• Autism and Botulinum Toxin
• The Use of Botulinum Toxin as a Pharmaceutical
• Risk of Allergenic Reactions from Genetically Engineered Foods

Briefly, when children with severe late-onset autism were treated with oral vancomycin, the devastating behavioral symptoms of regressive-onset autism diminished until the antibiotics were discontinued. Research at the University of California–Los Angeles by Dr. Finegold and collaborators implicated toxigenic clostridia as contributing to the autistic symptoms. Apparently, their colonization and elaboration of neurotoxins contributed to the behavioral symptomatology of autism.

Eric Johnson's laboratory has begun a collaboration with Drs. Finegold at UCLA and Rolfe at the University of Texas to try to isolate and characterize neurotoxins from clostridial strains that were obtained from the autistic children. The strains have been sent to our laboratory and we are evaluating them for production of neurotoxins and will also use the infant-botulism mouse model test for neurotoxic effects on colonization of the strains in the gastrointestinal tract of infant mice.

From a medical perspective, it is clear that botulinum toxin can be used to treat more syndromes than muscle spasms. It has been successfully used to treat pain including tension and migraine headaches, myofascial pain and fibromyalgia. It has also been used to treat a disorder characterized by excessive sweating of the palms, face, underarms, and other areas containing sweat glands. The toxin has been used for cosmetic purposes, diminishing wrinkles, frown lines, flaring nostrils, and other "disorders", thus helping to beautify humans who can afford the treatments. It has also been used to assist people who have difficulty swallowing or speaking, as well as those who have severe constipation due to tightness of the anal sphincter. In an extraordinary case, the toxin successfully relieved a woman with vaginismus, or inability to relax the vagina, and the treatment enabled conception and birth of a child. Botulinum toxin has even seen use as a treatment to silence barking dogs, to keep peace in the neighborhood.

As is characteristic with the more traditional syndromes that have been treated with botulinum toxin, such as hemifascial spasm and blepharospasm, the relaxation of muscles and even pain is long lasting, having a duration of several weeks to months. The duration of action depends upon the serotype administered, with type A having by far the longest duration compared to serotype B and E. The explanation for the varying length of duration is not fully clear, but it appears to correlate with the severity of botulism in humans who contract the disease from contaminated foods.

A limited number of drawbacks have become apparent during the past decade of medical use. Certain individuals appear to be refractory to treatment, and in most cases this appears to be due to the formation of neutralizing antibodies when an immunogenic threshold is reached. Another drawback is the diffusion of the toxin to neighboring nerves, causing a transient ptosis in the surrounding muscles. Interestingly, the antibody problem and the regional ptosis appear to be related to particular batches of toxin and their method of purification and formulation. Thus, the methods used in manufacturing are paramount to achieve a high quality pharmaceutical with minimum side effects.

After more than a decade of use, it is becoming apparent that potential improvements could be made in the preparation of the toxins to increase their efficacy. For example, toxins could be designed to be less immunogenic, or to have a longer duration of action. Improved formulations and delivery systems could improve the pharmaceutical properties. Our laboratory continues to conduct research on many different aspects of botulinum toxin including issues in food safety, control of intestinal botulism, improved methods for detection of the toxin, and development of more efficacious toxin preparations for medical treatment of humans.

Currently, the exact characteristics of proteins which induce allergic reactions in humans are not fully known. Heat stability and resistance to digestive conditions are some of the potential characteristics, but this is not a universal phenomenon. Recent studies from Japan (Yagami T et al., J. Allergy Clin. Immunol., 2000; 106:752–762) indicate that some allergenic proteins are easily digestible using in vitro methods and that this property alone is not predictive of the protein's allergenic potential.

When new proteins that have not previously been identified as allergens are introduced into foods, it is not possible to precisely predict their allergenic potential with our current scientific methods. When a known allergen is genetically engineered into a food, it is possible to assess the risk as was demonstrated by the previously reported study on soybeans that were transgenically modified with a Brazil nut allergen (Nordlee JA et al., N. Engl. J. Med. 1996; 334:688–692).

