35 opinions or conclusions contained therein do not necessarily represent the statements, opinion... more 35 opinions or conclusions contained therein do not necessarily represent the statements, opinions or 36 conclusions of NIEHS, NIH or the United States government. 37 38 This report has been reviewed by the Environmental Protection Agency"s Office of Research and 39 Development and Office of Pesticides Programs, and approved for publication. Approval does not signify 40 that the contents necessarily reflect the views and policies of the Agency nor does mention of trade 41 names or commercial products constitute endorsement or recommendation for use. 42 43 44 2 ABSTRACT (Word count = 199)
Regulatory toxicology and pharmacology : RTP, Jan 20, 2016
Genetically modified (GM) crops have achieved success in the marketplace and their benefits exten... more Genetically modified (GM) crops have achieved success in the marketplace and their benefits extend beyond the overall increase in harvest yields to include lowered use of insecticides and decreased carbon dioxide emissions. The most widely grown GM crops contain gene/s for targeted insect protection, herbicide tolerance, or both. Plant expression of Bacillus thuringiensis (Bt) crystal (Cry) insecticidal proteins have been the primary way to impart insect resistance in GM crops. Although deemed safe by regulatory agencies globally, previous studies have been the basis for discussions around the potential immuno-adjuvant effects of Cry proteins. These studies had limitations in study design. The studies used animal models with extremely high doses of Cry proteins, which when given using the ig route were co-administered with an adjuvant. Although the presumption exists that Cry proteins may have immunostimulatory activity and therefore an adjuvanticity risk, the evidence shows that Cr...
The objective of this study was to evaluate the subchronic toxicity of a commercial fluoroalkylet... more The objective of this study was to evaluate the subchronic toxicity of a commercial fluoroalkylethanol mixture, which is an intermediate in the production of fluoroorganic compounds that are used as protectants and surfactants. The test substance was administered daily by gavage to Sprague-Dawley rats as a suspension in aqueous methylcellulose. The dosages were 0, 25, 100, or 250 mg kg(-1) day(-1). A 1- and 3-month recovery period was included to evaluate the reversibility of toxic effects. No test substance-related mortality or neurotoxicity occurred. Body weights and/or nutritional parameters were significantly reduced at 100 and 250 mg kg(-1) day(-1), and these effects were reversible. Broken and absent teeth were observed in rats dosed with 250 mg kg(-1) day(-1), and microscopic tooth lesions (ameloblast degeneration/disorganization) occurred at 100 and 250 mg kg(-1) day(-1) and persisted with decreased severity throughout recovery. Decreased red cell mass parameters occurred at 90 days in the 250 mg kg(-1) day(-1) group, but red cell counts were normal thereafter during recovery. A persistent elevation of liver weights was seen in groups given > or =100 mg kg(-l) day(-1). The increased weights correlated with microscopic hepatocellular hypertrophy only in males and females administered 250 mg kg(-1) day(-1). Hepatic beta-oxidation was increased in a dose-dependent manner and persisted through 1 month of recovery at 250 mg kg(-1) day(-1). Increased kidney weights were observed at 25 (females only), 100, and 250 mg kg(-1) day(-1). These elevated weights persisted in the high dose after recovery and correlated with microscopic tubular hypertrophy (males only). Thyroid follicular hypertrophy was present at 100 and 250 mg kg(-1) day(-1) but was not present after recovery. Total fluorine in whole blood increased with continuous dosing and achieved steady state in approximately 42 days. Both plasma and urine fluoride levels were elevated in a dose-dependent manner. Under the conditions of the study, the no-observed adverse effect level for this mixture was 25 mg kg(-1) day(-1) for subchronic toxicity.
A rigorous safety assessment process exists for GM crops. It includes evaluation of the introduce... more A rigorous safety assessment process exists for GM crops. It includes evaluation of the introduced protein as well as the crop containing such protein with the goal of demonstrating the GM crop is ''as-safe-as" non-transgenic crops in the food supply. One of the major issues for GM crops is the assessment of the expressed protein for allergenic potential. Currently, no single factor is recognized as an identifier for protein allergenicity. Therefore, a weight-of-evidence approach, which takes into account a variety of factors and approaches for an overall assessment of allergenic potential, is conducted [Codex Alimentarious Commission, 2003. Alinorm 03/34: Joint FAO/WHO Food Standard Programme, Codex Alimentarious Commission, Twenty-Fifth Session, Rome, Italy, 30 June-5 July, 2003. Appendix III, Guideline for the conduct of food safety assessment of foods derived from recombinant-DNA plants, and Appendix IV, Annex on the assessment of possible allergenicity, pp. 47-60]. This assessment is based on what is known about allergens, including the history of exposure and safety of the gene(s) source; protein structure (e.g., amino acid sequence identity to human allergens); stability to pepsin digestion in vitro . A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins. Regul. Toxicol. Pharmacol. 39,[87][88][89][90][91][92][93][94][95][96][97][98]; an estimate of exposure of the novel protein(s) to the gastrointestinal tract where absorption occurs (e.g., protein abundance in the crop, processing effects); and when appropriate, specific IgE binding studies or skin prick testing. Additional approaches may be considered (e.g., animal models; targeted sera screening) as the science evolves; however, such approaches have not been thoroughly evaluated or validated for predicting protein allergenicity.
