J. Steven Stanley

Molecular cloning and epitope analysis of the peanut allergen Ara h 3

Peanut allergy is a significant IgE-mediated health problem because of the increased prevalence, potential severity, and chronicity of the reaction. Following our characterization of the two peanut allergens Ara h 1 and Ara h 2, we have isolated a cDNA clone encoding a third peanut allergen, Ara h 3. The deduced amino acid sequence of Ara h 3 shows homology to 11S seed-storage proteins. The recombinant form of this protein was expressed in a bacterial system and was recognized by serum IgE from approximately 45% of our peanut-allergic patient population. Serum IgE from these patients and overlapping, synthetic peptides were used to map the linear, IgE-binding epitopes of Ara h 3. Four epitopes, between 10 and 15 amino acids in length, were found within the primary sequence, with no obvious sequence motif shared by the peptides. One epitope is recognized by all Ara h 3-allergic patients. Mutational analysis of the epitopes revealed that single amino acid changes within these peptides could lead to a reduction or loss of IgE binding. By determining which amino acids are critical for IgE binding, it might be possible to alter the Ara h 3 cDNA to encode a protein with a reduced IgE-binding capacity. These results will enable the design of improved diagnostic and therapeutic approaches for food-hypersensitivity reactions.

Engineering, Characterization and in vitro Efficacy of the Major Peanut Allergens for Use in Immunotherapy

Background: Numerous strategies have been proposed for the treatment of peanut allergies, but despite the steady advancement in our understanding of atopic immune responses and the increasing number of deaths each year from peanut anaphylaxis, there is still no safe, effective, specific therapy for the peanut-sensitive individual. Immunotherapy would be safer and more effective if the allergens could be altered to reduce their ability to initiate an allergic reaction without altering their ability to desensitize the allergic patient. Methods: The cDNA clones for three major peanut allergens, Ara h 1, Ara h 2, and Ara h 3, have been cloned and characterized. The IgE-binding epitopes of each of these allergens have been determined and amino acids critical to each epitope identified. Site-directed mutagenesis of the allergen cDNA clones, followed by recombinant production of the modified allergen, provided the reagents necessary to test our hypothesis that hypoallergenic proteins are effective immunotherapeutic reagents for treating peanut-sensitive patients. Modified peanut allergens were subjected to immunoblot analysis using peanut-positive patient sera IgE, T cell proliferation assays, and tested in a murine model of peanut anaphylaxis. Results: In general, the modified allergens were poor competitors for binding of peanut-specific IgE when compared to their wild-type counterpart. The modified allergens demonstrated a greatly reduced IgE-binding capacity when individual patient serum IgE was compared to the binding capacity of the wild-type allergens. In addition, while there was considerable variability between patients, the modified allergens retained the ability to stimulate T cell proliferation. Conclusions: These modified allergen genes and proteins should provide a safe immunotherapeutic agent for the treatment of peanut allergy.

Chondroitin sulfate glycosaminoglycans as major P‐selectin ligands on metastatic breast cancer cell lines

The metastatic breast cancer cell line, 4T1, abundantly expresses the oligosaccharide sialylated Lewis x (sLex). SLex oligosaccharide on tumor cells can be recognized by E‐ and P‐selectin, contributing to tumor metastatic process. We observed that both selectins reacted with this cell line. However, contrary to the E‐selectin reactivity, which was sLex dependent, P‐selectin reactivity with this cell line was sLex‐independent. The sLex‐Neg variant of the 4T1 cell line with markedly diminished expression of sLex and lack of sLea, provided a unique opportunity to characterize P‐selectin ligands and their contribution to metastasis in the absence of overlapping selectin ligands and E‐selectin binding. We observed that P‐selectin binding was Ca2+‐independent and sulfation‐dependent. We found that P‐selectin reacted primarily with cell surface chondroitin sulfate (CS) proteoglycans, which were abundantly and stably expressed on the surface of the 4T1 cell line. P‐selectin binding to the 4T1 cells was inhibited by heparin and CS glycosaminoglycans (GAGs). Moreover, Heparin administration significantly inhibited experimental lung metastasis. In addition, the data suggest that surface CS GAG chains were involved in P‐selectin mediated adhesion of the 4T1 cells to murine platelets and human umbilical vein endothelial cells. The data suggest that CS GAGs are also the major P‐selectin‐reactive ligands on the surface of human MDA‐MET cells. The results warrant conducting clinical studies on the involvement of cell surface CS chains in breast cancer metastasis and evaluation of various CS types and their biosynthetic pathways as target for development of treatment strategies for antimetastatic therapy of this disease.

Biochemical determinants of Adriamycin toxicity in mouse liver, heart and intestine.