Consumers, the scientific and medical community, and the regulatory agencies eagerly await the upcoming Environmental Protection Agency's decision on whether or not to release genetically modified corn for human consumption. Stay tuned.

I am originally from Baton Rouge, Louisiana, a city known for its great food, folks, and fun. I received a Bachelor of Science degree in Biological Sciences from Southern University and Southern A&M College. It was then that I decided to pursue graduate studies in Environmental Toxicology because of the numerous potentially environment-polluting facilities in the area. I have been a graduate student in Environmental Toxicology since the fall of 1998. After a year's rotations, I decided to join Professor Eric Johnson's laboratory for my graduate training. I am currently studying the regulation of carotenoid biosynthesis in the very interesting yeast Phaffia rhodozyma. The overall goal of my research is to determine genes and proteins involved in the regulation of the carotenoid astaxanthin in P. rhodozyma. I have begun to establish an experimental system in which astaxanthin biosynthesis is expressed in response to reactive oxygen species and ethanol. At the 2000 Food Research Institute Annual Meeting, I received the prestigious Edward J. and Katherine L. Schantz Fellowship. In the spirit of the Schantz Fellowship's theme of helping develop and train young scientists, I am currently a volunteer at Dane County's Boys and Girls Club as a tutor and have served as a mentor for several summer research programs at the University of Wisconsin–Madison.

John P. Price,  Ph.D. candidate

Wenyan Zhang, M.S., Research Specialist

I am from Yunnan Province in the southwest corner of China, a region that neighbors Myanmar, Laos, and Vietnam. I obtained my B.S. in Biochemistry from Sichuan University in 1998, then I worked for 4 years as a technician in the Institute of Medical Biology, Chinese Academy of Medical Sciences. My duties included producing and testing attenuated active hepatitis A vaccine and studying the effects of the vaccine in animal models. I went to Peking Union of Medical College for my graduate studies in 1992 and earned a Master's degree in Immunology. In 1995, I came to the United States to join my husband, who was a postdoctoral fellow at UW–Madison. After staying at home for 2 years to rear my first child, I worked in Dr. Richard Atkinson's laboratory in the Department of Nutritional Sciences, studying adenovirus-induced obesity. I started working in Dr. Eric Johnson's laboratory as a technician this April. My work involves testing a novel antimicrobial system for control of foodborne pathogens. More specifically, I have tested the effect of terpenoid compounds on the sensitivity of bacterial foodborne pathogens to various sanitizers. Also I am working with Marite Bradshaw to screen the mutants of transconjugants by transferring Tn916 transposons from Streptococcus faecalis CG110 to Clostridium botulinum 62A. I enjoy living in Madison with my husband Hong, our 4-year-old son Jerry and 10-month-old daughter Jessie. We love Madison for its beautiful scenery and nice people. As a person from a subtropical climate, Wisconsin's winter is a little too long, but we find the summer extremely beautiful.

I joined Dr. Yu's research team on November 1, 2000. I have worked in the molecular genetics of Fusarium mycotoxin biosynthesis at the National Center for Agricultural Utilization Research of USDA in Peoria, Illinois, for a year and half. There, I identified and characterized four structural genes (FUM6, 7, 8, and 9) involved in the fumonisin biosynthetic pathway.  I received a Ph.D. degree in Agricultural Science in Seoul National University (SNU) in February 1998 with the title of my thesis, "Isolation and characterization of C-type fumonisins produced by Fusarium oxysporum O-1890 and production of fumonisins by Fusarium species." My research led to purification of four new fumonisin derivatives, and determined their structures using various analytical methods. I also analyzed mycotoxins such as trichothecenes and fumonisins from corn, barley and solid cultures of Fusarium species from various sources. Now my research goal in Dr. Yu's lab is to investigate regulatory mechanisms of fungal growth, sporulation and mycotoxin production in Aspergillus and Fusarium. The proposed model of Aspergillus nidulans will be tested in major Fusarium species, F. graminearum and F. verticillioides. Initially the major genetic elements (G-protein subunits and Regulator of G-protein signaling) will be identified using reverse genetics approaches. These major genetic elements will be used to clarify the signal pathways in Fusarium species, hopefully contributing to our understanding of the mechanisms controlling growth, development, and mycotoxin biosynthesis in Fusarium. I have been married for 10 years and have a 9-year-old son and a 4-year-old daughter. My husband is working in agrochemical marketing at the Novartis company (changed to Syngenta). I am interested in painting beautiful nature by pastel and watercolor. I also love music and sports. I plan to enjoy living in Madison and working with Dr. Yu. Hopefully we can make progress in fungal molecular genetics and mycotoxin research, with a long-term goal of eliminating mycotoxin contamination and toxic fungal infection from the field.