Toxicology Mechanisms and Methods 2006 16 101 19, 2006
Recent advances in genomics-based identification of gene families and gene polymorphisms associat... more Recent advances in genomics-based identification of gene families and gene polymorphisms associated with immune system dysfunction have answered basic questions in immunology and have begun to move forward our understanding of immune-related disease processes. In toxicology, "omic" technologies have the potential to replace or supplement current immunotoxicological screening procedures, to provide insight into potential mode or mechanisms of action, and to provide data suitable for risk assessment. The application of omic technologies to the study of the immune system also has great potential to appreciably impact the diagnosis and treatment of immune-related diseases. This review focuses on the use of omic technologies in immunopharmacology and immunotoxicology, specifically considering the potential for these technologies to impact chemical hazard identification, risk characterization and risk assessment, and the development and application of novel therapeutics. The state of the science of omics technologies and the immune system is addressed in terms of a continuum of understanding of how omics technologies can and cannot yet be applied in the various aspects of immunopharmacology and immunotoxicology. Additionally, information gaps are identified that, once addressed, will move each area further down the continuum of understanding.
The goals of this project were to determine the conditions for optimal immune assessment followin... more The goals of this project were to determine the conditions for optimal immune assessment following in utero exposure to lead (Pb) and to evaluate the roles of age of assessment, gender, and genetic strain of rat on the observed immunotoxicologic outcomes following exposure. A combined delayed-type hypersensitivity response (DHR) to keyhole limpet hemocyanin (KLH) and anti-KLH IgG antibody were assessed as potential biomarkers for developmental immunotoxicity with age, gender, site of antigenic challenge, and strain as variables. Studies were conducted using both male and female Crl:CD (SD)BR or Fisher 344 rats. The heavy metal, Pb, was used as a known developmental immunotoxin. IgG antibody against KLH antigen was measured using an enzyme linked immunosorbant assay (ELISA). Following sensitization with KLH, animals received a challenge injection in either the earlobe or footpad with KLH and the DTH measured 24 hours later using a spring-loaded caliper. Sprague-Dawley (CD) weanlings produced lower levels of antibody and a decreased DHR compared with adults; only adult males had a significantly increased DHR while both male and female adults produced higher levels of antibody than both genders of weanlings. The DHR was greater in young animals when challenged in the footpad vs. the earlobe. Females had optimum antibody levels with a DHR challenge in the earlobe whereas males had optimum levels when challenged in the footpad. In a strain comparison between weanlings exposed in utero to control or 250 ppm Pb acetate in drinking water and rats examined at five weeks of age, the CD vs. F344 rats had higher antibody responses; in contrast, F344 rats exhibited an elevated DHR. Pb exposure in utero via the pregnant dams produced differential gender effects in the juveniles of both strains. Females had a significantly decreased Pb-induced DHR (p<0.5) whereas males did not. These results suggest that the DHR and anti-KLH IgG are suitable as biomarkers of developmental immunotoxicity and, based on the Pb results, assessment can be performed in juvenile rats. Furthermore, these studies provide evidence that there are differential gender effects after in utero exposure to Pb that can be detected in juveniles but also persist into adulthood. Additionally, at the KLH concentrations utilized, the balance of cell-mediated vs. humoral response differed among the two strains examined. The project was conducted during the course of one year and completed in 2000.
In phase one of an interlaboratory study, baseline values for rat splenic lympho-cyte populations... more In phase one of an interlaboratory study, baseline values for rat splenic lympho-cyte populations were established. In phase two, rat splenic lymphocyte popu-lations were evaluated using immunoŊ uorescent staining and Ŋ ow cytometry following exposure to the immunosuppressive ...
The gut mucosal immune system may be a primary target for many ingested chemicals. Methods have b... more The gut mucosal immune system may be a primary target for many ingested chemicals. Methods have been developed to examine the effects of chemicals on the systemic humoral immune response; however, studies to evaluate various methods of assessing the local gut mucosal immune response in a toxicology assay have been limited. The objectives of this study were to examine the effects of the known immunosuppressive compound, cyclosporine (CYS), on the generation of a cholera toxin (CT)-specific gut mucosal IgA response and evaluate the methods used to measure the gut IgA response. Groups of female B6C3F1 mice were left untreated or were treated daily, p.o., with corn oil (vehicle) or CYS at doses of 10 and 50 mg/kg for 20 days. On Days 3 and 13, mice were sensitized p.o. with CT. On Day 21, mice were terminated, gut washings were collected, and lamina propria lymphocytes were extracted from gut tissue with collagenase treatment. Cholera toxin-specific IgA in the gut washings was measured by an ELISA. The numbers of CT-specific IgA (CT-IgA) and total IgA antibody-forming cells (spot-forming cells, SFC) obtained from the lamina propria were determined by the ELISPOT method. A dose of 50 mg/kg CYS produced a significant decrease in the amount of CT-IgA in gut washings. This dose also decreased the number of cells recovered from the lamina propria by at least 50%. The amount of CT-specific SFC/million lamina propria cells decreased with a dose of 10 mg/kg CYS, whereas 50 mg/kg CYS did not alter the response.(ABSTRACT TRUNCATED AT 250 WORDS)
journal published by Elsevier. The attached copy is furnished to the author for internal non-comm... more journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Heat stability, its measurement, and its lack of utility in the assessment of the potential allergenicity of novel proteins a b s t r a c t Thermal stability has been reported as a shared characteristic among some of the major food allergens and appears to have originated from the ...
