘Biotherapy’ involves the use of normal healthy organisms, so managed that natural aspects of their behaviour are appropriately utilised. Leeches (hirudotherapy), maggots (maggot débridement therapy, MDT), and scavenger fish (ichthyotherapy), all have had a place in traditional medicine. In the ‘evolution’ of medical practice, all these branches of ‘Biotherapy’ are now being incorporated into modern medical practice, in a variety of ways, albeit mostly in a somewhat unstructured environment. To this list we can now add helminths (helminotherapy), and, in the recognition of disease, ‘sniffer’ rats and ‘cancer sniffer’ dogs (biodiagnostics). Hirudotherapy The use of leeches in medicine, for bloodletting, dates back to the ancient civilizations of India and Egypt. Bloodletting in ancient Greece was used to restore the balance of the four humors, blood, lymph, black and yellow bile. Throughout history the use of leeches this way has waxed and waned. In medieval Europe, religious and superstitious ideas caused a surge in the popularity of bloodletting. In 1833 alone France had to import ‘over 40 million leeches’!(2). With the development of microsurgical techniques leeches now have a vital place in plastic and reconstructive surgery, particularly where venous congestion hinders healing [Illustration slide 1]. An excellent example of this is the following case report: A patient was admitted to a plastic surgical unit having had his ear bitten off by a dog. The dog was caught and the 20 ear retrieved. The ear was then taken to hospital with the patient. Its surgical reattachment included finding a small divided arteriole and suturing it to restore arterial blood flow into the ear. But no veins of sufficient size could be found for such suturing. This left a precarious situation which, if left that way, would have led to engorgement of the reattached ear and failure. But leeches were applied to the rim of the ear for a sufficiently long period to allow capillary re-growth across the wound, with ultimate restoration of normal circulation [Illustration slide 2]. So this patient regained his ear, by dint of prompt action at the roadside, meticulous micro-vascular surgery, and the judicious application of hungry leeches (3). Modern hirudotherapy includes the use of leeches over arthritic joints, in venous ulcers, in a variety of ophthalmic and neurological conditions (4). A number of physiologically active biochemical agents in the leech saliva have been studied in the search for mechanisms for the beneficial effects of this treatment in these conditions. A word of caution must however be added. In spite of meticulous culture in dedicated laboratories it is impossible to fully eradicate bacteria from leech secretions, and appropriate antibiotic cover is advised during hirudotherapy. Maggot Débridement Therapy (MDT) ‘Harvested’ maggots have been used for centuries as cleansing agents in open wounds. They were first cultured in dedicated laboratories, for clinical use, in the 1930s (5). With the advent of antibiotics maggot therapy almost disappeared. But with the renaissance of this treatment by Sherman in the 1980s (6), and in the UK in 1995 (7), Maggot Débridement Therapy (MDT) has now not only been accepted internationally but has in many centres become an integral part of modern wound care [Illustration slide 3-6, or 7]. This development however is by no means uniform, due in part to negative or cautious attitudes of health care professionals and managers. It is also due to the difficulties of adequately monitoring and regulating the production and use of maggots this way. After extended deliberation FDA approval in the USA was granted in January 2004, and maggots have been available on prescription in the UK from February 2004. 21 Maggot feeding activity in wounds is characterised by at least the following factors: Enzymic digestion of decomposing organic material. Antibacterial action, in the wound and in the maggot gut. This includes the destruction of bacterial biofilm, and suppression of its formation. Enhancement of granulation tissue, presumably by the action of cytokines. Mobility. Maggots are dynamic and respond actively and appropriately to changes in the wound environment. pH. Maggot alkaline exo-secretions reverse acid wound pH. Wound warming. Maggot metabolism is exothermic, particularly so when they are closely packed, in group feeding. Enzyme ‘shelf life’. Maggot exo-secretions have a zero ‘shelf life’, being secreted directly from the salivary glands into the wound. A number of prospective studies have been undertaken or are under way, comparing MDT with other wound care products. However, this whole exercise, though essential for proper scientific assessment, presents considerable intrinsic challenges. There is no single manufactured product available than can simultaneously deliver all the factors listed above, and thus be compared with maggot activity. Equally, it is impossible to separate or isolate these various factors when healthy maggots are allowed to feed normally in open wounds. Ichthyotherapy This can be defined as the use of fresh water or marine organisms as agents of wound cleansing. The history of such treatment in traditional medicine is sparsely documented. In a museum near the River Kwai, recording the privations of prison camps, a sketch drawn by a prisoner showed him up to his waist in water, but with small fish attending to his leg ulcers (8). There is widespread use of such fish in India, particularly in rural areas. In Kerala State Professor Padmanabham PhD studied this phenomenon in detail (9) During the sixth International Biotherapy Conference in Turkey, in June 2003, the Kangal Balikli spa was visited. Warm water pools are 22 stocked with three species of small fish that scavenge lesions of the skin, particularly psoriasis [Illustration slide 8 ] (10). Helminthotherapy A number of diseases are associated with auto-immunity dysregulation. Typical of these are Crohn’s disease and ulcerative colitis. Acting on the hypothesis that the auto-immune response in these patients is due to lack of helminth antigenic stimulus in the alimentary tract, studies have been conducted giving such patients doses of helminth eggs orally. The chosen helminth, porcine whipworm, Trichuris suis, is not pathological to man, but has a positive effect on the host immunity, with reversal of symptoms (11). This work opens up a further horizon on the potential to use living organisms that interact with each other, particularly through immunological reactions, thereby maintaining healthy co-existance. Biodiagnostics Biodiagnostics can be defined as the use of living organisms in the detection of disease. Though modern medical screening systems are thorough, they are not universally available, and they are often still far short of requisite levels of sensitivity and specificity. There are still no reliable tests for the early detection of most cancers. National screening in the UK is currently only available for breast and cervical cancer. Trained sniffer-dogs are now used extensively in the detection of drugs and explosives, in forensic work, and in a variety of other ways such as the detection of dry rot and termites in houses. Anecdotal stories have been published of pet dogs that have apparently caused their owners to seek advice, and have an early cancer diagnosed and treated, with good result (12, 13) A controlled pilot study has shown that dogs can be trained to recognise cancer of the urinary bladder [Illustration slide 9] (14). A certain species of rat in Tanzania has been trained to recognise tuberculosis in samples from patients afflicted with this disease (15) 23 This presents us with a extensive field for similar studies, using any species with a well developed olfactory system that can be trained to recognise disease at high levels of specificity and sensitivity. The future of ‘Biomedicine’ Nature provides us with an almost limitless array of species that respond to chemical or other signals in their environment. There is thus a vast potential to harness or control such behaviour to give us models for the early recognition of disease, perhaps most readily when it is characterised by volatiles expressed by the patient. Working well with living organisms is exacting. It demands an understanding of their biology, and enough control of their ‘working’ environment to result in ‘optimal’ behaviour. Their use in the clinical arena is thus an art. With skilful and appropriate use, excellent clinical results can be obtained. It must be expected that all this ‘fuzzy-edged’ activity will become progressively more integrated into modern clinical practice. This already presents its own array of challenges for its requisite monitoring and evaluation, and the assessment of clinical outcomes, but guidelines for all this activity are being established. References: 1. Benford G. The Biological Century http://reason.com/9511/BENFORDfeat.shtml 2. El-Awady A. Maggots and leeches make a comeback. Science in Africa July-Aug 2003 http://www.scienceinafrica.co.za/2003/july/leech.htm 3. Teo TC. Personal case. Sherman RA. Maggot Therapy – The Last Five Years. ETRS Bulletin Vol 7 Issue 3. 2000 4. Gilyova O. Modern Hirudotherapy – a Review. http://www.scienceinafrica.co.za/2003/july/leech.htm 5. Lederle - Surgical Maggots. Council of Pharmacy and Chemistry. New and non-official remedies. JAMA 1932;98:401 24 6. Pechter EA, Sherman RA. Maggot therapy: the surgical metamorphosis. Plast Reconstr Surg 1983;72(4):567-570 7. Church JCT. ETRS working party consensus paper on wound debridement. Surgery 1995;13(10):228c-d 8. JCTC. Personal observations April 1998 9. Cohen J. Feeding the fish. BMJ 2000;320:181 10. Church JCT. 2003 Report on the Sixth International Biotherapy Conference, Turkey http://biotherapy.md.huji.ac.il/newsletter07.htm 11. Weinstock J, Summer WR. Will Helminths Become the Future Treatment for Inflammatory Bowel Disease? http://www.uihealthcare.com/news/currents/vol2issue1/1helminths.html 12. Williams H, Pembroke A. Sniffer dogs in the melanoma clinic? Lancet. 1989;1:734 13. Church J, Williams H. Another sniffer dog for the clinic? Lancet 2001;358:930 14. Willis CM, Church SM, Guest CM, Cook WA, McCarthy N, Bransbury A, Church MRT, Church JCT. Olfactory detection of human bladder cancer by dogs: proof of principle study. BMJ 2004 329: 712. 15. Balile D. Tanzania trains rats to detect tuberculosis. Science and Development Network December 28 2003 http://www.scidev.net/News/index.cfm?fuseaction=readNews&itemid=116 9&language=1 Acknowledgments. The author is grateful to Mr .T C Teo for illustrations nos. 1 and 2, Mr. A Jarvis for illustrations nos. 3-8, and to the Medical Illustration Department, Wycombe General Hospital for illustration no. 9. 25 Evidence Based Complementary and Alternative Medicine Edwin L. Cooper, Ph.D., Sc.D. Professor, Department of Neurobiology David Geffen School Of Medicine at UCLA, USA University of California, Los Angeles Time and contributions have moved rapidly since organizing the first International Congress, Complementary and Alternative Medicine, Kanazawa Japan in 2003. There we established the international peer reviewed journal: Evidence Based Complementary and Alternative Medicine (eCAM) published quarterly by Oxford University Press. Our journal, truly international with an impressive list of Editorial Board Members, seeks to create dialogue between and across disciplines in order to fully understand CAM and its numerous practices worldwide that include for example acupuncture and moxibustion. Kanazawa Japan was also the site for organizing and editing the Proceedings with Prof. N. Yamaguchi (Biomedical Approaches to Complementary and Alternative Medicine published by Plenum). We strongly believe that eCAM will flourish through imagination, scientific rigor and cooperative enthusiasm. The first objective in a serious approach to complementary and alternative medicine (CAM) should be to obtain a broad understanding, with a minimum of detail, of how CAM fits into the pattern of biology—of the way in which the neuroendocrineimmune system evolved, its function and coordination with other body systems, and its development from the embryo onwards. At the same time, such an outline should provide an adequate background for easy application of CAM ideas to the detail of practical CAM work in public health, clinical and medical practice, and yet not stray far away from the very biology that under girds it. CAM is organismic, inclusive and not reductionist and exclusive. Quality control is important for the safe use of natural products. Our eCAM is launched in a desire to ameliorate this situation, by encouraging the publication of original scientific papers based on sound scientific guidelines, but without prejudice against the possible efficacy of these new and ancient treatments. Turning to products from animals, particularly those from the sea, marine natural product bioprospecting has yielded a considerable number of drug 26 candidates. Most of these molecules are still in preclinical or early clinical development. For terrestrial animals, Lombrokinase is a product isolated from earthworms and marketed as a fibrinolytic agent. Their use dates to ancient Ayurvedic practices in India and Traditional Chinese Medicine (TCM) in China. Propolis from bees and antimicrobial peptides from maggots (fly larvae) are all effective CAM therapies. We must remember that such products are often associated with the immune systems of these creatures and that they evolved millions of years ago—thus their immune systems have been an effective survival strategy. And if it has worked for them, then humans should harness these as new-wave antibiotics or anticancer molecules, just to offer two biomedical (CAM) applications. Clinical Aspects of Modern Maggot Therapy Wim Fleischmann, MD Department of Trauma and Reconstructive Surgery Academic Hospital of the University of Heidelberg, Germany 1. Indications: When the orthopaedic surgeon William S. Baer presented his method and results of maggot therapy in 1931 his focus of interest was acute soft tissue and chronic bone infection. Later, it was mainly the work of Ronald Sherman which enlarged the scope of indications to chronic wounds, like stasis ulcers and bed sores. Presently, clinical emphasis is put to the problem of impaired wound healing as seen in diabetic wounds. 2. Mode of action: Maggots release digestive juices into the wound which contain growth factors, antiseptic substances and proteases. The high proteolytic activity liquefies necrotic tissue, which is the main food source of maggots. Clinically, there is an effective, atraumatic debridement of the wound and together with dissolved dead tissue its contaminating bacteria are removed as well. 3. Application of maggots: Maggots may either be applied directly to the wound surface or enclosed in 27 porous pouches which might be compared with tea bags. Using the latter the maggots are physically separated from the wound and the fluids streaming through the pores of the pouch convey active substances and nutrients. As it is superior to "free range maggots" in terms of patients` comfort, pain reduction, ease of maggot application, hygiene and aesthetics maggot pouch dressings could be regarded as an important innovative step in the logical progression of maggot therapy. In Germany pouch dressings account for more than 85% of sold maggot products. 4. Surgical technique: In superficial wounds like some bed sores, stasis and diabetic ulcers as a first measure mechanical “macro-debridement” (scalpel etc.) should be performed to remove dead tissue, mainly to accelerate healing and reduce costs of treatment. Maggots are then used for biological “microdebridement” and as an antiseptic agent. In deep soft tissue and bone infections the septic focus has either to be completely removed by surgery or at least extensively exposed by tissue incisions or excisions to give maggots and their secretions enough space to do their work properly. To prevent suffocation of maggots when put into deep wounds either textile spacer materials or polyvinyl-alcohol sponges are used to keep the wound edges apart. Profuse wound secretions need to be drawn off using drainage systems. To enhance the survival rate of maggots in particularly deep wounds special drains for ventilation purposes may be necessary. 5. Secondary measures: Having achieved a clean, well healing wound vacuum dressings (VAC) may be applied for acceleration of wound closure or external tissue augmentation procedures (EasApprox) are used for rapid closure of remaining wound defects (Biological-Mechanical Closure of Wounds, BMW). 6. Conclusion: Maggot Debridement Therapy is an excellent low-risk procedure for the treatment of wound infections and a variety of chronic wounds, given that this therapy is embedded in an effective concept of interdisciplinary wound management. 28 Hirudotherapy in Modern Medicine Olga Gileva, DDS Perm State Academy of Medicine, Russia In spite of the serious evolution of our scientific findings of the HTH performance, we haven’t totally covered its mechanism even now. From the modern biochemistry position we can speak about polyfactoral effects of leech saliva substances with anticoagulant, antithrombotic, antiaggregative, antiflogistic, vasodilatative activities from leech saliva, secreted to blood and tissues during the blood drainage. HTH has the positive influence on posttraumatic inflammation and regeneration, promoting antiexudative effect, improving the microcirculation and oxygen regimen, sells balance and etc. In the medical practice physicians have usually used Hirudo Medicinalis Oficinalis (HM), reared in special biofactories. All the biomaterial used in clinics has conformity certificate of regional centers of certification and quality control of drugs, and that permits doctors to use Hirudo Medicinalis in Curative purposes. Leech therapy may be used alone, as monotherapy or in addition to the main treatment, can be combined with pharmacotherapy. The usual treatment involves leeches applying on dermal or mucosal surfaces, with limited or unlimited time of hemoextraction, in non aspirative regimen - without hemoextraction and in aspirative regimen – with limited time of hemoextraction. The treatment course depends on the character of the disease and lasts from 3 days up to 5 weeks. HTH has low number of contraindications. The absolute ones are hemophilia, hemorrhagic diathesis, expressed and firm hypotension, expressed and firm anemia, sepsis. Clinicians also must remember about the cases of individual intolerance to leeches and should be very careful to prescribe leeching in pregnancy and during anticoagulant treatment. The lecture will be focused on a spectrum of clinical indications for HTH and it’s efficacy: - cardiovascular diseases (myocardial ischemia, postinfarction cardioscleroses, hypertension and it’s complications) 29 - - phlebopathology (chronic tromboflebitis and varicose dilatation of veins) leg ulcers, post- thrombotic symdrome dermatological diseases (psoriases, eczema, sclerodesmia, neurodesmite, ruber lichen planus) different forms of ophthalmology (inflammatory diseases of eyes – keratitis, uveitis, traumatic jnjuries of organ of vision, cataract, glaucoma) gynecology diseases (parametritis, mastitis, genital endometriosis) neurological pathology (radiculitis, neuritis, plexitis, chronic myofacial syndrome, infantile cerebral paralysis) lesions of oral cavity (glossalgia, facialgia, aphtose stomatitis, periodontitis, gingivitis) Practitioners emphasize the value of leeches in microvascular and reconstructive surgery). Side effects of HTH (local allergic skin reaction, hyperpimentation of the skin, regional lymphadenitis, subfebrile condition, headache, the formation post procedural cixatrix) will be analyzed in lecture. The majority of these side effects are transient, can be cupping rapidly and easily by means of pharmacotherapy. The wide array of bioactive products, combined with its minimal side effects led many clinicians and researches to apply leeches to a variety of illnesses with high efficacy. Ichthyotherapy for patients with psoriasis: first results and future aspects Martin Grassberger, MD, PhD University Medical Center Hamburg-Eppendorf, Germany Ichthyotherapy (therapy with the so-called “Doctorfish of Kangal”, Garra rufa) has been shown to be effective in patients with psoriasis and other 30 skin diseases in the Kangal hot springs in Sivas, Turkey. In a pilot study we set out to evaluate the efficacy and safety of ichthyotherapy in combination with short-term UVA sunbed radiation in the treatment of psoriasis under controlled conditions. Sixty-seven patients diagnosed with psoriasis who underwent 3 weeks of ichthyotherapy at an outpatient treatment facility in Lower Austria between 2002 and 2004 were analyzed retrospectively. Main Outcome Measures were (1) overall relative reduction in Psoriasis Area Severity Index score; (2) proportion of patients with an improvement in their PASI score of ≥75% (PASI-75) and ≥50% (PASI-50); (3) patient reported outcomes assessed with a custom questionnaire; (4) patient follow-up with a questionnaire sent out in March 2005. Safety was evaluated by reviewing adverse events and vital signs. Overall there was a 71.71% reduction in PASI score compared to baseline (p<0.0001). Of the 67 patients studied, 31 (46.3%) achieved PASI-75 and 61 patients (91%) achieved at least PASI-50. Patients reported substantial satisfaction with the treatment. The reported mean remission period was 8.58 months (95% CI, 6.05 to 11.11). 