Jeong-Ah Seo, Ph.D.,  Research Associate

Appointments

• Human Food Safety Panel, IFT

Ron Weiss, Research Program Manager of FRI, has been appointed to the IFT Human Food Safety Panel. The panel also includes former FRI faculty and staff members John Luchansky, Research Leader, Eastern Regional Research Center, USDA, and Assistant Professor Susan Hefle, Food Allergy Research and Resource Program, University of Nebraska, Lincoln. The panel produced the section on Food Safety in the IFT Expert Report on Biotechnology and Foods.
 
• Wisconsin Food Safety Task Force

Mike Pariza has accepted an invitation from the Wisconsin Department of Agriculture, Trade and Consumer Protection to participate in improving Wisconsin's food safety system as a member of Wisconsin's Food Safety Task Force. The Task Force is a partnership among the state and local food safety agencies, food businesses, academia, and consumers. The U.S. Food and Drug Administration serves in an advisory role and provides funding for travel and meeting facilities, and the Wisconsin Department of Agriculture, Trade and Consumer Protection, Division of Food Safety, provides coordination and administrative support for this effort.
The objectives of the Task Force are to:
 - Identify and prioritize food safety issues, problems, and concerns
 - Identify efforts in progress to improve the nation's food safety system
 - Build connections to and provide support for identified improvement effort
 - Build Wisconsin-specific improvements in areas determined to be unique and important for the State's food businesses and consumers

• Fish Oils for Improving Memory?
• Is Food a Source of Antibiotic-Resistant Pathogens?

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DHA (docosahexaenoic acid), an n-3 polyunsaturated fatty acid found primarily in fish oils and a few vegetable oils like mustard seed oil, has been shown to have beneficial cardiovascular effects. Experiments with laboratory animals and some human trials demonstrated that dietary DHA can reduce blood pressure and the incidence of thrombosis or internal blood clots.

Data from studies with rats and monkeys have shown that dietary DHA is rapidly absorbed from the gastrointestinal tract and incorporated into lipids in the brain. Further, higher DHA levels in the diet and in brain lipids have been correlated with improved learning and memory in laboratory animals. Some studies have found that brains of patients with vascular dementia or Alzheimer's Disease have significantly lower DHA concentrations, on average, than the brains of other older persons without dementia.

To determine whether DHA affects brain function, a Japanese research group examined cerebral blood flow in young adult (6-year-old) and aged (18-year-old) conscious monkeys that had consumed diets containing a placebo or 150 mg DHA/kg body weight/day for 1 or 4 weeks. When neurons in the cerebrum of the brain are activated by some stimulus, blood flow to that area is increased in response to the need for more glucose and oxygen. Such an increased blood flow can be detected by giving the monkeys radioactive water and using high resolution positron emission tomography (PET).

PET scans of control monkeys which did not receive DHA supplements indicated that cerebral blood flow in the younger animals increased to 141% while that of the older monkeys increased to only 116% of resting blood flow (considered as 100%) when the monkeys gripped a small vibrator with their forepaws. After 1 and 4 weeks of DHA supplements, cerebral blood flow in the aged monkeys increased to 127% and 133%, respectively, of the resting blood flow. These results demonstrated that, in old monkeys, dietary DHA improved cerebral blood flow elicited by a stimulus to nearly the level observed in younger animals. Such a positive physiological effect suggests that DHA supplements may improve the cholinergic neuronal system impaired in several diseases such as vascular-type and Alzheimer's dementia.