A group of thirty immunotoxicology experts from the U.S. and E.U. representing government, indust... more A group of thirty immunotoxicology experts from the U.S. and E.U. representing government, industry, and academia met in May 2003, in Washington, D.C., to reach consensus regarding the most appropriate methods to assess developmental immunotoxicology (DIT) for hazard identification, including under what conditions such testing might be required. The following points represent the major conclusions from this roundtable discussion: (1) the rat is the preferred model; (2) any DIT protocol should be based on immune assays already validated;
Exposure to 7,12-dimethylbenz[a]anthracene (DMBA) has been demonstrated by numerous investigators... more Exposure to 7,12-dimethylbenz[a]anthracene (DMBA) has been demonstrated by numerous investigators to result in suppression of both humoral and cell-mediated immune responses of mice and cultured splenocytes. The mechanism(s) of this DMBA-induced immunosuppression, however, is not well characterized. PAHs must be converted to reactive metabolites via cytochrome P450-dependent monooxygenase systems to exert their carcinogenic and mutagenic effects. Thus, we have hypothesized that immunosuppression seen upon exposure to DMBA may also be mediated by its reactive metabolites. The objective of this study was to determine if DMBA metabolites can suppress the in vitro, T-dependent humoral immune response to sheep red blood cells. Compounds were evaluated in the in vitro plaque-forming cell (PFC) response at concentrations of 10(-9) to 10(-5) M. DMBA and benzo[a]pyrene (B[a]P) were also evaluated for their ability to suppress the in vitro PFC response. Addition of either of these PAHs to spl...
Recent studies have demonstrated that macrophages are the cell types capable of metabolizing benz... more Recent studies have demonstrated that macrophages are the cell types capable of metabolizing benzo[a]pyrene (B(a)P) within the spleens of untreated mice. Since repeated exposure to B(a)P results in immunosuppression and B(a)P is known to induce cytochrome P450 levels, the first objective of this study was to investigate whether exposure of mice to B(a)P could increase the amounts of immunosuppressive B(a)P metabolites generated and/or alter the pattern of B(a)P metabolites formed by several different splenic cell types. Mice were dosed with a daily sc dose of 200 mg/kg B(a)P or vehicle for 4 days. Separation of splenocytes based on density by centrifugation through discontinuous Percoll gradients along with immunomagnetic negative selection or antibody-mediated complement lysis was used to obtain different splenic cell populations. Cells were incubated with [3H]B(a)P for 24 hr. High-pressure liquid chromatography was used to separate and quantitate B(a)P metabolites. Results indicat...
The objective of the present study was to determine which splenic cell type(s) of B6C3F1 mice was... more The objective of the present study was to determine which splenic cell type(s) of B6C3F1 mice was capable of metabolizing B(a)P. Separation of splenocytes based on density by centrifugation through discontinuous Percoll gradients along with immunomagnetic negative selection or antibody-mediated complement lysis was utilized to obtain highly enriched populations of splenocytes for B(a)P metabolism studies. Immunofluorescent cell staining in conjunction with flow cytometry and examination of Giemsa-stained cytospin cell preparations indicated that B- or T-cell populations of greater than 95% purity and an 80-90% pure population of splenic macrophages were routinely attained. Splenic cell populations were incubated with [3H]B(a)P for 24 hr. High-pressure liquid chromatography was used to separate and quantitate the B(a)P metabolites generated by the enriched splenic cell populations. The results of these studies demonstrate that the macrophage is the cell type responsible for the metab...
The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity i... more The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity is affected by physiochemical characteristics of proteins. The complexity created by the matrices of foods and the variability of the human immune system add additional challenges to understanding the relationship between sensitization potential and allergy disease. To address these and other issues, an April 2012 international symposium was held in Prague, Czech Republic, to review and discuss the state-of-the-science of sensitizing properties of protein allergens. The symposium, organized by the Protein Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, featured presentations on current methods, test systems, research trends, and unanswered questions in the field of protein sensitization. A diverse group of over 70 interdisciplinary scientists from academia, government, and industry participated in the symposium. Experts provided overviews on known mechanisms by which proteins in food may cause sensitization, discussed experimental models to predict protein sensitizing potential, and explored whether such experimental techniques may be applicable in regulatory settings. Three accompanying reviews address critical factors and methods for assessing allergic sensitization: 1) food-and protein-related factors; 2) host-specific factors and 3) screening methods, i.e., the ability of experimental models to predict the sensitizing potential of proteins and whether such models are applicable within regulatory settings.