87.5% of patients reported a more favourable outcome with ichthyotherapy, when asked to compare ichthyotherapy to other previously tried therapies. Sixty-five percent stated that after the relapse their symptoms were less severe than before treatment. There were no significant adverse events. The benefit demonstrated in this study along with the favourable safety profile suggests that ichthyotherapy combined with UV-phototherapy could provide a viable treatment option for patients with psoriasis. Additionally we conducted a morphological study on some features of the fish’s anatomy, shedding more light onto the mechanism of the observed therapeutic effect. A prospective controlled trial to further validate efficacy of this unusual treatment modality is currently in the planning phase. Keywords: Garra rufa, ichthyotherapy, Kangal fish, psoriasis treatment, ultraviolet A. 31 Introduction Psoriasis is a common skin disorder with a worldwide distribution; the average prevalence in Europe and the USA has been estimated at 2%.1 Whilst considerable advances have been made in the management of this disease in recent years, there is no cure, and no simple, safe and invariably effective treatment.2 The disease carries a substantial burden even when not extensive, and is associated with widespread treatment dissatisfaction. 3 A wide range of treatments is offered for psoriasis. Some patients rely on conventional pharmacological methods; others try alternative and complementary treatment modalities. Surely one of the most unusual alternative treatments is the so-called “Doctor Fish of Kangal” in the Central Anatolia region of Turkey. This treatment was first mentioned in The Lancet in 19894 but the details of the treatment were published only recently by Özcelik et al.5 According to the authors two different types of fish live in the pools of the Kangal hotspring: Cyprinion macrostomus and Garra rufa. Both fish are members of the carp and minnow family (Cyprinidae). Garra rufa is regarded as the main therapeutic. Garra rufa is normally a bottom dweller, where it adheres by suction to rocks with its ventral crescent-shaped mouth to feed on phytoand zooplankton.6 However, in the hot pools of Kangal, where phyto- and zooplankton are scarce, these fish feed on the skin scales of bathers, reportedly reducing illnesses such as psoriasis and atopic dermatitis.5 Whether this remarkable treatment is also effective outside of the Kangal hotspring in Turkey is unknown. Since there have been many unscientific and misleading names for this kind of therapy, we suggest the term “ichthyotherapy”, in accordance with other so-called biotherapy concepts such as maggottherapy (use of sterile fly larvae), hirudotherapy (use of leeches) and apitherapy (use of bee venom). In this retrospective study, we set out to evaluate the efficacy and safety of ichthyotherapy when used in combination with short term UVA radiation in patients with psoriasis vulgaris in an outpatient treatment facility. 32 Patients and Methods Patients We retrospectively analysed 67 patients diagnosed with psoriasis who underwent 3 weeks of ichthyotherapy combined with a short course of UVA sunbed treatment at an outpatient treatment facility in Lower Austria between 2002 and 2004. All patients had moderate to severe chronic plaque psoriasis. Patients referred themselves to the treatment facility to find relief from their symptoms. The costs of this treatment were not covered by the patients’ health care insurance. All patients had signed informed consent and were under constant medical supervision throughout the course of treatment. Treatment regimen Treatment lasted three weeks. A daily two hour “fish bath” (Fig. 1) was taken in a bath tub at a comfortably warm temperature (36-37°C). Patients with no contraindication to UV exposure (reported history of UV-related skin malignancies) used a commercially available stand-up rapid-tan facility (Cyclone 60 with 60 Cosmolux VHR160 lamps, CMC Sun Capsule USA) for 3-5 minutes after each bath session according to skin type. The spectral emission of the lamps is shown in figure 2. After UV exposure, the patients applied a generic skin lotion (Pharmacy Neunkirchen, Austria) containing glycerine, Butyrospermum parkii (Shea butter) and Aloe vera extract. If psoriasis lesions severely involved the scalp, the patient’s head was shaved before treatment. Treatment tubs The treatment tubs, made from food-safe plastic (Chemo®, Weinstadt, Germany) had a capacity of 1100 litres, filled to approximately 80% capacity. Between 250 to 400 fish were used, depending on the size and severity of the skin lesions. The bath tubs were equipped with an elaborate fresh water system, the technical details of which will be published elsewhere. The water in the tubs was constantly filtered and sterilized (700 litres per hour) by a filter pump and a UVC water sterilisation device and was simultaneously enriched with oxygen. The water was exchanged completely 3 to 4 times a day. A thermostat was used to maintain a comfortable water temperature between 36 and 37°C. 33 The fish, reared in an adjacent breeding facility, ranged from 5 to 10 cm in length (approx. 1.5 years old) when used for treatment. In contrast to the Kangal hotspring, where they lack nutrients due to the high temperatures of the spring, the fish were fed commercially available fish food (TetraMin®, Tetra GmbH, Melle, Germany) daily after the treatment sessions. Water samples were tested monthly for Legionella species and Pseudomonas aeruginosa. The fish were examined for Aeromonas hydrophila, A. sobria, A. caviae, Mycobacterium marinum and M. piscium to rule out any risk for potential zoonotic infections. Since the fish were given only commercially available fish food, the risk of Diphyllobothrium latum (fish tapeworm) infestation could be ruled out based on the absence of an intermediate host. Each patient was allocated to a single bathing tub for the duration of the three-week treatment, and no two patients shared the same tub at any time during that period. After the three-week treatment, each tub along with the technical equipment was disinfected (2% solution Dodarcana® Rapid, Schülke & Mayr, Austria) for 1 hour. Assessment Clinical assessment. The primary efficacy outcome measure was the overall total reduction in Psoriasis area severity index (PASI) score and the proportion of patients with 50% and 75% improvement in PASI score (PASI-50 and PASI-75) at week 3, relative to baseline. The PASI is a physician-accessed score, recognized by the US Food and Drug Administration to assess efficacy of psoriasis therapies in clinical trials. The PASI score takes into account the extent of involved skin surface and the severity of erythema, desquamation, and plaque induration. The composite score ranges from 0 to 72, with higher numbers indicating more severe disease and a reduction in score representing improvement. The PASI-75 is the currently recognized benchmark of end-points used in psoriasis clinical trials. PASI-50 is also regarded as a clinically significant endpoint in the assessment of psoriasis.7 PASI was assessed on highresolution digital colour photographs taken at baseline and at the end of the 34 three-week treatment period. Baseline measurements were those made closest but prior to the beginning of treatment. Additionally, response to treatment was defined according to the rate of improvement in PASI score, as follows: complete response, more than 95% improvement; good or marked, 75% to 94%; moderate, 50% to 74%; slight, 25% to 49%, and none, less than 25%. Patient-reported outcomes were evaluated by a short custom questionnaire immediately after the three-week course of treatment (Table 1) and by a follow-up questionnaire sent to all patients after treatment in March 2005 to assess the duration of remission, the number of different treatment regimens prior to ichthyotherapy, the severity of a possible relapse and the personal satisfaction with ichthyotherapy when compared to previous treatments (Tables 2&3). At this point the time elapsed since the end of therapy was between 3 months to almost 3 years. Safety evaluation. The safety and tolerability of the therapy were evaluated by reviewing adverse events (assessed weekly), vital signs and a weekly physical examination. Statistical analyses The primary endpoint (efficacy of treatment) was evaluated by comparing the mean PASI score before and after three weeks of treatment. The PASI scores were analysed using a Wilcoxon signed rank test for paired comparison. P values ≤ 0.05 were considered statistically significant. Analysis was carried out using Prism 4 for Macintosh (GraphPad Software, Inc. San Diego, California). 35 Results Patients’ Characteristics Sixty-seven patients, 39 male and 28 female, were included in this retrospective study. All 67 patients completed the three-week treatment. Ages ranged between 10 and 75 years (mean 41.01 years, 95% confidence interval, CI 37.51, 44.52). The mean duration of psoriasis at baseline was 13.9 years (range 1 to 35 years; 95% CI 11.6, 16.21). Treatment Efficacy Physician-Assessed outcomes. At the end of the three-week treatment course, 31 of the 67 patients (46.3%) achieved PASI-75 and 30 other patients (44.8%) achieved PASI-50. The mean PASI score at baseline for the entire study cohort was 18.9 ± 12.37 (95% CI 15.89, 21.9). PASI score at the end of the treatment period was 5.34 (95% CI 4.27, 6.42). Overall there was a 71,71% reduction in PASI score compared to baseline (p<0.0001) (Fig. 3). Response to treatment was complete in 3 patients (4.5%), marked in 29 (43.3%), moderate in 29 (43.3%), and slight in 6 (8.9%). No patient failed to respond at all. Patient-Reported outcomes. In the short questionnaire given immediately after the three weeks of treatment, patients reported substantial satisfaction with the treatment (Table 1). Of the 67 follow-up questionnaires sent to the patients in March 2005, 64 were deliverable. Forty questionnaires were returned, giving a response rate of 60%. The mean time since the end of treatment was 21.8 (95% CI 18.99, 24.68). The reported mean remission period was 8.58 months (range: 1 to 30 months; 95% CI 6.05, 11.11) with two patients (5.1%) still in remission at the time the questionnaire was received (time elapsed since the end of therapy was 12 and 26 months, respectively). The mean number of previously used other therapies was 5 ± 3 (± SD) (range: 0 to 12; 95% CI 4.02, 5.93) (Table 2). When asked to compare their treatment results to all previously used therapies, 87.5% of patients reported a more favourable outcome with ichthyotherapy. 65.1% stated that after the relapse their symptoms were less severe than compared to baseline (Table 3). 36 Safety evaluation, Side effects No severe side effects were recorded during the treatment period. Mild, transient bleeding from open crusted lesions was reported by one patient with eczema, and UV-radiation-related erythema by two others. No clinically significant pattern of changes in vital signs were observed during the study period. Ichthyotherapy in combination with UVA-treatment was generally very well tolerated. Discussion This retrospective study indicates that ichthyotherapy used in combination with short term UVA radiation is an effective and safe treatment for psoriasis vulgaris. 46.3% of the 67 patients achieved PASI-75 and an additional 44.8% patients achieved at least PASI-50 after a three-week course of treatment with the “doctorfish of Kangal” Garra rufa (Fig. 4). It is well established that a 75% improvement in PASI score is a clinically meaningful endpoint for clinical trials, and there is strong evidence demonstrating that a 50% improvement in PASI score is also associated with a significant improvement in the patients quality of life. 7 These findings are further supported by the results of the questionnaire completed by the patients immediately after treatment (Table 1). The results of the follow-up survey indicate that most of the patients were more satisfied with ichthyotherapy than with any other previously tried treatment. This satisfaction might be explained, at least in part, by the rather long reported mean remission period of 8.6 months. The response rate of the survey is comparable to those of other mail-based surveys.8 However, we are aware that this approach leaves room for potential nonresponse bias; patients with unfavourable results (i.e., early relapse) may have been less likely than others to respond to our survey. In this study it was also shown, that ichthyotherapy combined with UVA radiation was generally well tolerated with only three patients experiencing mild side effects. Two of these three patients showed UVA treatment related erythema. Whereas a prolonged UVA treatment course, or frequently repeated courses, may have an, as yet, undefined risk with regard to melanoma and ageing changes, a recent study suggests that UVB but not UVA radiation initiates melanoma.9 However, we are aware of the risks as identified by the British Photodermatology Group Workshop 10, and 37 therefore suggest to further evaluate ichthyotherapy in combination with broadband UVB and narrowband UVB treatment. Several mechanisms have been suggested regarding the observed efficacy of ichthyotherapy in Turkey.5 One obvious mechanism is the physical contact with the fish, which feed on the desquamating skin, thus leading to a rapid reduction of the scales. This phenomenon was also consistently observed in our patients, who reported a pleasing micro-massage like feeling while the fish nibbled at their skin. The fish seem to prefer affected to healthy skin possibly it is easier to nibble at this surface. Another suggested mechanism is the direct effect of natural ultraviolet radiation due to the high altitude (1650m) of the Kangal spa.5 Phototherapy is a well recognised option for patients with widespread psoriasis lesions with climatotherapy being the simplest form.2 However, a simultaneous removal of scales by the fish probably facilitates the penetration of UV rays to the dermis. This exposure of the lesions may explain the better outcome of combined ichtyotherapy/UVA-treatment when compared to the poor results of UVA sunbed treatment alone.11 A third suggested mechanism of ichthyotherapy in Kangal is the presence of a high level of selenium (1.3 mg/L) in the hot spring water.5 While Özcelik et al.5 argue, that selenium constitutes an important factor to treatment success in Kangal, an analysis of the water used in our study revealed a selenium concentration of less than 5 µg/L. Therefore selenium is not likely to have contributed to the observed efficacy of ichthyotherapy reported in the present study. A fourth mechanism suggested by Özcelik et al.5 is the reverse Koebner phenomenon. The reverse Koebner phenomenon is seen when an area of psoriasis clears following injury.12 However, it had been shown that destruction of the dermal papillae within the injured tissue plays the key role in preventing epidermal hyperplasia of psoriasis.13 Therefore it seems unlikely that the reverse Koebner phenomenon contributes to the observed efficacy of ichthyotherapy. Finally, psychological factors like stress are regarded a causal or exacerbating factor in psoriasis.1 Thus, the two hour daily fish bath, which most patients referred to as relaxing and pleasing, might have contributed to the observed treatment effect by reducing patients’ stress and enhancing psychological well-being. There are two important key differences between this study and the study by Özcelik et al.5 concerning treatment protocol. First, in our study the 38 patients were required to stay in the treatment tubs for only two hours per day, whereas the mean stay in the pools in Kangal was 7.4 ± 1.1 hours a day.5 This shortened treatment time makes ichtyotherapy more acceptable for the patients and considerably improves compliance. Second, at the treatment facility described herein, each patient was allocated a personal bathing tub, and the fish only came into contact with a single patient. Conversely, in the pools of the Kangal hot spring patients are required to take their bath with 10 to 20 patients simultaneously (personal observation MG). This approach might be unacceptable for some patients and a possible hygienic risk cannot be entirely excluded. The present study is limited by the relatively small number of patients treated and by lack of a control group. Randomized studies would be needed to compare the ichthyotherapy treatment with controls (e.g. water with the UV-therapy alone) and to assess treatment with standardized health related quality of life questionnaires. Therefore it is evident that many questions remain unanswered concerning the optimal protocols for ichthyotherapy. In the absence of studies to address these issues, we recommend that the described protocol may be used for guidance. In summary, the benefit demonstrated in this study, along with the favourable safety profile, suggests that ichthyotherapy combined with a short course UVA treatment could provide a viable treatment option for patients with psoriasis vulgaris. Maggot therapy - mode of actions and clinical practice Hasenöhrl K. M.D., Wollina U. Prof. Department of Dermatology, Dresden-Friedrichstadt Hospital, Germany Biosurgery has received increasing interest for the debridement of chronic wounds. Maggot therapy employs the use of freshly emerged, sterile larvae of the common green bottle fly, Lucilia sericata and is a form of artificially induced myasis in a controlled clinical situation. How maggots remove necrotic tissue from the wound is currently investigated. 39 There are several proposed mechanisms: removing of necrotic tissue by their secretion and mechanical effects by attacking the tissue with their mouthhooks. Maggots secretions contain a rich soup of digestive and proteolytic enzymes while feeding. They produce for instance carboxypeptidase A and B, leucine aminopeptidase, collagenase and trypsin-like and chymotrypsin-like serine proteases. Serine proteinases exhibited degradation of extracellular matrix in its components laminin, fibronectin and collagen types I and III. Both effects, mechanical action and the secretion of digestive enzymes seem to be the clue in efficient wound debridement. Despite great success in clinical use and technology of breeding and application the knowledge about larval changes in the wounds is limited. Therefore our clinic investigated larval morphology before and after feeding in the wound by histopathology. These results demonstrate the changes in larval morphology during biosurgery. After removing necrotic tissue larvae encouraged wound healing and formation of granulation tissue. The enhanced tissue oxygenation was measured by remittance spectroscopy. Our results showed an improvement of tissue oxygenation as revealed by the characteristic oxygen doublet peak. Beside direct promotion of granulation tissue maggots has been observed to combat infection. The majority of wounds hosts a variety of both anaerobic and aerobic bacteria. Bacteria are directly killed in the alimentary tract of maggots and they produce several antibacterial factors. Also maggots are able to kill clinical isolates of MRSA. Our lecture gives further a brief review of medical indications of biosurgery and the practical handling of maggots. It is used in wounds which have previously failed to respond to conventional treatment like chronic venous or mixed leg ulcers, traumatic wounds, pressure sores, diabetic foot ulcers and malignant tumours of skin. We provide clinical data from literature and our own experience. 40 Studies of the Apitox (Apitoxin) Christopher MoonHo Kim, M.D. Visiting Professor, Biomedical Center, CHA University, Korea President, International Pain Institute, USA (1) Pharmacology Written Summary of the Apitox 1. Brief Summary The pharmacology profile of bee venom has been determined in in vitro systems and in various animal models. The results from some of these animal studies are summarized in the following sections and show that Apitoxin has biological activity in animal models that are suggestive of potential efficacy for the treatment of refractory osteoarthritic pain and inflammation in humans. 2. Primary Pharmacodynamics Adjuvant-induced polyarthritis in the rat has been established as an experimental model for human rheumatoid arthritis.i Arthritic symptoms are induced by administering a single SC injection of 1 mg Mycobacterium butyricum (Freund’s adjuvant or CFA) suspended in mineral oil into a hind paw of the rat. Other studies, including those in rabbits, dogs, and monkeys, are also described. The ability of bee venom (dose range: 0.01 to 1.0 mg/kg/day) to suppress adjuvant-induced arthritis was studied in rats following daily SC administration over a period of 17 days. ii Bee venom suppressed the development of adjuvant arthritis in a dose-related manner. A single SC administration of bee venom also suppressed the development of carrageenan-induced paw edema and, when administered SC the day before or on the day of injection of adjuvant, effectively suppressed the development of polyarthritis. This suppressive effect decreased progressively as dosing was delayed. Bee venom was found to be most effective when mixed and injected together with CFA, the disease-inducing agent. Similarly, antigens such as egg albumin, when incorporated into CFA and injected into the hind paw, prevented the development of arthritis. These results suggest that at least two mechanisms are involved in the antiarthritic action of bee venom: (1) alteration of the immune response, most likely via antigen competition, and (2) an anti-inflammatory action. 