Biochemical characteristics relevant to the differential susceptibilities of liver, heart, and intestine to acute Adriamycin toxicity were examined in female CD-1 mice with and without intravenous Adriamycin (dose range 23-30 mg/kg). The liver which, unlike heart and intestine, is relatively resistant to Adriamycin toxicity, had high levels of glutathione and glutathione peroxidase, and exhibited a sharp decline in non-protein thiol concentrations within 1-3 hr with rebound by 6 hr after Adriamycin. Covalent binding to Adriamycin or its metabolites could not account quantitatively for the loss of non-protein thiols, implicating an oxidative mechanism. No lipid peroxidation was observed in the liver, apparently due to effective utilization of antioxidant defenses. Adriamycin caused significant increases in cardiac lipid peroxides, indicative of oxidative tissue damage, which would be expected to exacerbate cardiotoxicity. However, non-protein thiol concentrations did not decrease in heart or in intestine in response to Adriamycin. Both heart and intestine had extremely low levels of glutathione peroxidase activity, which may limit glutathione utilization for protection against oxidative toxicity. The activity of DT diaphorase, which may have an activating role in Adriamycin metabolism, was high in heart and intestine and was induced 4-fold in liver in response to Adriamycin.

Isolation and characterization of a clone encoding a major allergen (Bla g Bd90K) involved in IgE-mediated cockroach hypersensitivity

Previous studies have established that atopic individuals living in cockroach-infested housing become sensitized to cockroach aeroallergens and produce IgE antibodies to a variety of proteins. We describe the isolation of a complementary DNA clone from an expression library, constructed with messenger RNA from German (Blattella germanica) cockroaches, which encodes a major allergen involved in mediating cockroach hypersensitivity. Approximately 0.2% of the clones from a lambda ZAP XR cDNA library bound IgE from a patient with cockroach sensitivity. A randomly selected subset of these clones revealed that they were either different isolates of the same gene or members of a closely related gene family. One of the largest clones (a 4 kb insert) from this subset, Bla g Bd90K hybridized to a single mRNA of approximately the same size. DNA sequence analysis showed that this gene consisted of seven 576 bp tandem repeats with a short unique region at either end. No significant sequence homologies were found between the cockroach clone and any other gene reported in the GenBank database. Serum from 17 of 22 (77%) patients with cockroach hypersensitivity identified IgE-binding recombinant protein expressed from clone Bla g Bd90K in Escherichia coli XL-Blue cells as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis/immunoblot analysis. This recombinant protein migrates with a molecular weight (90 kd) apparently similar to one identified in whole body extracts. We have identified and isolated a cDNA that encodes a major cockroach allergen (Bla g Bd90K) present in German cockroaches.

Biochemistry of food allergens

Summary and ProspectusThe proteins within foods that are allergenic represent a wide variety of proteins with functions that vary from storage to enzymatic activity. General characteristics of food allergens include an acidic isoelctric point, abundance in the food, stability to digestion, and the presence of multiple, linear IgE binding epitopes. However, there are exceptions even to these very general characteristics making it nearly impossible to predict whether a protein is a potential allergen. Since the development of an IgE response to an allergen involves a series of interactions among antigen-presenting cells (APCs), T-cells, and B-cells, it is important to continue to characterize both B-cell and T-cell epitopes of known allergens. With this information, it will be possible to improve diagnosis, develop new immunotherapeutic strategies, and eventually develop hypoallergenic plants for a safer food supply.

Proliferation of peripheral blood mononuclear cells causes increased expression of the sodium-dependent multivitamin transporter gene and increased uptake of pantothenic acid

Antigenic or mitogenic stimulation of peripheral blood mononuclear cells (PBMC) causes rapid cell proliferation. PBMC proliferation is associated with increased activities of pantothenic acid-dependent metabolic pathways, suggesting increased demand for pantothenic acid. We sought to determine whether PBMC respond to proliferation by increased cellular uptake of pantothenic acid and, if so, by what mechanism(s) the increased uptake is mediated. Uptake of pantothenic acid into PBMC was mediated by the sodium-dependent multivitamin transporter, SMVT, as judged by sodium dependency of uptake, substrate affinity and specificity, and RT-PCR of PBMC RNA. Proliferating PBMC accumulated two times more [3H]pantothenic acid than quiescent PBMC. Rates of [3H]pantothenic acid uptake paralleled rates of PBMC proliferation, as judged by uptake of [3H]thymidine. The increased uptake of [3H]pantothenic acid into proliferating PBMC was mediated by increased expression of SMVT (as judged by RT-PCR using total RNA from PBMC), leading to an increased number of transporters on the cell surface (as judged by maximal transport rates for pantothenic acid). We conclude that proliferating PBMC increase expression of the gene encoding SMVT to increase uptake of pantothenic acid.

BIOCHEMICAL ASPECTS OF FOOD ALLERGENS

The most common food allergies known to affect children are IgE-mediated reactions to cows milk, eggs, peanuts, soybeans, wheat, fish, and tree nuts. Approximately 80% of all reported food allergies in children are caused by peanuts, milk, or eggs. In adults the most common food allergies are caused by peanuts, tree nuts, fish, and shellfish. The consumer marketplace reflects widespread interest and concern about adverse reactions to certain foods and food additives. A recent consumer survey indicated that 30% of the people interviewed reported that they or some family member had an allergy to a food product. 47 This survey also found that 22% of the people avoided particular foods on the mere possibility that the food may contain an allergen. Whereas most childhood food allergies are outgrown, allergies to peanuts, tree nuts, and fish are rarely resolved in adulthood. Food allergies are on the rise worldwide, and the development of new treatments are required for this increasingly common disease. The development of any new treatment will require in-depth structural and biochemical knowledge of the food allergens that precipitate the clinical symptoms.