Soybean (Glycine max ) is a hugely valuable soft commodity that generates tens of billions of dol... more Soybean (Glycine max ) is a hugely valuable soft commodity that generates tens of billions of dollars annually. This value is due in part to the balanced composition of the seed which is roughly 1:2:2 oil, starch, and protein by weight. In turn, the seeds have many uses with various derivatives appearing broadly in processed food products. As is true with many edible seeds, soybeans contain proteins that are anti-nutritional factors and allergens. Soybean, along with milk, eggs, fish, crustacean shellfish, tree nuts, peanuts, and wheat, elicit a majority of food allergy reactions in the United States. Soybean seed composition can be affected by breeding, and environmental conditions (e.g., temperature, moisture, insect/pathogen load, and/or soil nutrient levels). The objective of this study was to evaluate the influence of genotype and environment on allergen and anti-nutritional proteins in soybean. To address genetic and environmental effects, four varieties of non-GM soybeans were grown in six geographically distinct regions of North America (Georgia, Iowa, Kansas, Nebraska, Ontario, and Pennsylvania). Absolute quantification of proteins by mass spectrometry can be achieved with a technique called multiple reaction monitoring (MRM), during which signals from an endogenous protein are compared to those from a synthetic heavy-labeled internal standard. Using MRM, eight allergens were absolutely quantified for each variety in each environment. Statistical analyses show that for most allergens, the effects of environment far outweigh the differences between varieties brought about by breeding.
FAO/WHO has recommended that IgE cross-reactivity between a transgenic protein and allergen be co... more FAO/WHO has recommended that IgE cross-reactivity between a transgenic protein and allergen be considered when there is greater than 35% identity over a sliding &amp;amp;amp;amp;quot;window&amp;amp;amp;amp;quot; of 80 amino acids. In a previous work, we evaluated the false positive and negative rates observed using the FAO/WHO criteria versus conventional, whole protein FASTA analyses [Ladics, G.S., Bannon, G.A., Silvanovich, A., Cressman, R.F., 2007. Comparison of conventional FASTA identity searches with the 80 amino acid sliding window FASTA search for the elucidation of potential identities to known allergens. Mol. Nutr. Food Res. 51 (8), 985-998]. A number of protein sequence datasets were used as queries against the FARRP 7 allergen database. Results indicated that conventional FASTA analysis produced fewer false positives then the &amp;amp;amp;amp;quot;sliding window&amp;amp;amp;amp;quot; search proposed by FAO/WHO. Further, both methods were able to identify the potential for cross-reactivity between the Bet v 1 family of proteins, indicating that the conventional FASTA search possessed sufficient sensitivity. Recently, collections of protein sequences from multiple crop species (corn, soy, barley, lettuce, sugar beets, and spinach) were subjected to the same screen against the FARRP7 allergen dataset. In all cases, the conventional FASTA search returned fewer above threshold matches than the sliding window search. Examination of the matches not recognized by the conventional search revealed two scenarios: (1) &amp;amp;amp;amp;quot;true&amp;amp;amp;amp;quot; false positives consisting of low statistical significance (as measured by E score, i.e., a measure of the potential random occurrence of aligned sequences used to evaluate the significance of an observed alignment) alignments not contained in the conventional FASTA outputs, and (2) above-threshold sliding window alignments that fell below the 35% identity threshold with the conventional FASTA analysis. Although some alignments within this second group were between regions of low sequence complexity, where there was little/no change in E score, the majority of the alignments displayed more significance (lower E scores) under the conventional FASTA algorithm, yet did not meet the threshold defined by FAO/WHO. These data question the utility of the FAO/WHO recommended sliding window FASTA compared to the traditional whole sequence FASTA analysis coupled with appropriate statistical analysis.
The production of antigen-specific antibodies represents a major defense mechanism of humoral imm... more The production of antigen-specific antibodies represents a major defense mechanism of humoral immune responses. Several assays have been developed to assess T-cell-dependent antibody responses (TDAR). Of these assays, the antibody forming cell assay (AFC) or plaque forming cell (PFC) assay and ELISA are the two most often used tests to assess immunotoxicity. Historically, the T-cell-dependent antigen of choice has been sheep red blood cells (SRBC). The SRBC AFC assay is considered the "gold standard" for TDAR based on extensive intra-and inter-laboratory validation in mice and has been utilized for over 35 years. The quantification of the primary AFC response (i.e., the specific IgM antibody-forming cell response) was found to provide one of the best predictors of immunotoxicity in mice. The SRBC-specific ELISA is relatively new, with the first publication of the method appearing in 1993. Data from the application of using both the SRBC specific AFC and ELISA for evaluation of potential immunotoxicity of chemicals in rodents and the pros and cons and associated issues of each method were presented. Specifically, the following was discussed: (1) studies investigating the incorporation of the SRBC-specific IgM ELISA in rats on standard toxicology study; (2) characterization of an approach to developmental immunotoxicology assessment in the rat using SRBC as the antigen; and, (3) data from an inter-laboratory study comparing the AFC assay and ELISA in outbred rodents using both cyclophosphamide and dexamethasone.