41 One of the earliest studies was conducted to evaluate the use of bee venom prophylactically (beginning 2 weeks prior to adjuvant injection) and therapeutically (beginning 1 week after adjuvant injection) to reverse adjuvant-induced arthritis in the rat.iii Bee venom (1 and 4 mg/kg injected SC three times a week for 4 weeks) was shown to prevent the arthritic syndromes (foot volume, secondary lesions, and reduction in inflammation units) both as a therapeutic and as a prophylactic (more pronounced effect). The molecular mechanisms of the anti-inflammatory effects of bee venom were investigated in the rat model of carrageenan-induced acute edema in the paw and the rat model of chronic adjuvant-induced arthritis.iv Bee venom at 0.8 and 1.6 µg/kg administered daily for 14 days into the plantar surface of the right hind paw reduced hind paw edema. These animal results were consistent with in vitro data that showed an inhibitory effect of bee venom at 0.5, 1, and 5 µg/mL and melittin (a major component in bee venom) at 5 and 10 µg/mL on lipopolysaccharide-induced expression of cyclooxygenase 2, cytosolic phospholipase A2, inducible nitric oxide (NO) synthase, generation of prostaglandin E2 and NO, and the intracellular calcium level, providing information on the mechanism of action of anti-arthritic effects of bee venom. The effect of chromatographic fractions (designated as Oa, Op, and the protease inhibitor) and pure proteins and peptides (melittin, apamin, and phospholipase A2) from bee venom were tested in the rat models of arthritis and inflammatory edema. v In rats with adjuvant-induced arthritis, the components of venom (100 µg/kg once daily for 20 days) caused a 15-25% inhibition of arthritic symptoms whereas phospholipase stimulated arthritic symptoms by 30%. Rats with hind paw edema induced by CFA were injected SC with various fractionated bee venom components (10 µg/kg) 24 hours and/or 0.5 hour (n = 6/group) prior to administration of the inflammatory agent. The inhibition of the carrageenan and prostaglandin E1 inflammation under the effects of Oa and Op fractions and the protease inhibitor were the strongest. Rats (n = 4/group) with induced adjuvant disease (which includes a severe and persistent polyarthritis) were treated for 21 days with SC administered bee venom (300 or 500 µg or 1.2 and 2 mg/kg for a 250 g rat) twice a day; saline (control); bee venom intraperitoneal (IP); or melittin, apamin, or 42 phospholipase A2 SC (at the amount contained in 1 mg of bee venom).vi Rats treated SC with bee venom developed little or no arthritis with the higher dose being more effective than the lower dose while rats injected IP with bee venom or with SC melittin, apamin, or phospholipase A 2 had no apparent benefit on the polyarthritis. When SC bee venom injections were initiated after moderate to severe polyarthritis had developed, rats did not have the course of the disease altered. The ability of a daily administration of bee venom (2 mg/kg/day) to suppress adjuvant-induced arthritis in rats was studied over a period of 24 days. The bee venom treatment suppressed but did not abolish the primary and secondary inflammatory responses to the adjuvant as monitored by decreases in the swelling of the left (where the adjuvant was injected) and right hind paws, respectively.vii The response was delayed, with statistically significant suppressive effects being noted 2-3 weeks following the administration of the adjuvant. A sex difference in the suppressive effect of bee venom was noted, with female rats demonstrating a more pronounced beneficial effect than male rats. The dosage used in this study (2 mg/kg) was rather high, as compared to 50-70 µg of solids in the average honeybee sting.viii,ix In a study performed to assess the clinicotherapeutic effect of bee venom, 90 rats were administered bee venom (1 bee [about 0.1 mg bee venom or 0.4 mg/kg for a 250 g rat], SC), prednisolone (10 mg/kg, orally), or saline control (0.1 mL, SC) every other day over a period of 14 days.x Clinical findings of lameness score, edema volume, hematological values, and histopathology (interphalangeal joint of the right hind paw) were observed during the treatment period. In the treatment groups, the development of inflammatory edema and polyarthritis was suppressed. No significant differences of hind paw edema volume and lameness score between the prednisolone and bee venom groups were observed during treatment. No differences in red blood cell count, hematocrit, or hemoglobin concentration were noted between the groups although significant leucocytosis was observed in the control group (p < 0.01). Erosions of articular cartilage and inflammatory cell infiltrations into the interphalangeal joint were effectively suppressed in treated groups. Thus, whole honeybee venom was found to suppress arthritic inflammation in the 43 rat. A study was conducted to determine whether some of the in vivo effects of bee venom may be mediated by alterations in lymphokine production and to observe the in vitro effect of lymphokines on reduced mitogenic responses of bee venom-treated rats.xi When 0.5 mg/kg/day bee venom was administered intramuscularly (IM) to rats over a period of 17 days, a reduction in interleukin (IL) production by splenocytes was observed. In vitro addition of IL-1 or IL-2 to the cultures resulted in an increase in responses to normal levels, suggesting that bee venom affects the production of IL-1 by macrophages. This finding suggests that the modifying effects by bee venom on inflammatory responses in local therapy may be due to interference with certain functions of inflammatory cells. In an attempt to explain the mechanism of bee venom’s anti-inflammatory action, the effect of bee venom on neutrophil O2- production was investigated. xii Neutrophil production of toxic oxygen radicals and metabolites are thought to play a role in chronic inflammation and tissue destruction in a wide variety of diseases. Using human peripheral blood leukocytes, the polymorphonuclear fraction was isolated and used in in vitro assessments on the ability of melittin and other bee venom peptides to affect the production of O2-. The results showed that melittin, but not other bee venom fractions, inhibited O2- production both pre- and poststimulation, suggesting the melittin may have a role in the in vivo regulation of radical production and suggesting a possible mechanism of action for this major bee venom component. The effect of bee venom on inflammatory cell function was studied following injection of immune complexes into rabbit knee-joints. xiii Treatment with single SC injections of bee venom (1.2 to 20 µg/kg) significantly reduced the leukocyte counts in the immune complex challenged joints as compared to untreated rabbits (p < 0.02). Bee venom at a dose of 12 µg/kg decreased leukocyte counts 3 and 6 hours after challenge with immune complexes but this effect was not noted at 9 hours. Significant effects on leukocyte random migration, chemotactic responsiveness, or phagocytosis were not observed, indicating that bee venom did not interfere with normal phagocyte motility and ingestion. The 44 modifying effects by bee venom on the inflammatory response to immune complexes in vivo was, therefore, thought to be most likely due to interference with other components of the inflammatory response. In a study designed to investigate the possible role of the soluble fraction of bee venom in producing the anti-arthritic actions of bee venom acupuncture, whole bee venom was extracted into two fractions according to solubility: a water soluble fraction (BVA) and an ethylacetate soluble fraction (BVE). xiv Subcutaneous BVA injection (0.9 mg/kg/day) into the Zusanli acupoint was found to dramatically inhibit paw edema and radiological change (i.e. new bone proliferation and soft tissue swelling) caused by CFA injection. The BVA treatment also reduced the serum IL-6 increase caused by rheumatoid arthritis induction to levels observed in the non-arthritic animals. In addition, BVA therapy significantly reduced arthritis-induced nociceptive behaviors (i.e. nociceptive scores for mechanical hyperalgesia and thermal hyperalgesia). Finally, BVA treatment significantly suppressed adjuvantinduced Fos expression in the lumbar spinal cord at 3 weeks post-adjuvant injection. In contrast, BVE treatment (0.05 mg/kg/day) failed to show any anti-inflammatory or antinociceptive effects on rheumatoid arthritis. These results demonstrate that BVA is the effective fraction of whole bee venom responsible for the antinociception and anti-inflammatory effects of bee venom acupuncture treatment. Further study is necessary to clarify which constituents of the BVA fraction are directly responsible for these antiarthritis effects. Previous studies in experimental animals suggested that the therapeutic effect of bee venom on arthritis is dependent on the site of administration. In particular, local injection of bee venom near the site of inflammation, such as the hind limb in the rat model, is more effective in inhibiting the development of adjuvant-induced arthritis than injections into a more distant site (e.g. on the back).vii,ix Because of this potential site specificity, a study was designed to evaluate the anti-nociceptive effect of bee venom injections into a specific acupoint (Zusanli) compared to a non-acupoint in the rat model of chronic arthritis.xv Subcutaneous bee venom treatment (1 mg/kg per day) was found to dramatically inhibit paw edema caused by CFA injection and significantly reduced arthritis-induced nociceptive behaviors (i.e. the nociceptive scores for mechanical hyperalgesia and 45 thermal hyperalgesia). These anti-nociceptive/anti-inflammatory effects of bee venom were observed from 12 days through 21 days post-bee venom treatment. In addition, bee venom treatment significantly suppressed adjuvant-induced Fos expression in the lumbar spinal cord at 3 weeks postadjuvant injection. Finally, injection of bee venom into the Zusanli acupoint resulted in a significantly greater analgesic effect on arthritic pain as compared to bee venom injection in to a more distant non-acupoint. The study demonstrated that bee venom injection into the Zusanli acupoint has both anti-inflammatory and anti-nociceptive effects on CFA-induced arthritis in rats. These findings suggest that bee venom acupuncture may be an effective therapy for the treatment of rheumatoid arthritis. The antinociceptive and anti-inflammatory effects of bee venom pretreatment on carrageenan-induced inflammation in the rat was studied in order to determine if bee venom, which is nociceptive under normal conditions, serves as an anti-inflammatory and/or antinociceptive agent under conditions of inflammation.xvi Rats were injected SC with bee venom at 0.8 mg/kg (n = 8) or 0.08 mg/kg (n = 6) 30 minutes prior to carrageenan-induced acute paw and thermal hyperalgesia. At 0.8 mg/kg, bee venom significantly suppressed carrageenan-induced edematous paw volume (p < 0.001) and thermal hyperalgesia (p < 0.01) and induced expression of Fos positive neurons in the spinal cord (p < 0.01). These results suggested that bee venom may be useful in the treatment of pain and edema associated with chronic inflammatory diseases. The effect of bee venom on cortisol levels and increase in activity in dogs with hip dysplasia was investigated.xvii The 24 dogs, 8 of which had hip dysplasia and 16 of which were normal, were divided into 4 treatment groups. Groups I and II included 8 normal dogs each and Groups III and IV each had 4 dogs with hip dysplasia. Groups II and IV received 1 mg (about 0.067 mg/kg for a 15 kg dog) of bee venom SC on Days 30, 37, 50, and 60 and then were crossed over to received saline control treatment on Days 90, 97, 110, and 120. Groups I and III began with saline control treatment and then were crossed over to the bee venom treatment. Dogs receiving bee venom injections had increased plasma cortisol levels and arthritic dogs had increased daily cage activity. On day 90, the treatments were crossedover and again dogs receiving bee venom had increased plasma cortisol 46 levels and arthritic dogs had increased daily cage activity. The results suggest that bee venom stimulates the production of cortisol and enhances the cage activity of arthritic dogs. The effect of whole bee venom on plasma cortisol levels was investigated in the unanesthetized monkey following single SC injections of bee venom (1.0 to 100 mg) or melittin (1.0 to 10 mg).xviii Both bee venom and melittin injections produced marked and sustained elevations in plasma cortisol levels. These increases occurred at approximately 1 hour following SC injection and lasted for 2-4 days. The effect appeared to be dose-related with the higher doses of bee venom or melittin producing the earliest and most pronounced plasma cortisol elevations. When a second dose of bee venom (1 mg) and melittin (0.1 mg) was administered to 1 monkey each at 72 or 96 hours, respectively, there was an immediate and sustained rise in cortisol, which lasted for 20 to 30 days. Melittin appeared to be 10 times more potent than bee venom. Necropsy results from 4 monkeys receiving the highest doses of bee venom or melittin indicated no significant gross or microscopic tissue changes. Surgical removal of the pituitary gland from 4 monkeys prevented the effect, indicating that bee venom and melittin may be stimulating the production of cortisol from the adrenal gland, which may provide an explanation for the beneficial effects of bee venom therapy in a variety of disease conditions that respond to adrenal steroid therapy. Using HTB-94 human chondrosarcoma cells, gene expression profiles were determined following treatment with bee venom, lipopolysaccharide (LPS), or both.xix Of 344 genes profiled, 35 were down regulated by bee venom, 16 were up regulated and 7 down regulated by LPS, and 32 were down regulated by bee venom and LPS combined. Bee venom reversed the upregulation caused by LPS for some genes, such as the IL-6 receptor, matrix metalloproteinase-15 (MMP-15), tumor necrosis factor, superfamily-10, caspase-6, and tissue inhibitor of metalloproteinase-1 (TIMP-1). These results provided information for understanding the pharmacologic activity of bee venom for the treatment of arthritis. 3. Secondary Pharmacodynamics 1) General Pain In a study designed to evaluate the potential antinociceptive effect of bee venom pretreatment on formalin-induced pain behavior and its associated 47 spinal cord Fos expression, rats were administered a single SC injection of bee venom (dose range: 0.0016 mg/kg to 0.08 mg/kg) or saline control into the Zusanli acupoint (5 mm lower and lateral to the anterior tubercle of the tibia).xx Pretreatment with bee venom significantly decreased paw-licking time in the late phase of the formalin test. In contrast, bee venom injected into a non-acupoint in the back region did not suppress the paw-licking time. Bee venom pretreatment into the Zusanli acupoint markedly inhibited spinal cord Fos expression induced by formalin injection. These findings indicate that bee venom pretreatment into the Zusanli acupoint has an antinociceptive effect on formalin-induced pain behavior. In a follow-up study to the one describe above, an abdominal stretch assay in mice and rats and formalin test in rats were used to further investigate bee venom’s antinociception effect.xxi Bee venom was administered SC to the mice at a 1 to 100 or 1 to 1000 dilution and to rats at a 1 to 1000 dilution in 20 µL of saline. Bee venom at the 1 to 100 dilution into an acupoint or non-acupoint produced antinociceptive effects while bee venom at the 1 to 1000 dilution was effective only when dosed into an acupoint, indicating that bee venom may be a promising method for the relief of pain. 2) X-Irradiation Protection The response of animals to whole-body X-irradiation in the lethal range can be modified by certain changes in their physiological state if induced prior to exposure. The ability of bee venom to produce a degree of physiological stress in animals, thereby eliciting a neuroendocrine response (pituitaryadrenal stimulation) that would increase radiation resistance, was studied in mice.xxii In a series of experiments, pre-treatment with bee venom (IP dose range: 1.1 to 1.24 µg; SC dose range: 4.3 to 5.6 µg) resulted in greater 30day survival rates as compared to a saline control. Bee venom administered SC afforded the greatest protection (70-80% 30-day survival rate). When melittin, a major component of bee venom, was separated and injected SC at 5.4 µg, the 30-day survival rate was 7%. Based on these results it was proposed that at least 3 mechanisms of action may account for the radioprotective effect of bee venom in mice: 1) it has a stressor-like action that elicits an “adaptation syndrome,” 2) it produces changes in the hematopoietic system, or 3) it has antibacterial properties. In a separate study, melittin provided statistically significant x-irradiation 48 protection to mice that received an SC injection doses up to 60 mg/kg 24 hours prior to the irradiation. xxiii Whole bee venom did not show conclusive evidence for the same protection. Pearson CM, Wood FD. Studies of arthritis and other lesions induced in rats by the injection of mycobacterial adjuvant. VII. Pathologic details of the arthritis and spondylitis. Am J Pathol. 1963 Jan;42:73-95. Chang YH, Bliven ML. Anti-arthritic effect of bee venom. Agents Actions. 1979 Jun; 9(2):205-211. Lorenzetti OJ, Fortenberry B, Busby E. Influence of bee venom in the adjuvant-induced arthritic rat model. Res Commun Chem Pathol Pharmacol. 1972 Sep;4(2):339-52. Park HJ, Lee SH, Son DJ, Oh KW, Kim KH, Song HS, Kim GJ, Oh GT, Yoon do Y, Hong JT. Antiarthritic effect of bee venom: inhibition of inflammation mediator generation by suppression of NF-kappaB through interaction with the p50 subunit. Arthritis Rheum. 2004 Nov;50(11):3504-15. Shkenderov S. New pharmacobiochemical data on the anti-inflammatory effect of bee venom. Animal, Plant, and Microbial Toxins. 1976;2:319-336. Zurier RB, Mitnick H, Bloomgarden D, Weissmann G. Effect of bee venom on experimental arthritis. Ann Rheum Dis. 1973;32:466-70. Eiseman JL, von Bredow J, Alvares AP. Effect of honeybee (Apis mellifera) venom on the course of adjuvant-induced arthritis and depression of drug metabolism in the rat. Biochem Pharmacol. 1982 Mar 15;31(6):1139-46. Lichtenstein LM, Valentine MD, Sobotka AK. A case for venom treatment in anaphylactic sensitivity to hymenoptera sting. N Engl J Med. 1974 May 30;290(22):1223-7. Hadjipetrou-Kourounakis, Yiangou M. Bee venom and adjuvant induced disease. J Rheumatol. 1984 Oct;11(5):720. Kang SS, Pak SC, Choi SH. The effect of whole bee venom on arthritis. Am J Chin Med. 2002;30(1):73-80. Hadjipetrou-Kourounakis, Yiangou M. Bee venom, adjuvant induced disease. J Rheumatol. 1988 Jul;15(7):1126-8. Somerfield SD, Stach JL, Mraz C, Gervais F, Skamene E. Bee venom melittin blocks neutrophil O2- production. Inflammation. 1986;10(2):175-80. Thomsen P, Bjurtsten LM, Ahlstedt S, Bagge U, Bjorksten B. Inhibitory effect of bee venom on immune complex mediated leukocyte migration into rabbit knee-joints. Agents and Actions. 1984; (14(5/6):662-66. 49 Kwon YB, Lee HJ, Han HJ, Mar WC, Kang SK, Yoon OB, Beitz AJ, Lee JH. The water-soluble fraction of bee venom produces antinociceptive and anti-inflammatory effects on rheumatoid arthritis in rats. Life Sci. 2002 May 31; 71(2):191-204. Kwon YB, Lee JD, Lee HJ, Han HJ, Mar WC, Kang SK, Beitz AJ, Lee JH. Bee venom injection into an acupuncture point reduces arthritis associated edema and nociceptive responses. Pain. 2001 Feb 15;90(3):271-80. Lee JH, Kwon YB, Han HJ, Mar WC, Lee HJ, Yang IS, Beitz AJ, Kang SK. Bee venom pretreatment has both an antinociceptive and anti-inflammatory effect on carrageenan-induced inflammation. J Vet Med Sci. 2001;63(3):251-59. Vick JA, Warren GB, Brooks RB. The effect of treatment with whole bee venom on daily cage activity and plasma cortisol levels in the arthritic dog. Inflammation. 1975-1976;1(2):167-74. Vick JA, Mehlman B, Brooks R, Phillips SJ, Shipman W. Effect of bee venom and melittin on plasma cortisol in the unanesthetized monkey. Toxicon. 1972;10:581-6. Yin CS, Lee HJ, Hong SJ, Chung JH, Koh HG. Microarray analysis of gene expression in chondrosarcoma cells treated with bee venom. Toxicon. 2005;45:81-91. Kim HW, Kwon YB, Ham TW, Roh DH, Yoon SY, Lee HJ, Han HJ, Yang IS, Beitz AJ, Lee JH. Acupoint stimulation using bee venom attenuates formalin-induced pain behavior and spinal cord expression in rats. J Vet Med Sci. 2003;65(3):349-55. Kwon YB, Kang MS, Kim HW, Ham TW, Yim YK, Jeong SH, Park DS, Choi DY, Han HJ, Beitz AJ, Lee JH. Antinociceptive effects of bee venom acupuncture (apipuncture) in rodent animal models: a comparative study of acupoint versus non-acupoint stimulation. Acupunct Electrother Res. 2001;26:59-68. Shipman WH, Cole JL. Increased resistance of mice to X-irradiation after the injection of bee venom. Nature. 