In many cases, the same, or structurally similar, antibiotics are used in agriculture as in human medicine. There has been increasing concern that the antibiotics incorporated into animal feed each year are inducing the development of antibiotic resistant bacteria that may become a serious human health problem. Resistant bacteria which are human pathogens may cause diseases that are difficult to treat because of the limited number of available, effective antibiotics. Even if the resistant bacteria are not human pathogens, they may still be dangerous because they can share genetic information and transfer their antibiotic resistance genes to other bacteria that are pathogenic.

Campylobacter spp. are commonly found in food-producing animals, and the human pathogen C. jejuni, the most common cause of foodborne illness in the U.S.A., has been detected in as many as 80% of broiler chicken carcasses in some surveys. Recent data from a 1999 survey by NARMS (National Antimicrobial Resistance Monitoring System) of 180 chickens purchased at grocery stores indicated that Campylobacter was present on 44% of them. Antibiotic resistance was common among these isolates, with 24%, 32%, and 65% resistant to ciprofloxacin, naladixic acid, and tetracycline, respectively. The introduction of fluoroquinolines (such as ciprofloxacin) into feed for chickens in 1995 was controversial because these antibiotics had been saved for human infections that are not treatable by more common antibiotics. Molecular subtyping has demonstrated an association between resistant C. jejuni strains from chickens in Minnesota and infections in Minnesota residents. NARMS is now preparing a risk assessment on the human health impact of fluoroquinoline-resistant Campylobacter associated with the consumption of chicken.

Recently a 12-year-old child in Nebraska was diagnosed with a Salmonella typhimurium infection, and the causative strain was resistant to 13 antibiotics including ceftriaxone. Analysis of this isolate and of 4 other S. typhimurium isolates from local cattle by pulsed gel electrophoresis revealed that the human strain was indistinguishable from one of the cattle isolates which also had the same extremely rare pattern of resistance to the same 13 antimicrobial compounds. Although the child did not consume meat or milk from these cattle, it appears very likely that this infection was acquired from the cattle.

Enterococci have emerged as human pathogens in the past decade and have also been found to be resistant to multiple antibiotics. Another survey by NARMS examined isolates of enterococci from chickens and from human stool samples. Of the strains tested, 2% of human and 68% of chicken were resistant to high-level gentamicin doses, and 1% of human and 52% of chicken isolates were resistant to quinupristin-dalfopristin (QD). Both gentamicin and virginiamycin (an analog of QD) are frequently used for disease prevention and growth promotion in poultry in the U.S.A. Since 84% of the chickens from grocery stores had detectable enterococci, there is certainly a significant potential for transfer of drug-resistant enterococci from chicken meat to human consumers.

Another case of antibiotic resistance of concern is vancomycin resistance observed in Enterococcus faecium. Vancomycin is considered a drug of last resort in treating some serious human infections. In Europe a structurally related compound, avoparcin, has been used as a growth promoter in poultry and pigs. For example, during 1994 doctors in Denmark used 24 kg of vancomycin to treat human illness while 24,000 kg of avoparcin were added to animal feed. Early in 1995, over 80% of E. faecium isolated from poultry in Denmark were resistant to avoparcin, but after a ban on the use of this antibiotic less than 10% were resistant in 1998. However, despite a ban on avoparcin use in 1995 in Norway, vancomycin-resistant enterococci were found to persist in broiler flocks.

In 1969 a governmental body in the U.K., the Joint Committee on the Use of Antibiotics in Animal Husbandry and Veterinary Medicine, issued a report (Swan Report) which warned of the potential threat to human health caused by the use of antibiotics in farm animals. Sweden, in 1986, was the first country to prohibit use of antimicrobial drugs for growth promotion in livestock. In 1998, virginiamycin was banned as a feed additive in Denmark, and in 1999 the European Union banned the use of bacitracin zinc, spiramycin, tylosine phosphate, and virginiamycin in animal feeds. Many European countries are concerned enough about the potential transfer of antibiotic-resistant bacteria from poultry and other livestock to humans to recommend that the use of antimicrobial growth promoters be terminated or rapidly phased out. In the U.S.A., there are indications that the FDA will soon act to restrict the use of some antimicrobial compounds used for growth promotion in animal agriculture.