35 opinions or conclusions contained therein do not necessarily represent the statements, opinion... more 35 opinions or conclusions contained therein do not necessarily represent the statements, opinions or 36 conclusions of NIEHS, NIH or the United States government. 37 38 This report has been reviewed by the Environmental Protection Agency"s Office of Research and 39 Development and Office of Pesticides Programs, and approved for publication. Approval does not signify 40 that the contents necessarily reflect the views and policies of the Agency nor does mention of trade 41 names or commercial products constitute endorsement or recommendation for use. 42 43 44 2 ABSTRACT (Word count = 199)
Regulatory toxicology and pharmacology : RTP, Jan 20, 2016
Genetically modified (GM) crops have achieved success in the marketplace and their benefits exten... more Genetically modified (GM) crops have achieved success in the marketplace and their benefits extend beyond the overall increase in harvest yields to include lowered use of insecticides and decreased carbon dioxide emissions. The most widely grown GM crops contain gene/s for targeted insect protection, herbicide tolerance, or both. Plant expression of Bacillus thuringiensis (Bt) crystal (Cry) insecticidal proteins have been the primary way to impart insect resistance in GM crops. Although deemed safe by regulatory agencies globally, previous studies have been the basis for discussions around the potential immuno-adjuvant effects of Cry proteins. These studies had limitations in study design. The studies used animal models with extremely high doses of Cry proteins, which when given using the ig route were co-administered with an adjuvant. Although the presumption exists that Cry proteins may have immunostimulatory activity and therefore an adjuvanticity risk, the evidence shows that Cr...
The objective of this study was to evaluate the subchronic toxicity of a commercial fluoroalkylet... more The objective of this study was to evaluate the subchronic toxicity of a commercial fluoroalkylethanol mixture, which is an intermediate in the production of fluoroorganic compounds that are used as protectants and surfactants. The test substance was administered daily by gavage to Sprague-Dawley rats as a suspension in aqueous methylcellulose. The dosages were 0, 25, 100, or 250 mg kg(-1) day(-1). A 1- and 3-month recovery period was included to evaluate the reversibility of toxic effects. No test substance-related mortality or neurotoxicity occurred. Body weights and/or nutritional parameters were significantly reduced at 100 and 250 mg kg(-1) day(-1), and these effects were reversible. Broken and absent teeth were observed in rats dosed with 250 mg kg(-1) day(-1), and microscopic tooth lesions (ameloblast degeneration/disorganization) occurred at 100 and 250 mg kg(-1) day(-1) and persisted with decreased severity throughout recovery. Decreased red cell mass parameters occurred at 90 days in the 250 mg kg(-1) day(-1) group, but red cell counts were normal thereafter during recovery. A persistent elevation of liver weights was seen in groups given &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; or =100 mg kg(-l) day(-1). The increased weights correlated with microscopic hepatocellular hypertrophy only in males and females administered 250 mg kg(-1) day(-1). Hepatic beta-oxidation was increased in a dose-dependent manner and persisted through 1 month of recovery at 250 mg kg(-1) day(-1). Increased kidney weights were observed at 25 (females only), 100, and 250 mg kg(-1) day(-1). These elevated weights persisted in the high dose after recovery and correlated with microscopic tubular hypertrophy (males only). Thyroid follicular hypertrophy was present at 100 and 250 mg kg(-1) day(-1) but was not present after recovery. Total fluorine in whole blood increased with continuous dosing and achieved steady state in approximately 42 days. Both plasma and urine fluoride levels were elevated in a dose-dependent manner. Under the conditions of the study, the no-observed adverse effect level for this mixture was 25 mg kg(-1) day(-1) for subchronic toxicity.
A rigorous safety assessment process exists for GM crops. It includes evaluation of the introduce... more A rigorous safety assessment process exists for GM crops. It includes evaluation of the introduced protein as well as the crop containing such protein with the goal of demonstrating the GM crop is ''as-safe-as" non-transgenic crops in the food supply. One of the major issues for GM crops is the assessment of the expressed protein for allergenic potential. Currently, no single factor is recognized as an identifier for protein allergenicity. Therefore, a weight-of-evidence approach, which takes into account a variety of factors and approaches for an overall assessment of allergenic potential, is conducted [Codex Alimentarious Commission, 2003. Alinorm 03/34: Joint FAO/WHO Food Standard Programme, Codex Alimentarious Commission, Twenty-Fifth Session, Rome, Italy, 30 June-5 July, 2003. Appendix III, Guideline for the conduct of food safety assessment of foods derived from recombinant-DNA plants, and Appendix IV, Annex on the assessment of possible allergenicity, pp. 47-60]. This assessment is based on what is known about allergens, including the history of exposure and safety of the gene(s) source; protein structure (e.g., amino acid sequence identity to human allergens); stability to pepsin digestion in vitro . A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins. Regul. Toxicol. Pharmacol. 39,[87][88][89][90][91][92][93][94][95][96][97][98]; an estimate of exposure of the novel protein(s) to the gastrointestinal tract where absorption occurs (e.g., protein abundance in the crop, processing effects); and when appropriate, specific IgE binding studies or skin prick testing. Additional approaches may be considered (e.g., animal models; targeted sera screening) as the science evolves; however, such approaches have not been thoroughly evaluated or validated for predicting protein allergenicity.