1967;215:311-12. Ginsberg NJ, Dauer M, Slotta KH. Melittin used as a protective agent against X-irradiation. Nature. 1968 Dec;220:1334. 50 (2) PREVIOUS HUMAN EXPERIENCES OF APITOX Several studies have been conducted with Apitox(Apitoxin), including a Phase 3 clinical trial that formed the basis of the marketing approval of Apitox in Korea. These studies are summarized below. 1. Studies in Healthy Normal Human Volunteers Fourteen healthy male (10) and female (4) subjects between the ages of 26 and 52 years of age were enrolled in and completed this study. Among the male subjects, 3 were beekeepers who were included in order to obtain data from the maximum end of the spectrum without having to administered venom in high dosages (they received 10-350 stings per week during the course of their normal work). Injections were administered subcutaneously twice weekly, with each dose being dependent upon tolerance to the previous dose. All subjects were administered a maximum dose of 0.07 mg of Apitox and maintained on that dose for a week before continuing with an arthritic injection schedule. Each subject received 13 doses of Apitox according to the following schedule: Concentration per Injection Injection # 1 2 3 4 5 6 7 8 9 10 11 12 13 1× 1× 2× 2× 3× 3× 4× 4× 5× 5× 5× 5× 5× 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 + 0.05 + 0.05 + 0.05 + 0.05 Total Dose (mg) 0.07 0.12 0.14 0.19 0.21 0.26 0.28 0.33 0.35 0.35 0.35 0.35 0.35 Skin tests, vital sign measurements, and blood and urine laboratory evaluations were performed. One subject who became ill and was 51 subsequently hospitalized was shown to have contracted an adenovirus infection. A female subject had a delayed local reaction: 18 hours after injection, she developed a 4+ wheal with a very large flare. A repeat injection of the same concentration and all subsequent injections were normal. None of the subjects had a systemic reaction and there were no changes noted in vital sign measurements. None of the subjects reported significant pain. In a second study, 20 healthy (10 male, 10 female) subjects between the ages of 23 and 45 years of age were enrolled and 15 completed the study. Five subjects dropped out of study because of failure to keep study appointments, not following study directions, or due to consumption of alcohol during the study. Each subject received an initial test dose of 0.05 mL and then 12 doses of Apitox, starting with 0.1 mL with the first intradermal injection and increasing to 0.2 mL (second injection), 0.25 mL (third injection), 0.3 to 0.7 mL (fourth through twelfth injections). Injections were administered 2 to 3 times per week over a period of 4 to 6 weeks. Physical examination, blood and urine laboratory evaluations, and vital sign measurements were performed. There were no significant changes from baseline noted. Localized itching was the most common adverse experience (11/15). Edema (5/15), pain at injection site (2/15), and blister at injection site (1/15) were also reported, but no serious adverse experiences were reported. Thus, it was concluded that Apitox can be safely administered to humans when applied in therapeutic doses. 2. Studies in Patient Populations Studies evaluating the safety and efficacy of Apitox have been conducted in patients with chronic pain and inflammation (due to rheumatoid arthritis, osteoarthritis, fibromyositis, or peripheral neuritis) and those with osteoarthritis. 1) Chronic Pain and Inflammation Study (United States) In the chronic pain and inflammation study, 180 subjects were randomized to receive, over a period of 6 weeks, twice weekly injections of either Apitox (1 mg/mL) or histamine phosphate (0.275 mg/mL). The number of injections increased with each subsequent visit (e.g. 3, 6, 9, 12, 15, 18, 52 20, 20, 20, 20, 20, and 20 injections). The injections were administered to the area of pain first and then beginning with the fifth session, as the number of injections increased, to the corresponding dermatomal area of the spine. The visual analogue scale (VAS) and McGill Pain Questionnaire (MPQ) were used to assess the level of pain. Thermographic evaluations and physical examinations (to evaluate swelling, tenderness, and range of motion limitations) also were performed. Both groups had reductions in pain scores following treatment, with the Apitoxin treatment group demonstrating a greater improvement (pain scores: control 57 vs. Apitox 18). In addition, there were significant differences between control and the Apitox treatment group at the 6-month follow-up visit (pain scores: control 83 vs. Apitox 29). The Apitoxin treatment group also demonstrated greater improvement in the physical examination and thermographic findings. 2) Pain and Inflammation of the Osteoarthritis Study (Korea) This was a randomized, active-controlled (nabumetone) study in which 101 subjects with osteoarthritis of the knee or spine were given twice weekly injections of Apitox (maximum doses of 0.7 mg [Group A], 1.5 mg [Group B], or 2.0 mg [Group C]) or an oral once daily dose of the control (1000 mg nabumetone, Group D) over a period of 6 weeks. A 4-point Likert-like symptom severity rating scale was used to assess pain, disability, and physical signs. A 5-point scale was used for subject self-evaluation. Safety was assessed through observation of adverse experiences and blood and urine laboratory measurements. A total of 81 subjects completed the study. Those subjects assigned to an Apitox treatment group demonstrated a statistically significant greater improvement than those in the nabumetone group (p < 0.01). Within the Apitox groups, Groups B and C demonstrated greater improvements than Group A (p < 0.01). The most common adverse experiences reported were injection site itching and generalized body aches. 3) Other Experience (Korea) Following approval of Apitox in Korea, a post-marketing survey was conducted in November 2005. Included in this survey were 1,596 patients who received Apitox therapy. 53 Patients, without any compensation, voluntarily filled out the survey forms. The information collected included personal information, present illness, past history, and present medications. The physicians recorded the treatment records, including the diagnosis, treatment dates for 12+ sessions, doses, and adverse experiences (including a detailed description). A complete blood count was performed before treatment and after the last treatment. The physician immediately notified the pharmaceutical company and Korean FDA in the event of a serious adverse experience. According to the survey, no major adverse experiences were reported. In addition to this survey, The Pain Center, PC University Medical Center located in Korea has documented the use of Apitox in 3, 679 intractable medical condition and autoimmune disease patients between September 2003 and November 2005. No major adverse experiences were reported. Minor adverse experiences included itching (injection site), swelling (injection site), pain, low-grade fever, flushing, headache, and diarrhea. Antibacterial Properties of Whole Body Extracts and Haemolymph of Maggots of Lucilia sericata Kosta Y. Mumcuoglu1, Lea Huberman2, Nathan Gollop2, Colin Bloch3 & Rachel Galun1 1 Department of Parasitology, Hebrew University-Hadassah Medical School, Jerusalem, Israel; 2Department of Food Sciences, The Volcani Center, ARO, Beit Dagan, Israel; 3Clinical Microbiology Unit, Hadassah-Hebrew University Medical Centre, Jerusalem, Israel The aim of this study was to partially characterize maggot-secreted antibacterial substances and determine their range of activity against different bacteria. Sterile and non-sterile maggots maintained in the laboratory and taken from wounds of treated patients were used. Whole body extracts and haemolymph was fractionated and their range of activity 54 against bacteria was tested by the zone inhibition assay. The mode of action of bacterial destruction was examined by viable counts, influx of K+, changes in the membrane potential by scanning electron microscope (SEM). Extracts of sterile and maggots non-sterile showed an activity of 200 arbitrary units (AU)/ml and 400AU/ml respectively. Maggots removed from chronic wounds had an activity of 1200AU/ml. Injuring sterile maggots with a sterile needle doubled the antibacterial activity within 24 hours, while the antibacterial activity of haemolymph increased fourfold after injuring with a sterile needle and sixteenfold with an infected needle. The fractions with a molecular weight of <1kDa and 3–10kDa showed antibacterial activity against Gram-positive and Gram-negative bacteria including Pseudomonas aeruginosa, Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA) isolated from wounds. The fraction with a MW <1kDa lysed over 90% of the bacteria within 15 minutes by causing an influx of K+ and changing the membrane potential of bacteria. The nature of the antibacterial materials extracted from maggots indicates their potential significance in wound healing in addition to the actual ingestion of the necrotic tissue on the wound. The Use of the Medicinal Leech, Hirudo medicinalis, in the Reconstructive Plastic Surgery Kosta Y. Mumcuoglu1, Rivka Cohen1, Andre Ofek2, Howard Lipton2 and Carole Pidhorz2 1 Department of Parasitology, Hebrew University-Hadassah Medical School, Jerusalem, Israel 2 Department of Plastic Surgery, Hadassah Medical Center, Jerusalem, Israel The medicinal leech, Hirudo medicinalis, is being used to salvage compromised microvascular free-tissue transfers, replanted digits, ears, lips and nasal tips due to venous congestion. Twenty-three patients (14 males and 9 females), 8-79 years old average: 35.9 years) with devascularized or amputated fingers, open wounds after accidents or surgical wounds after scar revision were included in the study. Of the 15 fingers treated by leech 55 therapy, 10 fingers were saved (4 out of 9 replanted fingers and 6 out of 6 revascularized fingers), while out of 18 flaps treated by this treatment modality, 17 were salvaged (3 out of 4 free flaps and all 14 island and random flaps). Fifteen patients received 1-13 units of packed blood cells (average 2.9). The patients with revascularized or replanted fingers were treated in average of 2.5 days and each finger was treated with an average of 5.7 leeches. The 15 patients with flaps were treated in average of 3.4 days and each flap was treated with an average of 9.2 leeches. In conclusion, leech therapy should be considered as an integral part of the armamentarium used in reconstructive surgery. It improves greatly the success rate of the surgery in cases of post-operative venous congestions, allowing blood drainage until angiogenesis is established. Elimination of Symbiotic Bacteria from the Intestinal Tract of the Medicinal Leech, Hirudo medicinalis Kosta Y. Mumcuoglu1, Colin Block2, Lea Huberman1, Rivka Cohen1, Violetta Temper2 & Rachel Galun1 1 Department of Parasitology, Hebrew University-Hadassah Medical School, Jerusalem, Israel and 2Department of Clinical Microbiology and Infectious Diseases, Hadassah-Hebrew University Medical Centre, Jerusalem, Israel In recent years, recognition of a number of antithrombotic substances, a vasodilator and other factors in the saliva of the medicinal leech, Hirudo medicinalis, has generated renewed interest in its use in promoting venous drainage in tissues whose vitality is threatened by venous congestion and obstruction, especially in plastic and reconstructive surgery. Hirudotherapy has been complicated by infections, occasionally of extreme severity, caused by Aeromonas spp., which are considered endosymbionts of the leech. Therefore, antibiotics such as cephalosporins or fluoroquinolones are given prophylactically during treatment. Up till now, methods of eliminating these bacteria prior to treatment have not proved successful. The purpose of this study was to use a unique method of inducing leeches to ingest a blood-free antibiotic solution in an attempt to eliminate the 56 aeromonads, thus possibly eliminating the need for chemoprophylaxis. Eighty control leeches were examined bacteriologically for colonization by aeromonads at 1-2 week intervals before the definitive experiment. Cultures were taken from the external surface and then by dissection from the crop and intestine. Test animals were fed in an artificial feeding chamber using an arginine solution in physiological saline as a phagostimulant: 63 received the solution with ciprofloxacin 100μg/mL; 54 of these were given blood meals at intervals after treatment, to evaluate whether the drug influenced their ability to take blood; 7 controls were not given blood. After treatment, leeches were held in sterilized chlorine-free tap water containing 20μg/mL ciprofloxacin. Aeromonas spp. were grown from 57/80 control leeches (71.25%). Readiness to take a blood meal was unaffected in treated animals. All 54 treated animals that were subsequently given blood meals were free of detectable aeromonads when cultured at intervals from 1 week to 2 months. All 7 treated leeches that were not fed blood meals were still alive after 2 months. Other environmental bacteria and some filamentous fungi were isolated from 28.6% of treated animals that had taken blood meals, but were not fully characterized in this phase of the study. These preliminary results indicate that our strategy reduced the population of leech-associated aeromonads to undetectable levels for extended periods, and that treated leeches are likely to take a blood meal from patients undergoing hirudotherapy. This might permit the avoidance of antimicrobial prophylaxis in these patients. Outstanding questions to be resolved are, among others: 1) the maximum time leeches can survive with undetectable quantities of their aeromonad symbionts, 2) the amount of time that will be needed between treating the leeches and their becoming ready to take a blood meal, 3) the nature and potential clinical significance of the microbial population remaining after the elimination of the aeromonads and 4) the true role of these organisms in leech biology. 57 58 59 60 61 Autoblood from medicinal leeches as a means of active nonspecific 62 immunotherapy in oncology V.A. Savinov and O.A. Kursakova Moscow Alliance of Hirudotherapeutists, Russia Autoblood from medicinal leech contains symbiotic bacterium Aeromonas hydrophila which is a nonanamnestic antigen for man and is therefore used with the purpose of active nonspecific/adjuvant immunotherapy. Autoblood diluted 1:10 with normal saline is administered intradermally in the amount of 0.1 mL, or 0.5 mL of the native autoblood – subcutaneously, or 1.0 mL of autoblood diluted in 100.0 mL of normal saline – intravenously in droplets. Intracutaneous administration is accompanied by development of the delayed-type hypersensitivity reaction with the formation of a macrophagal-lymphocytic infiltration, while subcutaneous administration is followed by fever and hyperdermia up to 41°C during 5 to 6 hours with no subsequent negative aftermath, which is related to invasion of the human body with the symbiotic bacterium. From 1992 to 2002, in the complex with the standard therapeutic methods autoblood from medicinal leech was used for treatment of 60 patients with morphologically verified diagnosis: 23 – prostatic cancer, 16 – urinary bladder cancer, 14 – breast cancer, 5 – lymphgranulomatosis, 2 – colorectal cancer. Improved quality of life and elevated level of the immune status were noted. Maggot Therapy - Reflections from the Past, Foretelling the Future Ronald A. Sherman, M.D. University of California, U.S.A Purpose To explore the history and current status of maggot therapy world-wide, in order to predict and better guide its future. Methods 63 Review of published and unpublished records of maggot therapy use, research, and regulatory intervention. Results Several trends were identified: Within the past 12 years, the use of maggot therapy has spread throughout the world. Some communities have adopted maggot therapy more readily than others. In most countries where it is used, it is now accepted, if not embraced, by the medical establishment. Factors inhibiting more widespread use include concerns about cost recovery, ready access, hassles associated with dressing application, emotional distress of patients, and lack of scientific studies. Some of these concerns are legitimate impediments; others are groundless. All must be addressed and overcome. Conclusions Some communities have adopted maggot therapy more quickly and completely than others. Understanding the reasons for such trends may help us to overcome the impediments to access. Frequently asked Questions in Apitherapy Dr. Stefan Stangaciu President of “Apitherapy Consulting & Trading International” President of the German Apitherapy Society • What are the best bee products and the best Apitherapy protocols to improve health, beauty and strength? The best bee products for us are the ones that clearly can improve our health, strength or beauty. To know how to choose the best bee products we need to follow the following guidelines: * the best bee products are the fresh ones, collected and processed in the areas where we live; these products can usually ensure the maximum of nutrients and active compounds that our body needs; if the local bee products are not available in enough amount, we can order other ones, but ideally also from our region; the reason is that the bee products are collected by the bees from plants and trees that are perfectly adapted to 64 the climatic conditions where we also live; the plants that lives in the mountains secretes more bio-flavonoids than the plants that are at the sea level, due to the higher radiation level in the mountains; as a result, the honey and propolis made in the mountains protects better the people that lives also in the mountains area, than the products made/collected at the sea level; same kind of example is valid also for the people suffering from pollen allergies (hay fever); if the bee pollen is collected from the same area where the patient lives, and administered in very small doses at the beginning, with slow increase of the administered amount, after meals, the chance to desensitize the patient of that allergy is higher; the explanation is in the fact that the pollen allergens that are present in the atmosphere of that city/area will be present for sure, in very small amounts, in the bee pollen collected by the bees from the same area too, but with greater probability will be not present in same amount in the bee pollen collected more than 30 Km. away * once we decided to use the local bee products and we can get them in enough amount, we need to ask our local medical doctors and pharmacists what are the best administration forms to use. Here the doctors will need to ask the local pharmacists to prepare specifically for each patient (if necessary) the exact preparation/product that can reach faster the affected area. If we have conjunctivitis (eye inflammation) for example, we should use mainly eye drops and not suppositories… Here below we insert a table that can orient you in asking your local doctors/pharmacists for a more specific administration method: System/Organ/Tissue to be treated Nervous system Best pharmacological forms Administration method Oral forms: fresh collected Per oral (products that can bee products, or at least fresh frozen, in any form that can be swallowed (honey-pollen mixtures, tablets, capsules, powder, granules, tincture, syrups, in form of liquids, juices, 65 be swallowed). Injectable bee venom solution and/or live bee stings applied on the painful spots and/or on the acupuncture points/meridians. etc.); bee venom in solution form for injection on acupuncture points; ointments and/or creams to stimulate the blood flow in the head and vertebral column area Endocrine system Same as above. The Same as above, but the preparations that can be acupuncture points to be kept under the tongue for stimulated will differ minimum 4-5 minutes according with the location should be preferred of the endocrine gland that is currently treated Immune system Face Same as above + Propolis Same as above, but based suppositories acupuncture points to stimulated will specifically elected by specialist in apipuncture Creams, ointments and/or Local application following masks with all bee products + oral forms + bee venom solutions for micro-apipuncture Eyes the be be the the cosmetic related rules, ideally on the facial related acupuncture points; swallowing the oral forms after chewing and/or sucking of the freshly collected (frozen) raw bee products Eye drops + ointments and Eye drops creams + oral conjunctival in sac the + pharmacological forms that ointments and creams that can be swallowed can be applied on the eye lids and on the surrounding acupuncture points + oral forms that can be swallowed Nose Nose drops + solutions for Dripping the nose drops 66 inhalations with honey, propolis extract and essential oils + sunflower or olive oil based propolishoney mixtures + ointments + oral forms + bee venom solution solution in small amount + inhaling the vapours from the solution specially made for inhalation + superficial inserting the creamy-oily mixtures in the nostrils (not too deep) + stimulation of the local acupuncture points with ointments/creams and/or micro-injectable bee venom for microapipuncture + swallowing the oral forms (ideally, except the capsules, after sucking them for minimum 3 minutes) Ears Sunflower or olive oil inserting gently the creamybased propolis-honey oily mixtures in the external mixtures + solutions for ears entrance, in very small inhalations with honey, propolis extract and essential oils + ointments/creams + oral forms + solutions bee venom solution + ear candles amount + stimulation of the local acupuncture points with ointments/creams and/or micro-injectable bee venom for microapipuncture + inhaling the vapours from the solution specially made for inhalation + swallowing the oral forms (ideally, except the capsules, after sucking them for minimum 3 minutes) + specific application of beeswax ear candles Mouth Various honey-pollen- Local application inside the propolis-royal jelly mouth + swallowing of the 67 mixtures + water extract of propolis + propolis tincture + chewing-gum + tooth paste with propolis + oral forms + bee venom solution Teeth oral forms + acupressure and/or micro-apipuncture on the local bioactive points + injectable bee venom on distant acupuncture points that are related energetically with the mouth tissues Liquid forms + injectable Ask your dentist to apply soft propolis extract + crystallized honey (small amount) mixed with propolis extracts, royal on and under the gums surrounding the affected tooth/teeth mixtures of bee products, according to the jelly and fresh bee pollen + local condition + swallow oral forms the oral forms + stimulate the teeth related acupuncture points Head Propolis and/or Bee venom stimulate the head and neck ointments + oral forms acupuncture points with the ointments + through microapipuncture + swallow the oral forms (after sucking them for minimum 3 minutes, except for the capsules) Neck Same as above Back Bee venom and Propolis Stimulation of the painful Creams and Ointments + raw honey + beeswax, propolis cataplasms + bee venom solutions + bee stings + oral forms Chest Same as above, but use the neck related acupuncture points spots + the acupuncture points + honey massage + local application of warm beeswax-propolis cataplasms + eating raw, fresh (frozen) bee products Bee venom and Propolis Same as above, but no 68 Abdomen Upper and lower limbs Creams and Ointments + raw honey + beeswax, propolis cataplasms + oral forms injections of bee venom solutions, due to the higher sensitivity of the skin (exception: microapipuncture) + eating raw, fresh (frozen) bee products Same as above Same as above Bee venom and Propolis Same as above; the best Creams and Ointments + acupuncture points are beeswax, propolis located on the “Yang”, cataplasms + injectable bee more hairy areas. venom solutions + oral forms Foot soles Bee venom and Propolis Ointments + beeswax, propolis cataplasms + oral forms Use of creams and/or ointments on the reflexologic areas (reflexotherapy = Fußsohlenreflexmassage) Cardiovascular apparatus All oral forms + ointments Swallowing of the oral + injectable solutions + bee forms after minimum 4-5 + Blood stings + suppositories minutes of chewing and/or sucking; stimulation of the acupuncture points with ointments, injections and/or (micro) bee stings +/- use of suppositories or ointments in cases of hemorrhoids Digestive Apparatus All oral forms, used Liquids or feshly made according with the distance “cocktails” for treatment of to the distance to the mouth, esophagus, stomach “target” + injectable bee and small intestine; venom solutions + capsules, solid propolis suppositories “beans” and suppositories for the large intestine, rectum and anus areas + stimulation of the 69 acupuncture points according to the location of the affected organs Respiratory Apparatus Solutions for inhalations + creams/ointments + cataplasms + injectable solutions + chewing-gum with propolis + oral forms, especially butter-propolis extract in tuberculosis Uro-genital Apparatus cases Inhalation through the nose; stimulation of the nose, chest and back areas (between shoulder bladders) with the ointments and/or injectable of bee venom solutions + swallowing the oral forms For women: Suppositories + vaginal suppositories + creams and ointments + vaginal solutions with propolis and/or royal jelly + injectable solutions + oral forms, especially propolis extracts + fresh Local use of suppositories (intra-rectal) + local stimulation of the acupuncture points with the creams and/or ointments + solutions to be used by women intra-vaginally. frozen royal jelly. Ingesting of the oral forms For men: Suppositories + creams and ointments + injectable solutions + oral forms, especially propolis extracts + pollen preparations and products Osteo-articular and muscular system Bee venom and Propolis Local stimulation of the ointments + injectable bee affected tissues and venom + bee stings + oral acupuncture points forms + propolis-beeswax including through microwarm/cold cataplasms apipuncture + gently “inject” the bee venom and propolis ointments through methods specific to physiotherapy (like electro- 70 and phonophoresis) + application of the cataplasms according to the local signs and symptoms + swallowing of the oral forms, especially propolis extracts Skin Raw, fresh collected Local application according Honey, Propolis, Royal to the symptoms and signs jelly and Pollen in various extracts and cocktails + Ointments and Creams + oral forms + (micro) bee of the Medicinal wounds Tincture stings + shampoos, soaps dermic and lotions with bee lesions products according to your skin type and/or disease affected area. honey for deep and Propolis for superficial and epidermic • What are usually the best doses? The ones that bring relief, which improves immediately or on a long term our health, strength and beauty. It is always recommended (except for the emergencies like burns or scalds) to use the bee products initially in very small doses (to test for a possible allergy or intolerance), than to increase gradually until a clear improvement occurs (see the time/quantity relationship) • What are the best pharmacological preparations and/or products for me? The ones that reach faster and in enough amount the affected tissues, organs or systems in your body. Ask always your local health practitioner and your pharmacist what could be the best forms. Use also, as guidelines, the above table. • What is the best way to administer the bee products for my health, strength and beauty? Ideally is to administer as many as possible pharmacological forms (see 71 above), to be sure that you will reach the affected area with enough nutrients, oxygen and active healing compounds. • What are the best doses and administration methods for children and old people? For children under 3 years ¼ of a dose of an adult. For children between 3-8 is better to use 1/3 of the adult dose and for the children between 8-12 use half (1/2) of the adult dose. • What is the best time/quantity relationship for me when I use bee products for health? How much time and in what amount shall I take the bee products? For those diseases that have less symptoms than usually (like blood diseases or cancers in initial stages) the level of “smile” can be obtained through specific laboratory analysis. After complete healing/improvement pauses can be taken, according to the medical advice. 72 • Are there any adverse reactions when I take the bee products together with other remedies or practice simultaneously Apitherapy with other healing methods? Usually not, but in some cases the use of bee venom therapy is restricted for those taking heart related drugs. Also, rheumatic and multiple sclerosis patients are usually advised to refrain from taking steroidal and non-steroidal anti-inflammatory drugs during bee venom therapy. In cases when your medical doctor prescribed you mandatory some drugs, you can use though the “soft” bee products like honey, bee pollen, royal jelly and propolis, and once the situation is improved, with the agreement of your doctor, you can start also bee venom therapy. • What persons can practice bee venom therapy? Only the licensed medical doctors (MD) and the Naturopathic Doctors (HP) can inject various bee products extracts and/or bee venom in solution form. Any person can though use propolis and bee venom ointments but respecting the leaflet recommendations. • How can we improve the efficacy of bee products through other remedies and/or healing methods? Is my diet and lifestyle important in Apitherapy? Any other method or remedy that can improve the absorption of the bee products related nutrients and/or active compounds should be used. Also, any other healing method that detoxifies the body, mind and spirit should be used. Once the necessary nutrients, the water and the oxygen have reached the affected area in perfect quantity and quality, the regeneration/improvement of that area should start. In this phase, a very good local or general systemic relaxation (sleep) can make wonders. Learn and use any technique of relaxation, Yoga, Taiji Quan recommended by your local psychotherapist in order to get faster the results you wish. All our living cells from our body needs besides nutrients, water and oxygen, also a warm enough environment; so learn to use daily massage and calisthenics (gymnastic) techniques. Our health depends 50% of our diet, 40% of our lifestyle and only 10% of 73 the treatments we make, so be sure to have also a very good diet, specific to your body needs and to your constitution. Here, the specific advises of your nutritionist (ideally specialized in Ayurveda too) can help you also tremendously. • What are the major counter-indications and limits of bee products? The allergies (especially the bee venom allergy), the intolerances (especially to honey and pollen) and the excessive (non-controlled) use of honey in cases of diabetes type 1. If the dose is correctly selected and the administration method is proper, even the people having normally counter-indications to the use of bee products can use them, but of course ONLY under strict medical control. • Where do I find more specific information? Ask for help your local health and Apitherapy practitioners registered in the nearest to your place Apitherapy Society and you will receive it! Morphologic Changes of Larvae during Biosurgery U. Wollina1, N. Kunath1, G. Haroske2 1 Department of Dermatology, Dresden-Friedrschstadt Hospital, Germany 2 Institute of Pathology, Dresden-Friedrschstadt Hospital, Germany Maggot therapy or biosurgery is discussed with increasing interest not only by scientists but also by the public. Maggot therapy is based upon the use of living larvae of necrophagous flies to treat chronic non-healing wounds and leg ulcers. While there is great success in clinical use and the technology of breeding and applying larvae, the knowledge about the changes of larvae within the wounds is still quite limited. For this reason we investigated the larval morphology before and after the use thereof in chronic ulcers by light microscopy. The results demonstrate dramatic changes in larval morphology during the biosurgical process. Introduction 74 The therapy of chronic wounds and ulcers with maggot (larvae of Lucilia sericata) was widely used during 1920s and 1930s, as no antibiotics for their treatment existed. [1,7,9] This therapy has experienced a renaissance in the last 10~15 years. Clinical studies have shown that the larvae were remarkably successful and efficient in the case of debridement of chronic wounds such as leg ulcer, diabetic gangrene, or decubitus. [ 10,12,13,17 ] Based on the level of present knowledge, the mechanism of healing supported by larvae is to be described as follows : * Larvae separate the proteolytic enzyme, which dissolves the necrotized tissues. * The mixture of tissue and fluid is soaked up by larvae. * In the presence of larvae the amount of the produced exudate of wounds increases, and as a result the bacteria will be washed out. * Through the secretion of larvae the pH-value of the wounds changes. * Effective antibacterial substances in the digestive tract of the larvae reduce the number of germs. * The secretion of larvae promote the healing process. [ See Table 1. ] * The movement of larvae inside the wounds promotes granulation. Thus the digestive tract plays the central role for the clinical activities of larvae. During antibacterial activities the separation of enzymes and Zytokinen appears to be important. [ 3,14,15 ] In one particular study, haemolymph, digestive fluid, and skinning (sloughing ??) hormone ( 20-Hydroxyecdyson ) of Lucilia sericata were brought together, and their effect on human Fibroblasten was tested. And it was confirmed that each of these extracts stimulated the growth of Fibroblasten cultivation, albeit merely by 12 %. The epidermal growth factor alone can achieve that much stimulation. A significant increase in the build-up of Fibroblasten occured, when the extracts were used together with epidermal growth factor. The digestive fluid had significant effects on the Fibroblasten, when they were combined with Zytokin Interleukin 6. [11] In the following investigation, we analyzed the morphological changes of larvae under the light microscope during the clinical biosurgical process. Material and Method 75 It was the larvae of Lucilia sericata species that were utilized by Biomonde, a Hamburg-based firm. A few of them were conserved in formalin, colored with Hematoxylin-Eosin (HE) and were fixed on the microscope slides for investigation. The remaining larvae were used for biosurgery on chronic leg ulcers. After 5 days of activities on the ulcer, these larvae were collected, colored likewise with HE. They were placed on the slides, and examined under the microscope. The morphology was then compared with the unused larvae. Outcome The larvae freshly delivered from the company are small and agile. Their average length amounted to about 6.2mm, with maximum diameter 0.34mm (Fig. 1 a). They were covered with finely folded skins (“Kutikula”). Around the head area, there were mouth-like tools with a hook visible. (Fig. 1 a & b ). The saliva-glands were well developed. At the rear end of larvae “Vakuolen” (= cellular cavity) were to be observed. The connective tissue existed only in narrow areas, although the “Malphigi vessel” were enlarged (Fig. 1 d). The intestines of the larvae were empty (Fig. 1 e). After the biosurgical usage, that is, after 5 days of application to chronic wounds, during which the larvae feed themselves with fluid necrotic materials, they grew up (Fig. 2b). The length was now about 1415 mm, and its diameter was grown up to 0.77 mm. “Kutikula” was now thicker, and the fat tissue was distinctly increased. (Fig 2b). The hooks of the mouth-like tools were not recognizable any more, and the saliva-glands had also disappeared. The connective tissues revealed themselves more strongly, and the Lipid- and Saccharid-filling were now visible (Fig 2c & d). Diameter of Malphigi-vessel had become smaller(Fig 2d). Discussion Lucilia sericata larvae form, in their natural living environment, amorphous mass of maggots, which protects each individual larva and offers the optimal conditions for growth. The larvae are necrophilia, which dissolve and take in the necrotized tissues through secretion of Lipasen and Proteasen. In the middle section of these larvae’s intestines, the materials thus taken experience a further corrosion of enzyme. Around the end of larvae stages the size of each individual maggot gets increased by up to 100 times. The length of the larva’s digestive tract 76 amounts to something like 5 times its body length, and is longer than the fully-grown flies. The digestive tract is divided into three parts. In the first (fore intestine) and the second (middle intestine) segments Proteasen – and perhaps Lipasen, too – are secreted. The pH-value in these areas is sour, standing at 2.4 up to 4.8. The most active segment is the middle one. Following this is the hinter segment, i.e., the end of intestine, which leads to the rectum. [3, 14, 15] The larvae finish their taking up of nutrition and digestion in 5~6 days, and prepare themselves for “metamorphosis” or “pupation”. Malphigi-vessels are an absorptive organ at the end of the intestine, which helps principally the excretion. They contain calcium carbonate in the form of small stones. The calcium is required in order to ensure the mechanical strength of the cocoons during the pupation process. [6] The reason Malphigi-vessel becomes smaller seems to be related to the fact that, around the end of the nutrition take-up phase, the end of intestine is empty. On the other hand, the fat deposits become notably bigger (Fig. 2 b), in order to satisfy the demand of energy during the pupation process. In the case of biosurgery for chronic wounds, the larvae achieve principally debridement and decontamination, and demand the formation of granulation tissues. The bacterial strains of chronic wounds are clearly diminished through the intra-intestinal anti-bacterial activities. The larvae are effective against a wide spectrum of “pathogenen” germs such as, for example, MRSA. [ 2, 3, 6, 11, 14, 15 ] The volume of bacteria thus taken will reduce itself very clearly, as they pass through middle- and end-intestines. [10] In a clinic study on the maggot therapy for the Osteomye patients, a much higher effectiveness in the case of the wounds infected with Gram-positive bacteria was confirmed, corresponding to the in-vitro results. [14] However, the Gram-negative bacteria are cultivated out of the wounds after maggot therapy more often than before. In the case of wounds infected with Gram-positive bacteria the opposite effect was to be noted. In “Vivo” the maggots appear to be less effective against the wounds which are infected with gram-negative bacteria. The authors of the study are of the opinion that, not only for the larger wounds but also for the wounds with larger scab on the surface, more maggots are needed, and also for the wounds infected with gram-negative bacteria. [14] 77 The Sezenierung of the substances looking like growth factors (?) stimulates the healing of the wounds through the larvae (Interleukin-6 and Interleukin-10, substances which are similar to the epidermal growth structure and insulin-like growth factor, as well as Interferon-y). [11] The excretive-secretive products (ESP) of the larvae clearly reduce the in-vitroadhesion of Fibroblasten to Fibronektin, and in closer environment, to collages. The expansion of Fibroblasten cells was equally reduced, although the cells were capable of surviving. As far as the expansion as well as the adhesion was concerned, the heat-treated ESP was essentially less active than ESP not so treated. Compared with ESP-Blank, the activity was far more considerable. ESP seems to indirectly change the adhesion of Fibroblasten, and in fact, on the way over a proteolytic fragmentation of the surface of the Fibronektin. Observed altogether, the secretion of Lucilia sericata larvae changes the adhesion of the Fibroblasten and the expansion of extra-cellular matrix over the protein surface, through which the cells nevertheless remain capable of surviving. Here the proteolytic activity of ESP plays an important role. Transferred to the situation of a wound, the amended interactions of Fibroblasten with the extra-cellular matrix can require the formation of new tissues. [8] As was displayed in a remissionspectroscopic way, the micro-circulation gets improved after the biosurgery, but the exact reasons for this are not yet known. It follows, from our own results as well as from the literature, that complex morphological changes take place with the maggots – either on a skin damage or in natural field – which is necessary, in order for the larvae to be able to pupate. The physiological properties of Lucilia sericata support the healing of chronic wounds. Their ESP and digestive tracts are essential factors of these medically desirable effects of biosurgery. 78 ABSTRACTS for Contributed Papers Maggot debridement therapy (MDT) in a diabetic foot wound patient suffering from severe gout A. Andersen1, 2, B. Joergensen2, T. Karlsmark2 ,K. Kirketerp-Moeller2, K. A. Krogfelt1. 1. Statens Serum Institut, Copenhagen Dk, 2. Copenhagen Wound Healing Center Dk. Purpose/Background: 57 year old male diabetic, through 25 years suffering from severe gout (arthritis urica AU), was confined to a wheelchair due to wounds on both feet. The severity of the AU in this patient makes him a unique case in Denmark. With the occurrence of large sloughy and necrotic diabetic wounds on both feet, distending from the big toes covering approximately 35 cm2 down both forefeet. The excessive deposition and release of monosodium-urate crystals, mm to cm in size made wound debridement difficult. The removal of the crystals was accompanied by excruciating pain for the patient and required lots of brute force from the nurses. Furthermore, bacterial colonization and the crystals in combination caused the wounds to be extremely malodorous. Methods: The patient was treated with MDT on both feet during two consecutive periods of two times 3 days with three weeks pause between 79 the two periods. 