Food Allergy: Modifying the Allergic Response

Allergists/immunologists who treat food-allergic patients are interested in modulating the immune response to food allergens. It is well known that the initiating event is the production of IgE antibodies to food proteins. Previous attempts to "desensitize" patients with conventional immunotherapy (similar to that used to treat bee sting allergy) have proven unsuccessful or caused serious side effects. Thus, a number of new approaches are being investigated. Although not inclusive, these studies involve administration of humanized monoclonal anti-IgE antibodies, plasmid DNA-based vaccines, and immunotherapy with mutated major food allergenic proteins.

Anti-IgE therapy appears to hold a great deal of promise for treatment of serious food-allergic reactions. A murine monoclonal antibody has been developed which binds to human IgE antibodies and prevents their fixing to mast cells (the source of histamine and other mediators involved in allergic reactions). The monoclonal antibody has been engineered to minimize the chance of eliciting its own immune response. Anti-IgE therapy has been extensively studied in the treatment of asthma and is currently being used in the treatment of peanut allergy. The treatment appears to be safe, but would need to be given on a monthly basis throughout the lifetime of the individual. Potentially, it could be used to minimize the adverse reactions to more conventional "desensitization" as a long-term treatment.

Plasma DNA-based vaccines, or variants thereof, are being investigated in the laboratory. In this approach, plasmid vectors containing so-called "immuno-stimulatory sequences" (ISSs) are coupled to cDNA encoding for an allergenic food protein. Injection of the plasmid DNA conjugate or ISSs conjugated to the allergenic protein results in a switch from an IgE antibody to an IgG antibody response. This approach can be used (theoretically) to switch off an established allergic response or prevent its development.

Unfortunately, recent studies showed in the case of peanut sensitivity that some strains of mice had a heightened IgE response to certain peanut allergenic proteins. Further, the long-term safety of such therapy is not known. Although it may not be possible to genetically engineer foods to exclude allergenic proteins, modification of the protein to eliminate IgE antibody binding epitopes on the molecule may be useful as immunotherapy vaccines. As described, one of the limitations of current "desensitization" is the use of extracts prepared from foods (e.g., peanut) which contain the allergenic (IgE-binding) proteins. By mutating the protein in vitro one can create a vaccine which does not lead to an allergic reaction, but "switches off" the production of IgE antibodies to that protein. Laboratory studies indicate the feasibility of this technique. The approach is limited by the fact that foods often contain 3 or more major allergenic proteins. This means a laborious search for the appropriate mutations given current technology.

For the allergist/immunologist, the advances in biotechnology represent an exciting potential for the eventual "cure" of food allergy. Clearly, a better understanding of the molecular biology of food allergens and the human immune response to these proteins is sorely needed.

• Flow Cytometry for the Rapid Detection of Salmonella
• Interactions Between Hemolysin BL, Phosphatidylcholine Phospholipase C, and Sphingomyelinase from Bacillus cereus
• Survival of Campylobacter jejuni — Influence of Protection Systems and Lipids
• A Neutron Makes a Difference

Traditional culture-based methods for the detection of Salmonella in foods require up to 5 days to yield a result, and current rapid tests require a minimum of 2 days. Currently our work is centered on the development of flow cytometric methods for the specific detection, enumeration and viability assessment of Salmonella spp. in foods. To achieve this goal, we are integrating methods developed in three separate areas, including a rapid means of cell concentration and recovery, fluorescence-based determination of cell viability and nucleic acid-based detection of target cells. Advantages of the finished work will include the ability to detect fewer target cells than present rapid methods and the ability to detect small numbers of target cells against a high background of competing microflora.

In recent work we have been using fluorescently labeled Peptide Nucleic Acids (PNAs) for rapid nucleic acid-based detection of Salmonella spp. PNAs are wholly synthetic nucleic acid mimics constructed by grafting adenine, guanine, thymine and cytosine bases onto a repeating backbone of amide-linked N-(2-aminoethyl)-glycine units.