Toxicology Mechanisms and Methods 2006 16 101 19, 2006
Recent advances in genomics-based identification of gene families and gene polymorphisms associat... more Recent advances in genomics-based identification of gene families and gene polymorphisms associated with immune system dysfunction have answered basic questions in immunology and have begun to move forward our understanding of immune-related disease processes. In toxicology, &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;omic&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; technologies have the potential to replace or supplement current immunotoxicological screening procedures, to provide insight into potential mode or mechanisms of action, and to provide data suitable for risk assessment. The application of omic technologies to the study of the immune system also has great potential to appreciably impact the diagnosis and treatment of immune-related diseases. This review focuses on the use of omic technologies in immunopharmacology and immunotoxicology, specifically considering the potential for these technologies to impact chemical hazard identification, risk characterization and risk assessment, and the development and application of novel therapeutics. The state of the science of omics technologies and the immune system is addressed in terms of a continuum of understanding of how omics technologies can and cannot yet be applied in the various aspects of immunopharmacology and immunotoxicology. Additionally, information gaps are identified that, once addressed, will move each area further down the continuum of understanding.
The goals of this project were to determine the conditions for optimal immune assessment followin... more The goals of this project were to determine the conditions for optimal immune assessment following in utero exposure to lead (Pb) and to evaluate the roles of age of assessment, gender, and genetic strain of rat on the observed immunotoxicologic outcomes following exposure. A combined delayed-type hypersensitivity response (DHR) to keyhole limpet hemocyanin (KLH) and anti-KLH IgG antibody were assessed as potential biomarkers for developmental immunotoxicity with age, gender, site of antigenic challenge, and strain as variables. Studies were conducted using both male and female Crl:CD (SD)BR or Fisher 344 rats. The heavy metal, Pb, was used as a known developmental immunotoxin. IgG antibody against KLH antigen was measured using an enzyme linked immunosorbant assay (ELISA). Following sensitization with KLH, animals received a challenge injection in either the earlobe or footpad with KLH and the DTH measured 24 hours later using a spring-loaded caliper. Sprague-Dawley (CD) weanlings produced lower levels of antibody and a decreased DHR compared with adults; only adult males had a significantly increased DHR while both male and female adults produced higher levels of antibody than both genders of weanlings. The DHR was greater in young animals when challenged in the footpad vs. the earlobe. Females had optimum antibody levels with a DHR challenge in the earlobe whereas males had optimum levels when challenged in the footpad. In a strain comparison between weanlings exposed in utero to control or 250 ppm Pb acetate in drinking water and rats examined at five weeks of age, the CD vs. F344 rats had higher antibody responses; in contrast, F344 rats exhibited an elevated DHR. Pb exposure in utero via the pregnant dams produced differential gender effects in the juveniles of both strains. Females had a significantly decreased Pb-induced DHR (p<0.5) whereas males did not. These results suggest that the DHR and anti-KLH IgG are suitable as biomarkers of developmental immunotoxicity and, based on the Pb results, assessment can be performed in juvenile rats. Furthermore, these studies provide evidence that there are differential gender effects after in utero exposure to Pb that can be detected in juveniles but also persist into adulthood. Additionally, at the KLH concentrations utilized, the balance of cell-mediated vs. humoral response differed among the two strains examined. The project was conducted during the course of one year and completed in 2000.
In phase one of an interlaboratory study, baseline values for rat splenic lympho-cyte populations... more In phase one of an interlaboratory study, baseline values for rat splenic lympho-cyte populations were established. In phase two, rat splenic lymphocyte popu-lations were evaluated using immunoŊ uorescent staining and Ŋ ow cytometry following exposure to the immunosuppressive ...
The gut mucosal immune system may be a primary target for many ingested chemicals. Methods have b... more The gut mucosal immune system may be a primary target for many ingested chemicals. Methods have been developed to examine the effects of chemicals on the systemic humoral immune response; however, studies to evaluate various methods of assessing the local gut mucosal immune response in a toxicology assay have been limited. The objectives of this study were to examine the effects of the known immunosuppressive compound, cyclosporine (CYS), on the generation of a cholera toxin (CT)-specific gut mucosal IgA response and evaluate the methods used to measure the gut IgA response. Groups of female B6C3F1 mice were left untreated or were treated daily, p.o., with corn oil (vehicle) or CYS at doses of 10 and 50 mg/kg for 20 days. On Days 3 and 13, mice were sensitized p.o. with CT. On Day 21, mice were terminated, gut washings were collected, and lamina propria lymphocytes were extracted from gut tissue with collagenase treatment. Cholera toxin-specific IgA in the gut washings was measured by an ELISA. The numbers of CT-specific IgA (CT-IgA) and total IgA antibody-forming cells (spot-forming cells, SFC) obtained from the lamina propria were determined by the ELISPOT method. A dose of 50 mg/kg CYS produced a significant decrease in the amount of CT-IgA in gut washings. This dose also decreased the number of cells recovered from the lamina propria by at least 50%. The amount of CT-specific SFC/million lamina propria cells decreased with a dose of 10 mg/kg CYS, whereas 50 mg/kg CYS did not alter the response.(ABSTRACT TRUNCATED AT 250 WORDS)
journal published by Elsevier. The attached copy is furnished to the author for internal non-comm... more journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Heat stability, its measurement, and its lack of utility in the assessment of the potential allergenicity of novel proteins a b s t r a c t Thermal stability has been reported as a shared characteristic among some of the major food allergens and appears to have originated from the ...