3 Biobags was applied to each wound. In the periods between the treatments the patient was treated with Biatain Ibu and standard conservative treatment by the home nurse. Following MDT the patient was admitted for Versajet, debridement of the deeper cavities and split thickness skingrafts on both feet. Results: MDT facilitated debridement of the wounds, odour and bacterial burden control and softening of the crystals, which could subsequently easily be rinsed from the wound. Versajet debridement finished the preparation of the wounds for skin grafts, which took nicely. Conclusion: Through the enzymatic action of the maggot secretions, the monosodium-urate crystals were softened and easily mechanically removed from the wounds. The wound balance was shifted as to allow skin grafting and the patient who had been confined to a wheel chair for almost one year walked out the Copenhagen Wound Healing Center supported by cane and prescription footwear. Maggot debridement therapy (MDT) in diabetic foot wounds – Quorum sensing dependent bacterial niches may promote infection or MDT failure A. Andersen1, 2, B. Joergensen2, T. Karlsmark2, T. B. Rasmussen3, T Bjarnsholt3, M. Givskov3, K. A. Krogfelt1. 1. Statens Serum Institut, Copenhagen Dk, 2. Copenhagen Wound Healing Center Dk. 3.CBM, DTU Lyngby Dk. Background: Many diabetic patients with foot wounds are ideal candidates for MDT. The maggots gently and thoroughly remove necrotic tissue by mechanical action and by proteolytic digestion. The maggots secrete antimicrobial peptides into the wound, kill ingested bacteria in their gut and alkalinize the wound. However a few reports show that MDT in some cases subsequently facilitates an infection, when specific bacterial species are present in the wound prior to treatment. MDT has also been observed to fail in some cases due to the maggot’s death in the wound environment i.e. Their bio surgical properties were not applicable. Wounds and particularly diabetic wounds are susceptible to infection due to the development of microbial communities within and around the wound environment. It is now realized that chemically based cell-cell 80 communication (denoted quorum sensing (QS)) and biofilm formation play important roles in a multitude of human infections. QS enables the bacteria to keep track of their numbers and pool their efforts in order to successfully cause disease. Bacterial biofilms show an inherent tolerance to a variety of antimicrobial treatments including the action of the immune system. This makes biofilm infections impossible to completely eradicate. Several pathogenic bacteria show QS mediated organisation and speculation regarding the role of QS and biofilm formation in wound healing is appealing. In this preliminary study we wish to investigate the possible role of QS in relation to infections related to MDT. Methods: In an in vitro setup using blood agar plates, we challenged L. sericata maggots with different concentrations of green fluorescent protein (GFP) tagged Pseudomonas aeruginosa wild type (PAO1 WT), a GFP tagged QS deficient mutant (PAO1 RR) and a Staphylococcus aureus. The survival of the maggots was determined and the clearing efficiency assessed during 3 day periods. The uptake of P. aeruginosa was determined using fluorescence microscopy. Results: The maggot survival and clearing efficiency was unaffected by S. aureus and PAO1 RR compared to negative controls, but maggot survival was significantly impaired by the PAO1 WT. Furthermore the florescence microscopy showed reduced uptake of PAO1 WT compared to the PAO1 RR. Conclusion: The results indicate that infections following maggot debridement therapy may originate from preferential feeding by the maggots creating micro niches in which bacteria survive and subsequently unchallenged cause infections and that this preference is QS dependent. Furthermore QS controlled virulence factors are toxic for the maggots. Therefore the presence of specific bacterial species may be counter indications for MDT. This suggests that QS inhibitors may be a useful future adjuvant for maggot debridement therapy in wounds harbouring specific bacterial species or as supplement to standard antibiotic therapy. MAGGOT IMMUNITY AND DRUG DEVELOPMENT 81 Sergey Chernysh1, Natalia Gordja1, Natalia Chernysh1, Dmitry Tulin1, Vadim Anikin1, Valery Pleskach1, Myung Jeom Ryu,2 Cheolju Lee2. 1 Laboratory of Insect Biopharmacology and Immunology, St. Petersburg University, St. Petersburg, Russia 2 Life Sciences Division, Korea Institute of Science and Technology, Seoul, Korea Purpose: To analyze mechanisms of Calliphora vicina maggots’ immunity with respect to drug development. Methods: Range of experimental immunology, peptide chemistry and microscopy methods has been used. Results: Septically injured maggots synthesize about dozen antibacterial and antifungal peptides of insect defensin, cecropin, diptericin and 3kDa peptide families. The peptides natural composition demonstrates antibacterial activity dramatically exceeding the activity of synthetic antibiotic, chloramphenicol. The maggot hemolymph contains cytotoxic hemocytes able to kill human tumor cells and soluble peptides stimulating cytotoxic activity of mammalian natural killer cells. The peptides referred to as alloferons were structurally characterized and subjected to full scale preclinical and clinical studies. Nowadays alloferon-1 is registered as antiviral drug to treat genital herpes and hepatitis B. Alloferon mode of action is mediated by NFκB signalling pathway and directed to boost recognition of viral and tumor antigens. Conclusion: Blow flies developed surprisingly effective immune system to survive in environments abundant in conventionally pathogenic microorganisms. Effector and regulatory molecules of maggot immunity demonstrate biological activities prospective for medical use. Alloferons are currently used as antiviral medicines and may have broader applications to treat viral, oncological and immunocompromized conditions in the future. The maggot antibacterial and antifungal peptides are other prospective drug candidates. Further research of maggot immunobiology may contribute new insight to the biotherapy and drug development. 82 Hepatoprotective potential of earthworm extract (Lampito mauritii, Kinberg) against paracetamol induced liver damage in rats Edwin L. Cooper, Ph.D., Sc.D. Professor, Department of Neurobiology David Geffen School Of Medicine at UCLA, USA Our pre-historic ancestors have been exploring natural compounds to cure their ills, improve and enrich their own lives. Most of these compounds were derived from plants and animals. The science of curing of various ailments by using therapeutics obtained from animals is known as Zootherapy. Animal-based medicines have been elaborated from parts of the animal body, from their products (secretions and excrements), or from non-animal materials (nests and cocoons). Research on traditional medicines has given a vital tool to the upcoming art of Bioprospecting for pharmaceuticals compounds. Totally 252 essential compounds have been selected by the World Health Organization, 11.1% derived from plants and 8.7% from animals. For thousand of years, the earthworm and its products, has been used for therapeutic benefits. The traditional medical knowledge of indigenous people internationally, more particularly in Asia: India, Myanmar, China, Korea and Vietnam has played a vital role in identifying, extracting and using biologically active compounds from earthworms. Earthworms have a dense nutritional content because they live in the soil. Various studies of the earthworm reveal several therapeutic properties: antipyretic, antispasmodic, detoxic, diuretic, antihypertensive, antiallergic, antiasthmatic, spermatocidal, antioxidative, antimicrobial, anticancer, antiulceral and anti-inflammatory (Cooper et al., 2004; Balamurugan, et al. 2007). Earthworm L.mauritii, found throughout India has been reported in Siddha medicine and is used mainly for antioxidative, antimicrobial, anticancer, antiulceral and anti-inflammatory. In our present study, the extract of L.mauritii was investigated for its hepatoprotective and antioxidant effects on paracetamol (2 mg/kg) induced acute liver damage in Wistar albino rats. Hepatoprotective activity was determined by using biochemical parameters such as serum alkaline phosphatase (ALP), serum 83 glutamate oxalate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), bilirubin and total protein. Extract at doses of 100, 200, 300 mg/kg produced significant hepatoprotective effect by decreasing the activity of serum enzymes, bilirubin and lipid peroxidation whereas it significantly increased the levels of glutathione (GSH) and super oxide dismutase (SOD) in a dose dependant manner. The effects of extracts were comparable to those of standard drug silymarin. These results suggest that earthworm extracts may have potential therapeutic value in treating certasin liver disorders, perhaps by antioxidative effect on hepatocytes. Balamurugan M., Parthasarathi K., Cooper, E.L. Ranganathan L.S. Earthworm paste (Lampito mauritii, Kinberg) alters inflammatory, oxidative, haematological and serum biochemical indices of inflamed rat. European Review for Medical and Pharmacological Sciences 2007 (11) 79-89.. Cooper, E.L., Hrzenjak, T.M. and Grdisa, M.. Alternative sources of fibrinolytic, anticoagulative, antimicrobial and anticancer molecules. Inter. J. of Immunopathol. And Pharmacol. (2004) 7(3): 237-244. Therapeutic Potential of Tunicates Edwin L. Cooper, Ph.D., Sc.D. Professor, Department of Neurobiology David Geffen School Of Medicine at UCLA, USA Biotherapy may be entering a new era with the recognition of the utility of looking to other sources of therapies especially by turning to products from animals, particularly those from the sea. In recent years bioprospecting using marine natural product has yielded a considerable number of drug candidates. Most of these molecules are still in preclinical or early clinical development but some are already on the market. Research into the ecology of marine natural products has shown that many of these compounds function as chemical weapons and have evolved into highly potent inhibitors of physiological processes in the prey, predators or competitors of the marine organisms that use them. Some of the natural 84 products isolated from marine invertebrates (sponges, mollusks, tunicates) have been shown, or are suspected to be of microbial origin and this is now thought to be the case for the majority of such molecules. Marine microorganisms, whose immense genetic and biochemical diversity is only beginning to be appreciated, may also become a rich source of novel chemical entities for the discovery of more effective drugs. This recognition offers a word of caution for maintaining the purity of the isolated products. With respect to products derived from complex multicellular organisms, we must remember that these products are often associated with the immune systems of these creatures and that they evolved millions of years ago—thus their immune systems have been an effective survival strategy. And if it has worked for them, then humans should harness these products as new-wave antibiotics or anticancer molecules, as offerings for biomedical applications, particularly those that may apply to complementary and alternative medicine (CAM). Many marine invertebrates are immobile, attached to the ocean floor and use highly evolved chemical compounds to attract food, block the growth of intruding neighbors or repel predators. Biologists, especially comparative immunologists in general and invertebrate immunologists in particular, believe that these survival demands triggered the evolution of a particularly abundant mixture of bioactive compounds. For example tunicates are one of the richest sources of interesting chemicals from the marine environment. They have an elaborate innate immune system and these various compounds some of them are being assayed as sources of anticancer molecules: lung, breast and ovarian cancer melanoma. Recently new work reveals the antiviral treatment of hepatitis B virus-transgenic mice using the sessile tunicate Styela plicata. Clearly the sea offers enormous potential for discovery of compounds that can be utilized as therapeutic agents. Cooper, E.L., Wright, R.K., Stein, E.A., Roch, P.J. and Mansour, M.H. 1987. Immunity in earthworms and tunicates with special reference to receptor origins. In Invertebrate Models, Cell Receptors and Cell Communication, ed. A.H. Greenberg, 79-103, New York, Karger. Cooper, E.L.1995. Immunology: A look toward the sea and what we have learned from tunicates. Aquaculture 132: 1-15. Cooper, E.L. 2004 Drug disvcovery, CAM and natural products. ECAM 1: 85 215-217. 86 The Use of Medicinal Leeches and Medicinal Preparation from them in the Complex Treatment of Skin and Mucosal Diseases in UST Health Resort 1 Sidorov B.B., 1Korobeinikova G.A, 2Gileva O.S. 1 UST Health resort, Russia 2 Perm State Academy of Medicine, Russia Hirudotherapy (HTH) – the use of the leeches for treatment – is one of the oldest practices in medicine. The past 3 decades have demonstrated a resurgence of interest in leeches among physicians and patients as a form of effective treatment. In spite of the serious evolution of our scientific findings of the hirudotherapy performance, we haven’t totally covered it’s mechanism even now. Among the important biological properties of medicinal leeches are blood – letting action and the production of a variety of active substances that suppress inflammation and pain, improve tissues microcirculation, metabolism and regeneration. Various skin and mucosal disorders and diseases still remain a serious medical problem. Leeches are now applied to treat a wide array of dermatological problems. Russian special literature of the last years contains isolated references on the dermatological aspects of leeching (Smirnoff A. V., 1994; Dyomina T.A. et al, 2003); the spectrum of indications for HTH of the skin pathology is wide enough: from the severe forms of dermatoses (psoriases, eczema, neurodermite, ruber lichen planus, lupus erithrematodes, sclerodermia) to traumatic and infectious lesions of the skin and mucosae (furuncule, carbuncle, purulent skin wound). Sometimes the leech can not be a comfortable medicinal preparation: in some clinical cases it is simply impossible to apply the leech to the focus of lesion, to standardize the curative effects of leeching, to avoid some side effects of HTH. That is why today the progress of hirudology is connected with creation and clinical aprobation of medicinal preparation with biological active components of leeches. Among them Pijavit – ecologically pure preparation with a complex of biological active substances produced by Hirudo Medicinalis. In the forms for external use it hasn’t shown any irritative, toxic and allergenic influence, standarts microcirculation in skin and mucosae, provide anti-inflammatory and 87 hydrotative effects. The purpose of the study was to assess the effectiveness of native HTH (with medicinal leeches) and hirudopharmacotherapy (HPTH) (medicinal preparation with biological active substances from Hirudo Medicinalis) in the complex treatment of dermal and mucosal diseases. Materials and methods: our own clinical experience in the treatment of different dermal disorders with the help of leeches (HTH and HPTH) we have cumulated in the department of Hiridotherapy of UST – Katchka Health Resort – one of the biggest balneological health resorts of Russia. The effect of the complex treatment with Piyavit use in the form of cream for external use was investigated in 36 children (6-13 years) with manifestations of neurodermite and atopic dermatitus, 17 patients (17-32 years) with herpetic chilitis and stomatitis. The skin, oral and lip mucosae were irrigated with antiseptics, dried and covered with thin layer of the cream twice a day. The duration of the treatment – 7-9 days. The native HTH (aspirative regimen) was used in 23 patients with ruber lichen planus (with dermal and mucosae lesions). The whole course consisted of 7 sittings over day. Results: in 63 % of clinical cases of lichen planus we saw positive results after 1-2 courses of HTH: the decrease of papul’s number, the decrease of their capacity, the healing of erosive arrears of the skin and mucosa, the diminish of edema, erythema and perifocal inflammation. Positive clinical dynamic was supported by means of polarographic method (increase of initial and maximal oxygen saturation, speed of oxygen saturation and etc.) . Results demonstrated the objective and subjective improvement just on the 1-2 days after the beginning of the treatment with Pijavit cream: diminish of the strain and pain, edema, induration and eczematisation of the derma, disappearance of the pruritus and xeroderma, recovery of salivation and etc. The results after the first course of HTH in patients with neurodermite and dermatitis were the following: completely healing of affected derma in 45% of cases, considerable improvement – in 37%, improvement – in 11% and condition without visible improvement - in 7% of the patients. The efficiency of the HPTH raised significally after the second course of treatment. Native HTH and the use of medicinal preparations from Hirudo 88 Medicinalis can be useful adjunct to the complex treatment of some types of skin and mucosal diseases. Induction of antibacterial activity in medicinal maggots by infected environment Takuya Kawabata Okayama university Graduate Sc, Japan Purpose: Maggot debridement therapy (MDT) has received increasing interest for the treatment of infected foot ulcers. It is reported that maggot has three effects on infected wounds; to debride necrotic tissue, to kill micro-organisms and to stimulate the wound healing. Previous studies on larval antibacterial activities in vitro have been performed using larvae grown up under sterile condition. But no attempt was made to investigate the antibacterial activity of larvae exposed to infected environment. The purpose of this study was to investigate the antibacterial activity of larvae exposed to infected environment. Methods: Larvae of Lucilia sericata were used. Pretreatment with bacteria was obtained by exposing sterile larvae to Staphylococcus aureus or Pseudomonas aeruginosa for 24 hours. Larvae were homogenized in sterile phosphate-buffered saline (PBS) and centrifuged. The supernatant was incubated with suspension of S. aureus of P. aeruginosa for 30 seconds, 3 hours or 6 hours at 37℃. Aliquots of the mixture were plated on nutrient agar and the number of Colony Forming Units (CFU) was counted after an overnight culture at 37℃. Results: Effect on growth of S. aureus The number of CFU in S. aureus and P. aeruginosa pretreated group was significantly lower than that of sterile and PBS-control group (n=8, p). 89 Treatment by Injection-Acupuncture with Bee-Venom(Apitoxin) and Apitoxin Combined by Chinese Herbal Medicine in Patients with Canine Hind Limb Paralysis Hyung-kyou Jun*, Se-kun Park*, Duck-hwan Kim*,1, Christopher Moon-ho Kim**, Chin-yuan Hsu***, Chin-ling Hsu***, Jim-cai Liao#, Hao-jen Chueh## and Han-wen Cheng### * College of Veterinary medicine, Chungnam National University, Daejeon, Korea ** Anapunsesang Clinic, Seoul, Korea *** Yeon Chang Veterinary Clinic, Miaoli City, Miaoli Province, Taiwan # Shan Qun Animal Hospital, Shang Cheng City, Taipei Province, Taiwan ## Zoo Animal Hospital, Taipei City, Taiwan ### Sino Union Animal Hospital, Young He, Taipei, Taiwan Purpose: The therapy by injection-acupuncture(AP) with bee venom (Apitoxin) and injection-AP with Apitoxin combined by administration of Chinese herbal medicine was applied in 2 cases with canine intervertebral disc disease(IVDD). Method: Case 1 was diagnosed as thoraco-lumbar IVDD (T11-T12, T12T13, L3-L4 and L4-L5) and case 2 was diagnosed as IVDD at T 10- T11 and T 12- T 13, respectively. Injection-AP with Apitoxin(total 200 ㎍ of Apitoxin, 0.1 ml /acupoint) plus physical exercise(walking with gocart, TID/day) and aquatherapy(swimming treatment, BID/week) were given to each patient. The used acupoints were GV20(Bai Hui), GB30(Huan Tiao), ST36(Zu San Li), GB34(Yang Ling Quan), ST40(Feng Long), ST41(Jie Xi) and BL40(Wei Zhong), the lesions, and trigger points. In addition, Chinese herbal medicine(Koda Pharmaceutical Co., Taiwan) including Zheng Gu Zi Jin Dan(正骨紫金丹: 1 g), Shiuh Duann(續斷: 0.2 g), Du Zhong(杜仲: 0.2 g), Mo Yao(沒藥: 0.2 g), Ru Xiang(乳香: 0.2 g) and Pyrite(自然銅: 0.2 g) were orally medicated BID for 9 days in case 2. Result: Walking was possible after session 11 for 4 weeks in case 1 and after session 6 for 2 weeks in case 2, respectively. Conclusion: The present cases were canine IVDD which showed favorable therapeutic responses to injection-AP with Apitoxin only and injection-AP with Apitoxin combined by Chinese herbal medicine. 