This unique chemical makeup gives PNA oligonucleotides several key advantages over DNA oligonucleotides, including faster hybridization kinetics, resistance to nucleases and the ability to bind to target sequences under high stringency, ribosome-denaturing conditions (e.g. low salt, high pH, high temperature). This latter property allows the use of PNA oligonucleotides for probing regions of the ribosome which may be inaccessible to conventional DNA probes due to higher order structure of the ribosome and protein-binding sites.

Additional work has focused on new methods for in situ amplification of target rRNA prior to hybridization with fluorescently labeled DNA or PNA probes. The resulting amplified signal may enable earlier detection of target cells as well as allowing unambiguous resolution of target cells from background microflora. Finally, we expect that the procedural innovations and refinements developed in this work will be directly applicable to the detection of other pathogens of interest.

Tissue necrosis is characteristic of B. cereus infections, therefore membrane-damaging factors most likely contribute directly to pathogenesis. We examined the modulation of lytic effects caused by various combinations of HBL, SM-PLC, and PC-PLC using erythrocytes from different species as models for membranes with varied phospholipid contents, as might occur in different tissues. The lytic potency of HBL did not correlate with phospholipid content, but lysis by the individual or combined enzymes did. SM-PLC alone lysed ruminant erythrocytes, which contain 46–53% SM. The cooperative action of PC-PLC and SM-PLC was needed to lyse swine and human erythrocytes (22–31% PC and 28–25% SM). SM-PLC synergistically enhanced hemolysis caused by HBL for all erythrocytes tested, which all contained >25% SM. PC-PLC enhanced HBL hemolysis only in cells containing significant amounts of PC (swine 22% PC; human 31% PC). Unexpectedly, PC-PLC inhibited HBL lysis of sheep erythrocytes (<2% PC) and enhanced the discontinuous hemolysis pattern that is characteristic of HBL in sheep blood agar.

Inhibition and pattern enhancement was abolished by washing PC-PLC-treated erythrocytes or by adding EDTA, suggesting that enzymatic alteration of the membrane is not involved, but that zinc in the active site is required, perhaps to facilitate binding. These observations highlight the potential for involvement of multiple toxic factors in B. cereus infections and suggest that the relevant interactions between factors during infections may vary in different tissues depending on membrane composition.

Cold-shock (15ºC, 2 h) and heat-shock (50ºC, 10 and 30 min) also did not enhance survival of C. jejuni in acid (pH 4.0) or NaCl (3%); however, heat-shock increased the survival of strain ATCC 33291 at 55ºC but not strain ATCC 29428. The lack of any detectable protection system that significantly improved survival led us to evaluate the role of food constituents on survival. The survival of two C. jejuni strains (ATCC 33291 and ATCC 43445) in whole and skim milk was greater at pH 3.0 than control cells in Brucella broth. In addition, C. jejuni suspended in oil and then challenged at pH 3.0 significantly enhanced (p<0.0001) survival, as viable cells were still recovered after 6 h of incubation. In more extreme acid conditions (synthetic gastric fluid, pH 2.0), the survival of strains ATCC 33291 and ATCC 43445 was significantly enhanced by suspension in chicken fat. While no enhanced protection to heat (55ºC) was observed by suspension in fat, these results suggest that C. jejuni survival in low-pH environments is enhanced by interactions with lipids.

In general, enzymatic processes prefer the heavier carbon. For example, plants can be divided into two major classifications, C₃ and C₄. C₃ plants (wheat, soybeans), when fixing atmospheric CO₂, discriminate against CO₂ that contains ¹³C, whereas C₄ plants do less discriminating against ¹³C. Hence, C₃ plants have less ¹³C than C₄ plants, and thus are considered "lighter." When an animal consumes the carbon in the plant, the animal's enzymes also discriminate against the heavier carbon molecules, hence the animal has more light carbon than the plants they consume. We fed laying hens diets made up of either C₃ or C₄ plants. We then looked at the carbon in different tissues and even the carbon deposited in eggshell and found that the ratio of the carbon isotopes matched the diet fed.