A group of thirty immunotoxicology experts from the U.S. and E.U. representing government, indust... more A group of thirty immunotoxicology experts from the U.S. and E.U. representing government, industry, and academia met in May 2003, in Washington, D.C., to reach consensus regarding the most appropriate methods to assess developmental immunotoxicology (DIT) for hazard identification, including under what conditions such testing might be required. The following points represent the major conclusions from this roundtable discussion: (1) the rat is the preferred model; (2) any DIT protocol should be based on immune assays already validated;
Exposure to 7,12-dimethylbenz[a]anthracene (DMBA) has been demonstrated by numerous investigators... more Exposure to 7,12-dimethylbenz[a]anthracene (DMBA) has been demonstrated by numerous investigators to result in suppression of both humoral and cell-mediated immune responses of mice and cultured splenocytes. The mechanism(s) of this DMBA-induced immunosuppression, however, is not well characterized. PAHs must be converted to reactive metabolites via cytochrome P450-dependent monooxygenase systems to exert their carcinogenic and mutagenic effects. Thus, we have hypothesized that immunosuppression seen upon exposure to DMBA may also be mediated by its reactive metabolites. The objective of this study was to determine if DMBA metabolites can suppress the in vitro, T-dependent humoral immune response to sheep red blood cells. Compounds were evaluated in the in vitro plaque-forming cell (PFC) response at concentrations of 10(-9) to 10(-5) M. DMBA and benzo[a]pyrene (B[a]P) were also evaluated for their ability to suppress the in vitro PFC response. Addition of either of these PAHs to spl...
Recent studies have demonstrated that macrophages are the cell types capable of metabolizing benz... more Recent studies have demonstrated that macrophages are the cell types capable of metabolizing benzo[a]pyrene (B(a)P) within the spleens of untreated mice. Since repeated exposure to B(a)P results in immunosuppression and B(a)P is known to induce cytochrome P450 levels, the first objective of this study was to investigate whether exposure of mice to B(a)P could increase the amounts of immunosuppressive B(a)P metabolites generated and/or alter the pattern of B(a)P metabolites formed by several different splenic cell types. Mice were dosed with a daily sc dose of 200 mg/kg B(a)P or vehicle for 4 days. Separation of splenocytes based on density by centrifugation through discontinuous Percoll gradients along with immunomagnetic negative selection or antibody-mediated complement lysis was used to obtain different splenic cell populations. Cells were incubated with [3H]B(a)P for 24 hr. High-pressure liquid chromatography was used to separate and quantitate B(a)P metabolites. Results indicat...
The objective of the present study was to determine which splenic cell type(s) of B6C3F1 mice was... more The objective of the present study was to determine which splenic cell type(s) of B6C3F1 mice was capable of metabolizing B(a)P. Separation of splenocytes based on density by centrifugation through discontinuous Percoll gradients along with immunomagnetic negative selection or antibody-mediated complement lysis was utilized to obtain highly enriched populations of splenocytes for B(a)P metabolism studies. Immunofluorescent cell staining in conjunction with flow cytometry and examination of Giemsa-stained cytospin cell preparations indicated that B- or T-cell populations of greater than 95% purity and an 80-90% pure population of splenic macrophages were routinely attained. Splenic cell populations were incubated with [3H]B(a)P for 24 hr. High-pressure liquid chromatography was used to separate and quantitate the B(a)P metabolites generated by the enriched splenic cell populations. The results of these studies demonstrate that the macrophage is the cell type responsible for the metab...
The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity i... more The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity is affected by physiochemical characteristics of proteins. The complexity created by the matrices of foods and the variability of the human immune system add additional challenges to understanding the relationship between sensitization potential and allergy disease. To address these and other issues, an April 2012 international symposium was held in Prague, Czech Republic, to review and discuss the state-of-the-science of sensitizing properties of protein allergens. The symposium, organized by the Protein Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, featured presentations on current methods, test systems, research trends, and unanswered questions in the field of protein sensitization. A diverse group of over 70 interdisciplinary scientists from academia, government, and industry participated in the symposium. Experts provided overviews on known mechanisms by which proteins in food may cause sensitization, discussed experimental models to predict protein sensitizing potential, and explored whether such experimental techniques may be applicable in regulatory settings. Three accompanying reviews address critical factors and methods for assessing allergic sensitization: 1) food-and protein-related factors; 2) host-specific factors and 3) screening methods, i.e., the ability of experimental models to predict the sensitizing potential of proteins and whether such models are applicable within regulatory settings.