90 Bee venom(Apitoxin) therapy on canine facial nerve paralysis Hyung-Kyou Jun*, Hyun-Uk Oh*, Hyun-Hwa Lee*, Ji-Won Han*, Cristopher Moon-Ho Kim** and Duck-Hwan Kim* * College of Veterinary Medicine, Chungnam National University, YusungGu, Daejeon 305-764, Korea ** Anapunsesang Clinic, Seoul, Korea Purpose: To elucidate the therapeutic effect by Apitoxin therapy for canine facial nerve paralysis(FNP), the present study was performed. Method: The 12 mongrel dogs (2-3 years old, 2.0-5.6 kg, BW) were used in this experiment. The experimental dogs were divided intocontrol(4 dogs), experimental group I(4 dogs) and experimental group II(4 dogs). FNP was induced by clamping of facial nerve with hemostatic forceps for 20 minutes under zoletil anesthesia. As for the treatment, control group was intramuscularly injected with saline into head muscle, and experimental group I was treated by injection-acupuncture(AP) with dexamethasone (Huons Co., Korea, 1 mg/ml/head) and experimental group II was treated with injection-AP with Apitoxin(Guju Pharmacological Co. and Apimeds, Inc., Korea, 1mg/bottle, 200ug of Apitoxin: 2 % lodocaine =1:1) after induction of FNP, twice per week for 2 weeks, respectively. The used acupoints were LI04, LI20, ST02, ST07, GB03, SI18, TH17 and GB34. The degrees of FNP improvement were evaluated as clinical scores(0: normal, 1: mild, 2: moderate and 3: severe) and serum creatine kinase(CK) activities were evaluated. Statistical significance was analyzed by student’s t-test. Result: The FNP symptoms were not improved at all until day 14 in control group, however, the FNP symptoms were much improved until day 14 in experimental group I and II, respectively. The significant differences of clinical scores were detected on day 14 in experimental groups, compared by those of control group (p<0.05). Maggot Therapy for severe diabetic foot ulcer H. Mitsui, T. Kawabata, S. Ugaki, S. Ohsawa, Y. Fujii, S. Sakurai, S. Kasahara, S. Sano 91 Institution: Okayama University Graduate School of Medicine and Dentistry, Department of Surgery, Division of Cardiovascular Surgery Purpose: Although effectiveness of maggot therapy for diabetic intractable ulcers has been advocated in Europe and United States, it has not been tried so far in Japan. Method: We started this therapy to treating diabetic foot ulcer for the first time in Japan and had experienced 43 cases during these 26 months. Result: The ulcers of 40 patients out of 43 patients were all healed after 3 months. During these treatments complications related to maggot therapy were not experienced. Failed 3 cases had the same co-mobidity (chronic renal failure and low cardiac output) and undergo major limb amputation because of sepsis. Conclusion: We concluded maggot therapy is effective to wound bed preparation of diabetic ulcer. Maggot therapy has some benefits: 1. No contraindication 2. Anesthesia is unnecessary 3. Cost-effective compared to conventional therapy. 4. Long history and enough evidence of effectiveness to diabetic ulcer treatment in western countries. Perspectives: We are now trying to promote maggot therapy to save diabetic foot which would have been amputated in Japan. In this congress we want to present the outcome of those 43 cases and discuss the indication and limitation of maggot therapy to diabetic foot ulcer. Also we want to show our special dressing for maggot therapy to make maggot to work effectively and comfortably on ulcer bed. 92 Effects of Bee Venom on Glioma Cells Byung-Soo Koo, O.M.D. Chief, Department of Neuropsychiatry, Dongkuk Oriental Medical School, Korea Objective: Bee venom (BV) has been used for the treatment of inflammatory diseases such as rheumatoid arthritis and relief of pain in Oriental medicine. The two main components of BV are melittin and phospholipase A2 (PLA2). Of these, melittin, the major active ingredient of BV, has been reported to induce apoptosis and to possess anti ‐tumor effects. Several studies have established that the agents inducing apoptosis in target organs suppress tumorigenesis. As the other component, PLA2 has been reported to induce neurite outgrowth in PC12 cells. However, there was no report about proliferative effect of BV in neuronal cells. In order to examine the effect of BV on glioma cell, human glioma cell line, U87 was used. Methods: Analysis of proliferation was confirmed by MTT assay. BV increased cell number through dose‐ and duration‐dependent manner and these effects are apparent at a concentration of 10 ug/ml. To observe which signaling molecules will be activated by BV, phosphorylation of Akt, MAPK, PYK2 or CREB were examined by Western blot analysis. To study the long term effect of BV in U87 cells, the image of cells treated with BV for 4 days were obtained. Results: The phosphorylation levels of PYK2 and Akt were increased at 5 min after addition of 10 ug/ml of BV and sustained to 2 hours. On the other hand, phosphorylation of MAPK and CREB were increased at 5 min, maximum at 10 min, and returned to 30 min. These imply that BV may activate two different signaling pathways, PYK2/Akt and MAPK/CREB. BV treated cells showed increased neurite number and length. These results propose that BV may induce differentiation as well as proliferation of U87 cells through the activation of PYK2/Akt and MAPK/CREB. Efficiency Maggot Debridement Therapy in Fournier’s Gangrene 93 Hugo E. Sarmiento Jiménez*, Jose Contreraz Ruiz**, Alicia Fonseca Muñoz*** *Department of Surgery, Hospital “San Jose” Oaxaca Oaxaca, ** Department of Dermatology, Hospital General "Dr. Manuel Gea Gonzalez," Mexico City, Mexico *** Department of Biology, Hospital “San Jose” Oaxaca Oaxaca, Mexico Maggot debridement therapy (MDT) has been shown to be a highly effective method of debridement of necrotic tissue. The speed and efficacy of this modality has made it a valuable aid in the treatment of chronic wounds. Purpose: To show the usefulness and safety of MDT in the treatment of a serious case of Fournier’s Gangrene. Method and Results: We present the case of a 59 year old male from Oaxaca, Mexico. Relevant history for diabetes for 30 years, hypertension for 5 years and renal insufficiency with peritoneal dialysis for the past 3 years. He was seen at the emergency room for necrosis of the scrotum and gas that had begun 15 days earlier. The diagnosis of Fournier’s gangrene was made and the patient underwent surgical debridement. After surgery and due to recurrent areas of necrosis sessions of MDT were applied and IV antibiotics continued. The patient had a favourable outcome with increased granulation tissue, and decreased exudate and odour. that allowed for treatment with honey-impregnated gauze and grafting. Conclusions: MDT has proved to be an effective debridement method and it can be used in Fournier’s gangrene after surgical debridement to increase the healing rate and improve outcomes. 94 Study of Maggot Debridement Therapy in necrotic pressure ulcers, Case Series. Hugo E. Sarmiento Jiménez*, Jose Contreras Ruiz**, Alicia Fonseca Muñoz*** *Department of Surgery, Hospital “San Jose” Oaxaca Oaxaca, México ** Department of Dermatology, Hospital General "Dr. Manuel Gea Gonzalez," México City, México *** Department of Biology, Hospital “San Jose” Oaxaca Oaxaca, México Purpose: To show the efficacy of MDT in four diabetic patients with necrotic pressure ulcers Method: We present a clinical trial with four diabetic patients with necrotic pressure ulcers in different areas: (2) ischiatic ulcers and (2) sacral area .The time of developed of them cover a period of (1-11 months) before MDT was applied. The cause of the development of wounds was hemiplegia. We treated four patients two women and two men. The average age was 60.83 years. The mean hospital stay was 18 days (range 7-45 days). Sterile maggots (100-1500) were administered to the wound two or three times per week and replaced every 24-48 hours. The patients were assessed daily by nurses in order to evaluate the maggots viability. Finally the wounds were treated with honey honey-impregnated gauze and grafting. Results: The complete debridement was achieved in three patients and partial debridement in one case. After debridation, 2 ulcers were treated with honey-impregnated gauze until they close and 2 ulcers required skin graft. Conclusions: In debridement of necrotic pressure ulcers MDT was effective and safety method and proved to be a cost-effective method in short time. 95 Water extracted propolis inhibits TNF- α release following LPS injection in the rat Myung-Sang Kwon, Ph.D. Department of Immunopharmacology, Kangwon University, Korea Purpose: Acute sepsis is a one of the most prevailing syndrome during inflammatory response, and is a leading cause of death. Tumor necrosis factor-α (TNF) is known to as a primary mediator of immune regulation and the inflammatory response. Some kinds of drug application of blunting the TNF response in sepsis have been attempted. The purpose of this study is to know the antiinflammtory effect of propolis on LPS induced acute inflammation. Materials and Methods: In order to compare how much blunting TNF between water extracted propolis (WEP) and pentoxifylline which inhibits the production of TNF as a positive control. WEP contains lots of flavonoids shown to inhibition of lipoxygenase. We infer this inhibition due to WEP may be down regulation of the production of TNF. In this study, we examined the effect of WEP in a rat model of lipopolysaccharide (LPS) induced acute septic shock. Results: Dose response was determined by two different doses injection of WEP and pentoxifylline. Rats were injected intraperitoneally with endotoxin. TNF was measured by ELISA. We also investigated the chemiluminescence of PMA(phorbol-1,2myristate-1,3-acetate) stimulated peritoneal macrophage of rat. Serum TNF highest elevation occurred after endotoxin injection with peak levels at 90 min. WEP and pentoxifylline treated rats had lower TNF levels and more diminish chemiluminescence than saline treated control rats at 90 min. We found that WEP is a potent inhibitor of LPS induced TNF levels and diminished this chemiluminescence. Conclusions : Our data suggest that propolis seems to contribute to alternative anti-inflammatory material for treatment of some kinds of fever inducing bacterial infection. 96 Water extracted propolis attenuates kainite-induced neurotoxicity via adenosine A1 receptor in the rat Myung-Sang Kwon, Ph.D. Department of Immunopharmacology, Kangwon University, Korea Purpose: It is well recognized that kainic acid (KA)-induced neurotoxicity is mediated, at least in part, by oxidative stresses. Because some antioxidants block KA-induced neurotoxicity, The purpose of this study is to know the antioxidant effects of propolis on seizure induced by kainic acid. Methods: we examined the effect of antioxidant propolis on KA-induced neurotoxicity in rats. Sprague-Dawley rats received water-extracted propolis (WEP) (50, 100, 200mg/kg, p.o.) five times at twelve times intervals. KA (10mg/kg, i.p.) was injected 1 hour after last propolis treatment. Results: Convulsing behaviors were significantly decreased in the seizing rat that pretreated with WEP in a dose-dependent manner. Malondialdehyde (MDA), protein carbonyl (CO) and glutathione (GSH) levels of rat hippocampus were examined as oxidative stress markers. MDA and CO levels were significantly increased 4 hours after KA administration. GSH levels were reduced by KA administration. Pretreatment with EEP blocked these changes in a dose-dependent manner. These neuroprotective effects of propolis were counteracted by adenosine A1 receptor antagonist, 8cyclopentyl-1,3-dimethylxanthine [CPT (1mg/kg, i.p.)] and adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine [DMPX (1mg/kg, i.p.)]. However, CPT was more efficacious than DMPX in counteracting protective effect of propolis. Conclusions: Consequently, these results suggest that antioxidant properties of propolis on KA-induced neurotoxicity are mainly via adenosine A1 receptor. 97 Maggot Excretions/Secretions Inhibit Multiple Neutrophil ProInflammatory Responses M.J.A. van der Plas1,2, A.M. van der Does1, M. Baldry1, H.C.M. DogteromBallering1, C van Gulpen1, J.T. van Dissel1, G.N. Jukema2 and P.H. Nibbering1 1 Dept of Infectious Diseases, 2Dept of Surgery, LUMC, Leiden Introduction: Maggots of the blowfly Lucilia sericata are used worldwide for the treatment of wounds that lack the tendency for spontaneous healing like diabetic foot ulcers. In such wounds, activated neutrophils and their products may precipitate tissue damage rather than contribute to healing. Since the beneficial actions of maggots are contained in their excretions/secretions (ES) we assessed the effects of maggot excretions/secretions (ES) on pro-inflammatory responses of activated human neutrophils. Methods: Neutrophils were isolated from venous blood. g/ml of ES (one maggotSubsequently, these cells were incubated with 0-100 g of ES/h) and stimulated with fMLP (syntheticproduces approximately 2 bacterial peptide) or PMA (activator of protein kinase C). The effects of ES on neutrophils were analyzed measuring chemotaxis, the production of H2O2 and the release of elastase. Furthermore, phagocytosis and killing of C. albicans by neutrophils was investigated. In an attempt to gain insight into how ES affects neutrophils, we measured the changes in intracellular concentrations of Ca2+ and cAMP. Results: ES inhibited elastase release and H2O2 production by fMLPactivated neutrophils in a dose-dependent fashion with maximal inhibition g of ES/ml inhibited neutrophilg of ES/ml. Already 0.5 seen with 5-50 g of ES/ml almost completely blocked it.chemotaxis towards fMLP and 100 Importantly, ES did not affect the ability of neutrophils to phagocytose and intracellularly kill C. albicans. Interestingly, ES did not affect the fMLP-induced rise in the [Ca++]i in neutrophils indicating that ES acts down-stream of the phospholipase C-mediated activation of protein kinase C. In agreement, ES inhibited the PMA-activated neutrophil responses. Furthermore, ES induced a rise in the intracellular cAMP concentration and pharmacological activators of cAMP-dependent 98 mechanisms mimicked its inhibitory effects. Conclusions: The beneficial effects of maggots on chronic wounds may be explained in part by inhibition of multiple pro-inflammatory responses of activated neutrophils by ES without affecting their antimicrobial activities. Although the mechanisms underlying these effects on neutrophils are not fully elucidated, activation of cAMP-dependent mechanisms may be involved. Maggot Excretions/Secretions are effective against biofilms of Staphylococcus aureus and Pseudomonas aeruginosa M.J.A. van der Plas1,2, A.M. van der Does1, M. Baldry1, H.C.M. DogteromBallering1, C van Gulpen1, J.T. van Dissel1, G.N. Jukema2 and P.H. Nibbering1 1 Dept of Infectious Diseases, 2Dept of Surgery, LUMC, Leiden, The Netherlands Introduction: Chronic wounds that lack a tendency of healing are common in patients with chronic conditions like diabetes mellitus. In many of such patients, the wound healing process is hampered by bacterial infection especially when the bacteria form biofilms protecting them from the actions of both antibiotics and the immune system. In this study we assessed the effects of maggot excretions/secretions (ES) on biofilms of Staphylococcus aureus and Pseudomonas aeruginosa, microorganisms that are clinically most relevant in this respect. Methods: g/ml ES (one maggotWe assessed the effect of 0-20 g of ES/h) on modulation of biofilms using microtiterproduces approximately 2 biofilm assays. Furthermore, antibacterial activity of ES was investigated using in vitro killing assays and radial diffusion assays. We also used specific reporter bacteria to investigate whether ES affects quorum sensing systems. Results: g of ES prevented S. aureus biofilmAlready 0.2 g of ES rapidly broke down established biofilms. In contrast,formation and 2 ES initially promoted P. aeruginosa biofilm formation, but after 10 h the 99 biofilms collapsed. Break down of established P. aeruginosa biofilms occurred after 10 h and required 10-fold higher amounts of ES than for S. aureus biofilms. Interestingly, boiling of ES abrogated their effect on biofilms of S. aureus, but not of P. aeruginosa, indicating that different molecules within ES are responsible for the observed effects. Furthermore, modulation of bacterial biofilms by ES did not involve bacterial killing or effects on bacterial quorum sensing systems. Conclusion: ES prevent S. aureus biofilm formation and degrade established S. aureus biofilms, but appear less effective against P. aeruginosa biofilms. Our results are consistent with clinical observations that maggots are effective against chronic, infected wounds although wounds infected with P. aeruginosa are more difficult to cure than wounds infected with S. aureus. Parasite role reversal Worms on trial in rhinitis and asthma University of Nottingham, NG7 2RD FINDINGS METHOD INTRODUCTION The allergic phenotype has a controlling influence over hookworm infection (IL5/eosinophilia/IgE)1-4 Group 1 ConA stimulation Therefore, hookworms may have a bystander effect on the development of allergic disease6 through the combined production of antiantiinflammatory molecules, and (possibly) the induction of regulatory T cell propulation5. 0 3 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 IFNIFN- 6 0 Weeks ILIL-5 pg/ml 400 300 300 200 200 100 100 0 0 0 3 0 3 6 Weeks CONCLUSION STUDY DESIGN GROUP 1 CD4/CD25 0.5 0.4 0.4 X 109 per litre X 109 per litre To determine, in normal volunteers, an infective dose of N. americanus which: 0.3 0.2 0.1 gave the desired infection intensity, equating to protection against respiratory wheeze, encountered in the study of Scrivener et al. (2001)6. We can administer a dose of N. americanus to patients exhibiting bronchial hyperhyper-responsiveness without adverse effects. GROUP 2 CD4/CD25 0.5 CD4/CD25 0.3 0.2 0.1 0.0 0.0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Weeks Weeks GROUP 1 GROUP 2 17.5 WBC Eosinophils 15.0 X 109 per litre X 109 per litre was asymptomatic, yet induced a measurable immunological response (as we will be looking in the longer term to induce protective levels of regulatory T cells). 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 12.5 10.0 7.5 WBC vs Eosinophils 5.0 did not cause adverse effects in the airways of patients demonstrating bronchial hyperhyperresponsiveness. Weeks We have now embarked on Phase 3 of the trial in asthmatics. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 REFERENCES Weeks 1.5 1.0 0.5 Airway responsiveness 0.0 -0.5 -1.0 -1.5 -2.0 These patients responded with a measurable immune response to the dose with data encouraging the belief that the immune system is sufficiently engaged to follow up with an investigation into regulatory T cell expansion. 2.5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Change in pd10AMP (doubling dose) To ensure that the safe dose selected in normal volunteers: During the first four weeks bronchial reactivity was greater (not significant) in the hookworm group compared with placebo. Therefore, there appears to be no deleterious effect on bronchial bronchial reactivity during pulmonary larval migration. 0.0 These data are published7. PHASE 2 – SAFETY 2 Whole blood was analysed for differential leucocyte counts. Changes in bronchial reactivity to AMP were measured. 6 Weeks PHASE I - SAFETY I Human peripheral blood lymphocytes (PBL) were isolated and stimulated with lectin (Concanavalin A) and compared with a medium control. After 4 days in culture, supernatants were assayed for cytokines (IFN (IFN and ILIL-5) using a multiplex assay system. PBL were labelled for CD4 and CD25 expression and analysed by flow cytometry. cytometry. Group 2 ConA stimulation 500 * 400 pg/ml 6 600 * 500 3 Weeks Group 1 ConA stimulation 600 A double blind, controlled clinical trial, will allow firm conclusions to be made. Whole blood was taken over a period of 12 weeks post treatment, treatment, processed as follows. Group 2 ConA stimulation 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 pg/ml pg/ml Hookworms modulate the allergic response to survive. Group 1 Group 2 1. 2. 3. 4. 5. 6. 7. Pritchard and Walsh (1995) Parasite Immunol. Immunol. 17, 605605-607. Pritchard and Brown (2001) Trends Parasitol. Parasitol. 17, 169169-172. Culley et al. al. (2002) Eur. Eur. J. Immunol. Immunol. 32, 13761376-1385. Quinnell et al. (2004) J. Infect. Dis. 190, 430430-438. Carvalho et al. al. (2006) Parasite Immunol. Immunol. 28, 525525-534. Scrivener et al. (2001) Lancet 358, 14931493-1499 Mortimer et al. (2006) Amer. J. Trop. Med. Hyg. Hyg. 75, 914914-920. ACKNOWLEDGEMENTS induced a measurable immune response. Clinical Immunology 100 J. Britton, J. Feary, Feary, A. Venn D. Pritchard, D. Hooi, Hooi, A. Brown A New in-vivo model for testing effects of topically biotherapy wound healing. Nasser M. Shahri Department of Biology, Azad University, Iran Studying in texts, documents and sources of Islamic and Iranian traditional Medicine suggests the point that taking care of wounds and injuries, is one of the special clinical subjects in their rapid healing. Most of the well-known ancient physicians of Iran have been aware of this matter by experience. They knew that by using medicines the rapid healing and recovery process can be made. In doing this research it has been tried to study, from a scientific viewpoint the efficacy of some of the traditional healing agents by conducting some experiments so that views can be made concerning the efficacy of these stuffs. Therefore, after making 200 regular holes (circular incisions with a diameter 8mm) on the ears of 30 rabbits, some of these holes were chosen as the test wounds and some of them, with the same anatomical position, were selected as evidence control wounds. The evaluation standards for quick diagnosis of the healing and recovery (reconstruction) procedures have been the measurement of the diameter of the test and control wounds, comparing them and also comparing the percentage of the recovered (reconstructed) levels in the test and evidence samples, during 7th-13th days after formation of the scar thus, based on the above-mentioned standards, the prepared charts and considering the evaluation standards of the rapid diagnosis of the healing process in following this research, in general it can be stated, with a scientific standpoint, that some of the traditional healing agents of animal origin (some of animals fats, in particular, cow butter oil and duck fat in some cases mixed with the ash of human hair); provided that they are noninfectious can interfere effectively in rapid healing and recovery/reconstruction of the damaged tissues of laboratory animals, in other words they can shorten the time necessary for healing and recovering. 