We found a real world application to this fractionation of carbon when Warren Porter in Zoology needed a means for determining if an animal caught in the wild was anabolic or catabolic. In past issues of the FRI Newsletter I have written about the catabolic consequence of the immune response, wasting muscle and general body weight loss (catabolism). We hypothesized that during immune-induced wasting, nutrients such as amino acids would be available for metabolism as they flowed from degrading muscle. These amino acids could either be redirected into making immune related proteins, or they could be used as fuel, ultimately being metabolized to CO₂. Since the amino acid charging of transfer RNA for protein synthesis involves considerably less metabolism steps than complete metabolism to CO₂, we predicted that the amount of ¹³C in breath would decrease and the amount in newly synthesized proteins would increase. Experimentation proved our prediction was correct.

Currently we are working to develop inexpensive stable isotope mass specs as a biosensor for human and animal health. This allows us to find evidence of immune stimulation (i.e., infection) within minutes by using breath. We believe that continuous on-line monitoring of breath could be of great value in detection of post-operative sepsis. Equally, the health of entire herds and flocks could be passively monitored for disease outbreaks.

Monitoring the fractionation of stable isotopes has value to the food industry as well. Historically, stable isotopes have been used to measure adulteration of foods, such as juices. Fractionation could be used in monitoring food processing such as fermentation or enzymatic treatments. Other applications involve the origin of foods, the source of fertilizer used on foods (such as manure) and the health status of an animal prior to slaughter. As new developments in stable isotope mass spectrometry move forward, cost effective means of measuring a neutron of difference will provide new opportunities for the food industry.

Sean Dineen, B.S.,  Research Assistant

I am currently an M.S. student in Dr. Eric Johnson's laboratory, having received a B.S. degree in microbiology from Cornell University in 1994. After graduating from Cornell, I continued working at the university in the Department of Food Science, then for a year and a half for the New York State Milk Quality Improvement Program, performing microbiological, chemical, and organoleptic analysis of NYS milk and dairy products. In 1996 I joined Dr. Kathryn Boor's Food Safety Laboratory at Cornell as a lab manager/technician. There I worked on several projects involving the survival of E. coli O157:H7 in dairy fermentation systems, molecular typing of dairy product spoilage Pseudomonas, and studying virulence factors of Listeria monocytogenes. I joined Dr. Johnson's laboratory in the summer of 1998. My Master's work involves the cloning, nucleotide sequencing and expression of the gene encoding boticin B, a bacteriocin produced by Clostridium botulinum strain 213B. I plan to defend this work this summer and continue on as a Ph.D. student in Dr. Johnson's laboratory. As a Ph.D. student I will work on the regulation of expression of type A botulinal neurotoxin. My wife, Melanie, is also a Cornell graduate and an M.S. student in the Department of Food Science at UW–Madison. We really enjoy living in sunny Madison (sunny compared to central New York, that is). We were blessed with the arrival of our first born, Maeve Elizabeth, in March of this year. At Cornell I was a member of the heavyweight crew team. So far the requirements of grad school and fatherhood have kept me off the water, but I hope to return some day.

A recent meeting here discussed projects at FRI of interest to the meat industry.  Those attending, rear, L to R: Andrew Milkowski, Senior Technology Principal, Oscar Mayer Division, Kraft Foods; Randall Huffman, Vice-President Scientific Affairs, American Meat Institute; Amy Wong, FRI, Ron Weiss, FRI; Charles Kaspar, FRI. Front: Michael Pariza, FRI; Margaret Dentine, Associate Dean and Executive Director of Research, UW College of Agricultural and Life Sciences; Eric Hentges, Director of Research, National Pork Producers Council.