Soybean (Glycine max ) is a hugely valuable soft commodity that generates tens of billions of dol... more Soybean (Glycine max ) is a hugely valuable soft commodity that generates tens of billions of dollars annually. This value is due in part to the balanced composition of the seed which is roughly 1:2:2 oil, starch, and protein by weight. In turn, the seeds have many uses with various derivatives appearing broadly in processed food products. As is true with many edible seeds, soybeans contain proteins that are anti-nutritional factors and allergens. Soybean, along with milk, eggs, fish, crustacean shellfish, tree nuts, peanuts, and wheat, elicit a majority of food allergy reactions in the United States. Soybean seed composition can be affected by breeding, and environmental conditions (e.g., temperature, moisture, insect/pathogen load, and/or soil nutrient levels). The objective of this study was to evaluate the influence of genotype and environment on allergen and anti-nutritional proteins in soybean. To address genetic and environmental effects, four varieties of non-GM soybeans were grown in six geographically distinct regions of North America (Georgia, Iowa, Kansas, Nebraska, Ontario, and Pennsylvania). Absolute quantification of proteins by mass spectrometry can be achieved with a technique called multiple reaction monitoring (MRM), during which signals from an endogenous protein are compared to those from a synthetic heavy-labeled internal standard. Using MRM, eight allergens were absolutely quantified for each variety in each environment. Statistical analyses show that for most allergens, the effects of environment far outweigh the differences between varieties brought about by breeding.
FAO/WHO has recommended that IgE cross-reactivity between a transgenic protein and allergen be co... more FAO/WHO has recommended that IgE cross-reactivity between a transgenic protein and allergen be considered when there is greater than 35% identity over a sliding &amp;amp;amp;amp;quot;window&amp;amp;amp;amp;quot; of 80 amino acids. In a previous work, we evaluated the false positive and negative rates observed using the FAO/WHO criteria versus conventional, whole protein FASTA analyses [Ladics, G.S., Bannon, G.A., Silvanovich, A., Cressman, R.F., 2007. Comparison of conventional FASTA identity searches with the 80 amino acid sliding window FASTA search for the elucidation of potential identities to known allergens. Mol. Nutr. Food Res. 51 (8), 985-998]. A number of protein sequence datasets were used as queries against the FARRP 7 allergen database. Results indicated that conventional FASTA analysis produced fewer false positives then the &amp;amp;amp;amp;quot;sliding window&amp;amp;amp;amp;quot; search proposed by FAO/WHO. Further, both methods were able to identify the potential for cross-reactivity between the Bet v 1 family of proteins, indicating that the conventional FASTA search possessed sufficient sensitivity. Recently, collections of protein sequences from multiple crop species (corn, soy, barley, lettuce, sugar beets, and spinach) were subjected to the same screen against the FARRP7 allergen dataset. In all cases, the conventional FASTA search returned fewer above threshold matches than the sliding window search. Examination of the matches not recognized by the conventional search revealed two scenarios: (1) &amp;amp;amp;amp;quot;true&amp;amp;amp;amp;quot; false positives consisting of low statistical significance (as measured by E score, i.e., a measure of the potential random occurrence of aligned sequences used to evaluate the significance of an observed alignment) alignments not contained in the conventional FASTA outputs, and (2) above-threshold sliding window alignments that fell below the 35% identity threshold with the conventional FASTA analysis. Although some alignments within this second group were between regions of low sequence complexity, where there was little/no change in E score, the majority of the alignments displayed more significance (lower E scores) under the conventional FASTA algorithm, yet did not meet the threshold defined by FAO/WHO. These data question the utility of the FAO/WHO recommended sliding window FASTA compared to the traditional whole sequence FASTA analysis coupled with appropriate statistical analysis.
The production of antigen-specific antibodies represents a major defense mechanism of humoral imm... more The production of antigen-specific antibodies represents a major defense mechanism of humoral immune responses. Several assays have been developed to assess T-cell-dependent antibody responses (TDAR). Of these assays, the antibody forming cell assay (AFC) or plaque forming cell (PFC) assay and ELISA are the two most often used tests to assess immunotoxicity. Historically, the T-cell-dependent antigen of choice has been sheep red blood cells (SRBC). The SRBC AFC assay is considered the "gold standard" for TDAR based on extensive intra-and inter-laboratory validation in mice and has been utilized for over 35 years. The quantification of the primary AFC response (i.e., the specific IgM antibody-forming cell response) was found to provide one of the best predictors of immunotoxicity in mice. The SRBC-specific ELISA is relatively new, with the first publication of the method appearing in 1993. Data from the application of using both the SRBC specific AFC and ELISA for evaluation of potential immunotoxicity of chemicals in rodents and the pros and cons and associated issues of each method were presented. Specifically, the following was discussed: (1) studies investigating the incorporation of the SRBC-specific IgM ELISA in rats on standard toxicology study; (2) characterization of an approach to developmental immunotoxicology assessment in the rat using SRBC as the antigen; and, (3) data from an inter-laboratory study comparing the AFC assay and ELISA in outbred rodents using both cyclophosphamide and dexamethasone.
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Papers by Gregory Ladics