101 Macroscopic and microscopic survey of the effectiveness degree of some of the biostimulators used in traditional medicine in the process of healing of sheep skin wounds. Dr. Nasser Mahdavi Shahri, Colleges of science, Ferdowsi University, Iran In view of the importance of the rapid healing of dermal (skin) wounds in veterinary medicine and taking into consideration that some of the stimulants of healing process, with animal origin in Iranian traditional medicine have been introduced as healing medicines, in the execution of this research, it has been tried to make studies on the degree of effectiveness of these materials in the healing process and recovery of sheep dermal (skin) wounds. For this purpose some wounds were made in forms of splits or by taking epidermis, dermis and hypodermis parts. Some wounds were selected as control and the other group as test, and both groups were almost at the same anatomical position. The wound of test group were locally treated once a day by traditional healing agents. Those used in this test were cow butter oil and duck fat. The principle of this research was to compare and evaluate control and test wounds both macroscopically and microscopically during 1-14 days. For this purpose , in macroscopic evaluations of the wounds , rapid formation of the wound crust , the wound extent (the distance between the wound edges) and the clinical appearance of the wounds, were the diagnosis basis, and in microscopic level has been possible. Cases: Rapid formation of epithelium, quicker omission of blood clots and damaged tissues, regarding the amount of the produced scar and the amount of the inflammatory cells, were the comparison basis. The result of the evaluations between the test and control samples showed that in all of the mentioned cases, the test wounds heal more rapidly than the control wounds. In making a general result, these cases in the test samples can be related to using the traditional healing agents. Using some of the synthetic healing ointment locally and by the comparison of their functions with the afore-mentioned traditional healing agents, the effectiveness of the traditional ones is proved in this research. 102 Evaluation of the efficacy of curcuma longa rizom ointment in healing process of burning wounds Nasser Mahdavi Shahri, H.Rakhshandeh, E.Nasrabadi Department of Biology, Ferdowsi University, Iran The application of curcuma longa has significant important in traditional medicine of eastearn asia and IRAN.in this study we used ointment of powder rizomCurcuma longa 0/010 on burned wound in pabbits skin. We collected 14 male rabbits and shaved dorsal part of them and then we burned wound part of them by chloidric for 50 seconds. We produced 6 burned wound on dorsal part of each rabbit and so we had 90 burned area in adition. We collected 4 wounds as test and i wound as control asd i wound as negative control. Every day and after bunting we used ointment of powder of rizom on test wound vazeline on control wounds. This was continued for 24 days. For historical studies we sampled from wounds in days 3, 6, 8, 16 after burning. The specimens were examined for the following elements of wound healing to determine a potential treatment response. Percent of wound epithelized epithelizal thickness (cell layer mm) and amound of scab formation every day, we measured tt of wound recovery. Result of clinical studies show that healing process in wounds that received ointment of powder rizom is faster than control wounds that received vazeline. Effect of a natural polymer "rabbit vitreous" to wounds therapy on an experimental model Nasser Mahdavi Shahri, S.samareh Mosavi Department of Biology, Ferdowsi University, and Department of Biology, Azad University, Iran Today for production of natural polymer, used different cells culture or isolation material from bioorganisms. There are much hyaluronic acid as natural polymer in ultrastructure of rabbit vitreous. It can adhesion 103 to surface of cells and led to cell immigration, regeneration of rabbit pinna punch hole. The rabbits that we used in this study were the Newzealand white rabbits. We used standard metal earpunched that marked for this purpose. We created well-circum scribed circuler surgical wound of 8mm in diameter. One of the rabbit pinna day-to-day treated with rabbit vitreaos and the other one was as control statistical analysis showed that deviation pinna nonregeneration tissue between vitreous treat and control was significant. However this deviation on day 15th after ear punching was maximum level of significance (p<0.05) vitreous treated wounds had an epithelial percentage of 76.16 and control had an epithelial percentage of 63.9 (day 14). Vitreous treated wounds and control had an epithelial percentage of 90.34, 79.1 respectively (day 30). Vitreous treated wound had an epithelial thickness of 59.16 pm and control had an epithelial thickness of 45 pm (day 14). Vitreous treated wounds and control had an epithelial thickness of 107.66, 94.54 pm respectively (day 30). MAGGOTS THERAPY IN SPECIAL WOUND TREATMENT Zohrabyan A. Sureni Surgical Department, Central Hospital, MOD, Yerevan, Armenia Purpose: To avoid from expanded surgical intervention on total removal of prolene mesh implant after plastics of a ventral hernial gate and shor tening of the treatment terms. Methods: Using medical maggots of Lucilia Sericata for wound debrim ent from necrotic tissue and infection. Results: The patient (43 year old gentlemen) was presented to the surgical department of Central Clinical Military Hospital with long-time functioning purulent fistula on a place of postoperative scar . In fight he was wounded in abdomen. After that case he was exposed to 104 numerous operations. Because of these numerous operations there was formed a huge ventral hernia. For closing this ventral hernia was done hernioplasty and the hernial gate was closed with mesh prolene implant of 30х30 cm size. Four years later purulent fistula was formed on the place of postoperative scar. Conservative treatment and modern medicines had not provided his recovery. Also he refused from total surgical removal of the implant. For this reason was offered maggot therapy. Narrow pass of a fistula was insufficient for the penetration of maggots, therefore the part of skin with subcutaneous and fibrous tissue and part of mesh were excised. Results from intraoperative cultures showed Pseudomonas aeruginosa without sensitivities to antibiotics. When the wound formation was completed two pots of maggots were applied. Three days later there was noticed improvement in the wound condition. The maggots were appl ied only two times. As a result the wound was cleared and implant was c overed with granulations. A month later on the place of the wound was generated scar. Conclusions: This case shows new and unexpected opportunities of th e maggot therapy. It was abundantly clear, that the maggots suppress ed wound infection so, that it had epithelized without removal of a fore ign body. MAGGOT DEBRIDEMENT THERAPY IN SLOVAKIA Takáč P, PhD.1 ;Čambal M, M.D.2; Krumpálová Z, PhD.1 Kozánek M, PhD.1. 1 Institute of Zoology,Slovak Academy of Sciences, Bratislava, Slovakia 2 1st Depatment of Surgery, University Hospital and Faculty of Medicine, Comenius University, Bratislava, Slovakia The maggot debridement therapy is used in Slovakia since August 2003. First sterile blowfly Lucillia sericata colony was established in the Institute of Zoology, Slovak Academy of Sciences (SAS) in Bratislava. First patients were treated on the 1st Department of Surgery, 105 University Hospital and Faculty of Medicine, Comenius University in Bratislava. The successful cooperation of scientific workers from SAS and physicians resulted in establishing of non-profit organization MEDALT (Medical alternative) settled in the building of the Technology Incubator built up with the European Union support. In 2005, MEDALT won the project supported by European Social Foundation. The main idea of the project is to develop research and development centre, which will be focused on the development and dissemination of the biomedical therapeutic methods in Slovakia. On the example of maggot debridement therapy, we would like to create some model applicable also for development of further biotherapeutic methods (i.e. hirudotherapy, apitherapy, helmintotherapy...) and introduce these methods to clinical practice. Up to now MEDALT has successfuly spread maggot debridement therapy in hospitals all around Slovakia. By straight application of Lucillia sericata larvae in wounds two layered dressing is used to prevent larvae escape. The bottom layer (cage layer) is fixed to the intact skin surrounding the wound. Its purpose is to keep maggots in the wound and to protect surrounding skin from aggressive maggots juice side effects. We use colostomy pads Coloplast. The second layer is chiffon, which is attached by a special disperse glue (developed in the Institute of Polymers, SAS, Bratislava) to the bottom layer. This layer allows maggots to breath oxygen and to drain out liquefied necrotic tissue and wound secretion. One day old sterile larvae are applied under plastered chiffon. Top layer is a dry gauze which is usually placed over the cage layer to absorb the liquefied necrotic tissue. Only this layer is regularly (every 4-6 hours) changed by the nursing staff. “Biobag” is our challenge. Simuntaneously we are designing a new “biobag” for maggot debridement therapy. By using biobags we didn´t notice any larvae escape. The application, removal and disposal is simple and practical. We can recommend this way of maggot’s application direct into the wound. 106 The optimal conditions for medicinal maggots before and during shipment Hitoshi Takase M.D. Biotherapy Medical Co., Ltd, Japan Purpose: To investigate the optimal conditions for medicinal maggots before and during shipments, such as period, feed, temperature, and ventilation. Method: Newborn sterilized maggots are put on the various conditions for certain periods, and then their medicinal effectiveness on wounds is assessed by their size, survival rate, and amount of food ingested. Result, conclusion: The experiment is now being conducted and the results are reported at the congress. Maggots to the Rescue: Using maggot therapy for wounds in hospice patients Aletha W. Tippett, M.D. Wound Consultant; Hospice Medical Director, Ohio, USA Purpose: Wounds are a problem that affect over 1/3 of hospice patients (1), and often these wounds are severe, with various causes: ischemic, pressure, diabetes, trauma and surgical. Many times debridement of the wounds is indicated, due to infection, sepsis, pain, necrosis, or gangrene, but patients usually are not candidates for surgical debridement, including amputation of limb. Often other debridement forms such as wet to dry, or enzymatic, are not appropriate due to issues of pain, cost, or time constraints (e.g. infection with sepsis needs urgent debridement). Sometimes debridement is not seen as consistent with palliative care. This paper presents a selection of case studies of the use of Phaenicia sericata larval therapy (maggots) for wound debridement in hospice patients. 107 Methods: A selection of case studies from wound treatment of hospice patients over a three year period to illustrate wound types, problems, and outcomes are presented. The cases were selected to be representative of the types of wounds encountered that could benefit from maggot therapy. Results: In all cases goals of therapy were achieved. Results included: resolution of sepsis, prevention of amputation, elimination of infection, reduced pain and odor, and in some cases, wound healing. Therapy was well tolerated in all cases. Conclusions: Maggot therapy is a very viable option for treating complex wounds in hospice patients when debridement is indicated. Maggot therapy is rapid, inexpensive, nearly painless, simple, quite gentle, and consistent with goals of palliative care. Standard protocols are published and easy to follow (2). Families and patients are very involved, and grateful for such innovative therapy, with newfound respect for one of nature’s often maligned creatures. Application of maggot therapeutics for repairing serious infective wound Jiangning Wang Dalian University Medical Acad, Dalian, China Objective: To research the application of maggot therapeutics for repairing serious infective wound. Methods: Two methods were used for getting clean maggot, one is flyblow asepsis, the other is larva antiasepsis. No bacterium or virus exist in the body of the maggots and the maggots are vivid. From 2005to 2007, we took use of maggot therapeutics to repair 4 cases serious infective surfaces. Fifty to one hundred aseptic maggots were put to the infective surfaces every time, aseptic dressing overlied the surface, nylon net covered outside the dressing. The maggots and dressings were changed every two days. Results: Average cure period was 7 days, all necrotic tissues of the wound were cleanup, fresh tissue developed. When bacterium culture of the wound surface shows no bacterium, the wound was covered with skin graft, all 108 results were satisfied. Conclusion: Maggot therapeutics is an effective biological therapy for repairing serious infective wound. The postoperative wound infection is still one of major complications of replantation. Failure to control infection can lead directly to vascular thrombosis and in turn loss of replanted extremity. The use of maggots for wound debridement has a long history and has been lately re-introduced for treatment of intractable wounds. In this report, we present the experience of successful debridement of a severe infected wound after forearm replantation with the maggot therapy. The results and mechanism of the maggot therapy are discussed. Today replantation has become clinical routine for salvage of amputated extremities with great success. However, since many cases of amputation are caused by agriculture and manufacture injuries, gunshot, and traffic accident with relative wound contamination, the postoperative wound infection is still one of major complications of replantation. The presence of infection can lead directly to vascular thrombosis, sepsis, and loss of replanted extremity. Treatment of infected wound needs good debridement and coverage with vascularized tissue, so we begin to choose maggots therapy for infected wound. The healing and antibacterial function research of the maggot secretion on the ulcer area Shouyu Wang Dalian Medical University No.1, Dalian, China Objective: Investigate influence of treatment with maggot secretion on wound healing of diabetes ulcer and to study the antibacterial function of maggot therapeutics for decubitus ulcer after spinal cord injury. Methods: (1) Some circle wounds were made in back of diabetes rats, The injured area spreads the maggot secretion. The area size were detected after injury with the transparent membrane method and carried on the pathology examination. (2) Using Staphylococcus aureus and Pseudemonas aeruginosa as the indicator organisms which from the infective wound of 109 decubitus ulcer after spinal cord injury, the hemolymph secretion of the larvae which was induced by the bacterium with pricking was collected, the antibacterial activity was tested with disc method, recorded the antibacterial effect, the hemolymph secretion of the larvae which was not induced by the bacterium and Cefoperazone Sodium as the contrast groups. 5 cases suffered from decubitus ulcer after spinal cord injury was treated with maggot therapeutics, observe the exchange of wound surface and checked the bacterium. Results: (1) The injured area achieved 100% recovery in latter 4 weeks, at the same time, wounds of diabetes big rats did not recover, and expanded unceasingly, in the pathology examination maggot secretion group regenerated epidermis , the small blood vessels and the fibrinogen cells. (2) The secretion of the larvae had antibacterial activity on Staphylococcus aureus and Pseudemonas aeruginosa. The secretion of the larvae which was induced by the bacterium had stronger antibacterial activity. Average cure period of 5 cases was 9 days, fresh tissue developed, necrosis tissue cleaned, no bacterium existed on the ulcer surface after bacterium culture. Conclusions: (1) The maggot secretion has the obvious promotion function to the ulcers. (2) Maggot therapeutics has effective antibacterial function on decubitus ulcer after spinal cord injury. Effectiveness of Maggot Therapy in Chronic Infected Wound with MRSA K.J. Yoon1, Y.B. Shim1, S.S. Ha1, H.T. Kim2, K.S. Kim, PhD2, N.H. LEE2 1 Department of neurosurgery, St. Peter’s Hospital; 2 Medilarvatech Purpose: There have been reports of using maggots to treat the bacterial infection that are resistant to antibiotics, especially, methicillin resistant staphylococcus aureus(MRSA) or to clean and diminish the size of the wounds for skin graft bed in burn or pressure sore patients. We produce the sterilized maggots and report the result of clinical applications of maggot therapy in the treatment of MRSA infection, pressure sore and tissue 110 defects. Material and Methods: From Aug 2005 to May 2007, 33 patients (7- 65 years old, mean 44.5, male 17, female 16) were treated: 7 MRSA infection cases( 3 pressure sore; 4 postoperative wound), 15 Diabetic foot, 6 crushing injuries, 5 acute burn wounds. We used sterilized, 1 day old larvae (1-2mm in length) of the flies, “Phaenicia sericata” (product by Medilarvatech). Maggot application was confined to the wounded area with a specially designed bandage and the larvae eat all the infected tissues. The bandage was removed after 3-4 days, when the larvae are mature enough and cannot consume the infected tissues anymore. The treatment was repeated 3 to 20 times (mean 8.3 times) depending on the severity of each wounds and follow-up period was 3 to 24 months (mean 12.5 months). Each treatment used 100-800 maggots. No antibiotics were used during the treatment. Results: A patient with crushing injury in the ankle gave up the treatment due to severe pain. In all the 7 MRSA cases, the discharge of the pus was stopped and wound was spontaneously healed at the end of the treatment. In all other patients with tissue defect, wound became clean and diminished in size, and secondary skin graft was performed successfully. There were no side effects. Conclusion: Maggot therapy is very effective and could be the new strategy in the treatment of MRSA infection. And also in cleansing and diminishing tissue defect in burn, pressure sore and crushing injuries to promote wound healing and get better outcome in skin graft 111 New development about medical leech therapy Yavuz Dinler, Ph.D. (Turkey) Leech therapy has been used in Turkey for at least 400 years. Today leeches are collected in large quantities from at least 600 lakes in Turkey. . More than 20 hospitals and clinics in this country practice hirudotherapy. Since 1994 our group has been using medical leech therapy and we have applied over 10.000 individual treatments. Dukin's method is used for the treatments and we have achieved with very good results for more than 10 different clinical conditions. Since 2006, we have also treated patients with glaucoma and other eye ailments. We intend to use this treatment modality in the future for skin cancer and leukemia. Effects of bee venom on melanogenesis and dendricity human melanocyte proliferation, Ai-Young Lee, M.D., Professor, Department of Dermatology, Dongguk University Hospital, Gyenggi-do, Korea Bee venom (BV) has been clinically used to control rheumatoid arthritis. In the present study, we examined the effect of BV on the human melanocyte proliferation, melanogenesis and dendricity, and its signal transduction in vitro. BV increased the number of melanocytes dose dependently through PKA, Erk, and PI3K/Akt activation. Melanocyte dendricity and the level of cAMP were also increased by BV treatment. Moreover, BV also induced MITF and tyrosinase expression by phosphorylation of CREB through activation of PKA and Erk. Overall, in this study, we demonstrate the effect of BV on melanocyte proliferation, melanogenesis and dendricity through complex signaling pathways in vitro, which suggest a possibility that BV may be a useful substance in the treatment of re-pigmentation. 112 113 114