E. M. Foster  Inducted in Wisconsin  Meat Industry Hall of Fame

Edwin M. Foster, emeritus professor of food microbiology and toxicology, and of bacteriology, and former director of the Food Research Institute, was inducted into the Wisconsin Meat Industry Hall of Fame in May. Dr. Foster was recognized for his research on vacuum-packaging of meat products, sodium nitrite, botulism, and for his leadership in guiding the Food Research Institute to its current status as the leading academic research unit nationwide to work on food safety. Foster, a former president of the American Society for Microbiology and a Charter Fellow of the Institute of Food Technologists, earned B.A. and M.A. degrees in biology at North Texas State College and the Ph.D. in bacteriology at the University of Wisconsin–Madison. After two years as instructor, first at Wisconsin and then at the University of Texas, Foster served in the U.S. Army. In 1945 he returned to the University of Wisconsin–Madison on the faculty in Bacteriology. In 1966 when the Food Research Institute moved from the University of Chicago to the University of Wisconsin–Madison, Foster became its director, a position he held until 1986. When the Food Research Institute achieved departmental status in 1975, Foster became the first chairman of the Department of Food Microbiology and Toxicology. He was named emeritus professor upon retiring in 1987.

Amy was born and raised in Hong Kong. She decided to explore the other side of the Pacific after high school and attended Wellesley College, earning a B.A. degree in molecular biology. She obtained the M.S. in bacteriology, and M.S. and Ph.D. in food science at the University of Wisconsin–Madison. She worked in the late Dr. Bergdoll's lab exploring the intricacies of staphylococcal enterotoxins and toxic shock syndrome toxin. Two years (1979–1980) were spent at the International Institute of Tropical Agriculture in Nigeria investigating the symbiotic relationship between soybeans and rhizobia. On completion of her doctorate she was employed by Playtex Family Products Corp. to set up a microbiology R&D lab and research on the role of tampons in toxic shock syndrome. Amy joined FRI in January 1989. Her research involves isolating, identifying and characterizing staphylococcal enterotoxins and other enterotoxins, and virulence factors from Bacillus cereus. She has also been investigating biofilm formation and control in food processing environments. She received the Pound Research Award from the College of Agricultural and Life Sciences at the UW–Madison in 1979 for her research accomplishments.

Amy Wong Promoted to Full Professor

Charles Kaspar Receives Pound Research Award

Charles W. Kaspar, Associate Professor of food microbiology and toxicology (Food Research Institute) and Associate Director of the Environmental Toxicology Center, University of Wisconsin–Madison recently received the Pound Award for Research. The award is given annually by the College of Agricultural and Life Sciences to a mid-career faculty member for excellence in research. Kaspar was honored for his work on Escherichia coli O157:H7, the cause of a major, and sometimes deadly, form of foodborne illness. Since 1992 when he started studies, Kaspar has become a nationally and internationally respected authority on this subject. He has tracked the spread of the bacterium among dairy cattle on Wisconsin farms and also has contributed to our understanding of the survival of the bacterium under acidic conditions. Dr. Kaspar earned the B.S. degree in biology from the University of Nebraska–Omaha (1980) and M.S. (1983) and Ph.D. (1986) degrees, both in microbiology, from Iowa State University. He did post-doctoral research at the University of Maryland (1986–1988) and then worked for the Food and Drug Administration (1988–1990) and the Cargill Company (1990–1992) before joining the faculty of the Food Research Institute at the UW–Madison in 1992 as an Assistant Professor. He was promoted to Associate Professor in 1998.

Elmer H. Marth, emeritus professor food science, bacteriology, and food microbiology and toxicology, has been chosen by the International Association for Food Protection to receive the 2000 NFPA Food Safety Award. The Association award notice reads: "We are pleased to inform you that you are the winner of the 2000 NFPA Food Safety Award. This award is given under the auspices of IAFP (International Association for Food Protection). The purpose of the award is to honor individuals or organizations that have a long commitment to improving the safety of food. The Award Committee noted that for over 45 years your exemplary and outstanding contributions to and leadership in all relevant areas of food safety, including but not limited to research, education, public health and regulatory policy, have served to significantly advance the state of the knowledge in the area of food safety for the benefit of all."

Elmer Marth Receives  the 2000 NFPA Food Safety Award

• Hormone Implants to Enhance Growth in Cattle
• Antioxidant Effect of Vitamin E

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Two comprehensive assessments of the safety of these hormone implants were published in 1999. A report by the FAO/WHO Joint Expert Committee on Food Additives (JECFA) concluded that implants containing the approved doses of hormones and inserted in cattle according to recommended procedures would not adversely affect human health. The Scientific Committee on Veterinary Measures of the European Commission (EU), on the other hand, concluded that elevated levels of hormones in meat from implanted cattle might present a hazard, particularly to children. Invoking the precautionary principle, the Committee decided that beef from implanted cattle should not be allowed in European markets.

A Sub-Group of the Veterinary Products Committee from the Ministry of Fisheries and Food (MAFF, UK) evaluated, point by point, a number of issues raised in the EU report and stated that it had "sufficient concerns about the scientific reasoning in a number of key areas" and could not support the conclusions of the EU Report. In a brief rebuttal to the JECFA analysis and the review by the MAFF group, the EU reaffirmed its concerns about the safety of hormone implants and recommended continuing the ban.

The controversy currently centers around the potential adverse effects of 17beta estradiol. Estrogens are naturally present in all mammals although the amounts vary with age, sex, diet, exercise, and, in females, with pregnancy and stage of the menstrual cycle. Estradiol 17beta is present in high levels in newborns but concentrations drop rapidly after birth so that young children of both sexes have very low concentrations of this compound. In adults, estradiol concentrations normally range from 2 to 4 ng/100 ml in males and from 4 and 18 ng/100 ml in females. After menopause, estradiol levels in females normally decrease to a mean of 1.2 ng/100 ml.

Although natural hormones are essential for various physiological processes in the body, excessive amounts may have adverse effects. Some epidemiological studies have demonstrated an increased incidence of breast cancer in post-menopausal women using hormone replacement therapy. These studies, in combination with research using laboratory animals, was considered by IARC (International Agency for Research on Cancer) to be sufficient evidence for the carcinogenicity of estradiol to humans. Carcinogenic effects were only observed in animals receiving a hormone dose which was sufficient to induce other hormone-related responses.

Based on these concerns, JECFA set an acceptable daily intake maximum of 0.05 mg/kg body weight for estradiol, based on reported no-hormone-effect-level (NHEL) of 5 mg/kg body weight per day. FDA set the allowable incremental increase in estradiol levels above those normally present in meat as 0.12 mg/kg. However, the European Union scientists question the sensitivity of assays to detect hormone levels and the relevance of the no-hormonal-effect level because some recent data indicate that estradiol is genotoxic, and it may have carcinogenic effects at concentrations below the NHEL.

Recent analyses by Fritsche et al. (1999) of beef from implanted and from unimplanted steers indicated that estradiol-17beta levels were 3.0 ng/kg in implanted steer meat and 2.8 ng/kg in beef from unimplanted animals. Metabolite (17alpha-estradiol and estrone) levels were higher in meat from the control (4.8 ng/kg) than in implanted animals (1.9 ng/kg). These levels would be well below the limits suggested by both FDA and JECFA.

In addition to considerations of slightly increased hormone levels in beef from implanted cattle, total dietary exposure to compounds with hormonal activity from other foods of animal origin (milk and eggs) and of plant origin should be considered in order to put the possibility of an increased exposure to hormones in beef from implanted cattle in context. The controversy over hormone implants in cattle appears to be affected by political as well as scientific concerns.

Free radicals are normally generated in substantial amounts by internal metabolic processes in aerobic organisms. Some reactive oxygen and nitrogen species are also produced in response to chemicals in the environment, such as cigarette smoke, air pollutants, ethyl alcohol, and halogenated hydrocarbons. These free radicals can attack biological macromolecules and alter their structure and function, causing oxidative stress. Many chronic diseases are believed to result from an accumulation of macromolecules, damaged by free radicals, which the body has not been able to repair.

Can the consumption of antioxidants in the diet protect us from free-radical-induced damage? Of all the antioxidant nutrients, vitamin E (alpha-tocopherol) has been studied more extensively and the evidence for its beneficial effects is more convincing than that for other antioxidants.

Dietary alpha-tocopherol is readily absorbed and transported to cell membranes and to intracellular sites. Its toxicity is very low with no mutagenic, carcinogenic, or teratogenic effects reported. Even in double-blind experiments utilizing doses of vitamin E as large as 3200 IU/day, no adverse side effects were reported. Since vitamin E does affect platelet aggregation, it can increase the tendency for bleeding in people with vitamin K deficiency.

Several large epidemiological studies indicate that there is an inverse relationship between reported intake of vitamin E and incidence of or death from coronary disease. Vitamin E supplements have also been reported to inhibit some oxidative changes associated with cardiovascular disease. Recently, evidence has been presented for several mechanisms by which this protective effect may be mediated. High plasma levels of oxidized LDL (low density lipoprotein) are an indicator of coronary artery disease, and intake of vitamin E supplements by people at risk for heart disease reduces the susceptibility of their LDL molecules to oxidation when tested ex vivo. A dose of alpha-tocopherol also decreased platelet aggregation and enhanced sensitivity to the platelet inhibitor, PGE1, which may prevent the formation of internal blood clots. In vitro studies demonstrated that vitamin E also inhibited the expression of adhesion molecules and production of inflammatory cytokines in aortic endothelial cells and monocytic cells. These changes may affect the buildup of plaque on the inside of artery walls.

Data from the Finnish ATBC (alpha-tocopherol, beta-carotene) clinical trial revealed that a daily dose of 50 mg alpha-tocopherol was correlated with a reduced incidence and mortality of prostate cancer in smokers, but did not affect risk for colorectal cancer. Other studies have also demonstrated an anticarcinogenic effect in smokers, but not in non-smokers. There is some evidence that vitamin E exerts a protective effect by inhibiting oxidative damage to DNA.

Efficiency of the immune system declines in many old people. Consumption of 200 mg vitamin E/day by a group of old men (65–75 years old) for three months was found to restore some measures of immune function to normal adult levels.

Neuropathological symptoms have been observed as consequences of dietary vitamin E deficiency and this has stimulated interest in the possible role of vitamin E in other neurological diseases. Lipoproteins from the brain and spinal cord of patients with Alzheimer's disease (AD) were found to be much more easily oxidized than lipoproteins from controls. However, analysis of data on 3,385 Japanese-American men participating in the Honolulu Heart Program indicated that regular consumption of vitamin E supplements did not have a protective effect against AD although it was correlated with a decrease in other types of vascular dementia.

Some recent data also indicate that high dietary levels of vitamin E or vitamin supplements may be useful in reducing sensitivity to UV light and reduce the risk for cataracts, though these results were not always confirmed in other studies.

According to the latest report by the Panel on Dietary Antioxidants and Related Compounds of the Institute of Medicine, the RDA (Recommended Daily Allowance) for alpha-tocopherol is 15 mg for men and women. The upper limit of intake of alpha-tocopherol was set at 1 g/day (1500 IU of "natural source" vitamin E or 1100 IU of synthetic vitamin E) based on the adverse effect of an increased tendency to hemorrhage.

Individual antioxidant supplements, such as vitamin E, when tested alone, sometimes do not appear to have protective or antioxidative effects. Fruits and vegetables are likely to be a better source of antioxidants because they contain multiple compounds with antioxidant properties as well as other beneficial substances, such as fiber.

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