Differences in allergenic potential of food extracts following oral exposure in mice reflect differences in digestibility: potential approaches to safety assessment.

An animal model for food allergy is needed to assess genetically modified food crops for potential allergenicity. The ideal model must produce allergic antibody (IgE) to proteins differentially according to known allergenicity before being used to accurately identify potential allergens among novel proteins. The oral route is the most relevant for exposure to food antigens, and a protein's stability to digestion is a current risk assessment tool based on this natural route. However, normal laboratory animals do not mount allergic responses to proteins administered orally due to oral tolerance, an immunologic mechanism which specifically suppresses IgE. To circumvent oral tolerance and evoke differential IgE responses to a panel of allergenic and nonallergenic food extracts, female C3H/HeJ mice were exposed subcutaneously or orally with cholera toxin as an adjuvant. All foods elicited IgE by the subcutaneous route. Oral exposure, however, resulted in IgE to allergens (peanut, Brazil nut, and egg white) but not to nonallergens (spinach and turkey), provided that the dose and exposures were limited. Additionally, in vitro digestibility assays demonstrated the presence of digestion-stable proteins in the allergenic food extracts but not in the nonallergenic foods. Our results suggest that the subcutaneous route is inadequate to distinguish allergens from nonallergens, but oral exposure under the appropriate experimental conditions will result in differential allergic responses in accordance with known allergenicity. Moreover, those foods containing digestion-resistant proteins provoke allergic responses in this model, supporting the current use of pepsin resistance in the decision tree for potential allergenicity assessment.     

Evaluation of the immunosensitizing potential of chlorogenic acid using a popliteal lymph node assay in BALB/c mice

It has yet to be established whether chlorogenic acid (CGA), a common xenobiotic with potential exposure risk to humans, is associated with immune-mediated hypersensitivity reactions (HRs). The primary limitation in evaluating this potential relationship is the lack of an effective animal model for use in predicting the immunosensitizing potential of low molecular weight compounds (LMWCs). Currently, the popliteal lymph node assay (PLNA) is considered a very promising tool for assessing immunosensitizing potential of LMWCs. To determine whether CGA may possess an intrinsic capacity to stimulate or dysregulate immune responses, and if so, what mechanisms may be involved, we characterized the popliteal lymph node reaction induced by CGA in naive female BALB/c mice using both a direct PLNA (d-PLNA) and a reporter antigen PLNA (RA–PLNA) method. Our results show that CGA failed to induce immunoreactivity following a single subcutaneous injection either alone or when combined with TNP–OVA or TNP–Ficoll. These results indicated that CGA lacks the intrinsic capacity to sensitize or stimulate immune responses in BALB/c mice. Moreover, these results suggest that exposure to CGA may not represent a safety concern for humans and that removal of CGA from Traditional Chinese Medicine Injections may not significantly decrease the prevalence of HRs.     

Characterization of antibody responses induced in rodents by exposure to food proteins: influence of route of exposure

There is a growing interest in the development of methods to characterize the allergenic properties of novel proteins, particularly those expressed by transgenic crop plants. Approaches to the direct evaluation of allergenic potential have focused generally on the ability of proteins to induce antibody (particularly IgE antibody) after systemic (intraperitoneal; i.p.) or gavage administration to high IgE responder strain rodents. To date there has been no systematic comparison of the reliability, sensitivity or selectivity of these approaches. We have, therefore, compared antibody (IgG and IgE) responses induced in Brown Norway (BN) rats by daily gavage administration and in BALB/c strain mice following intraperitoneal or gavage exposure to food proteins of varying allergenic potential. Animals were exposed to the allergens peanut agglutinin and ovalbumin (OVA) or to a crude potato protein extract (PPE) containing acid phosphatase activity, a common foodstuff which appears to be of low allergenicity. All test proteins were clearly immunogenic when administered by gavage to BN rats, with measurable, and in some cases very vigorous, IgG antibody responses recorded for all animals. Identical exposure of BALB/c strain mice also stimulated detectable IgG antibody responses, with particularly high titers recorded following treatment with peanut agglutinin and somewhat less vigorous responses induced by OVA and PPE. Despite these high titer IgG antibody responses, however, none of the proteins provoked detectable IgE antibody following gavage administration to BN rats. In contrast, in BALB/c mice oral exposure to peanut agglutinin elicited high titer IgE antibody, although IgE antibody responses to both OVA and PPE were much weaker. Parenteral (i.p.) treatment of BALB/c strain mice with each of the test materials induced relatively high titer IgG antibody and a differential potential to stimulate IgE antibody was observed. High titer IgE responses were provoked by i.p. administration of peanut agglutinin and OVA, whereas PPE stimulated little or no detectable IgE antibody. It would appear, therefore, that while it is possible to elicit robust IgE responses by gavage exposure of BALB/c strain mice to some protein allergens, such as peanut agglutinin, such responses are generally weaker and less consistent than those provoked by i.p. administration. Furthermore, gavage treatment failed to induce detectable IgE responses in the BN rat, suggesting that the ability these animals to mount IgE responses is somewhat variable. Following i.p. administration to BALB/c strain mice, these proteins displayed immunological properties consistent with what is known of their allergenic potential in humans, suggesting that, following further evaluation with a wider range of proteins, this method may provide one approach to the identification of potential protein allergens.     

Oxazolone and diclofenac-induced popliteal lymph node assay reactions are attenuated in mice orally pretreated with the respective compound: potential role for the induction of regulatory mechanisms following enteric administration

The murine popliteal lymph node assay (PLNA) was examined as a preclinical assay with the potential to identify low-molecular-weight compounds (LMWCs) that are likely to be associated with immune-mediated drug hypersensitivity reactions (IDHRs) in humans. We hypothesized that the contact sensitizer oxazolone (OX) would cause a strong PLN reaction in naive mice and that the PLN reaction would be attenuated in mice orally pretreated with OX due to the induction of oral tolerance. In naive mice, OX induced a strong PLN reaction and caused dose-dependent increases in PLN size, weight, cellularity, percentage of CD4(+) PLN T cells, and percentage of PLN B cells, with a concomitant decrease in the percentage of CD8(+) PLN T cells. Next, the PLNA was conducted in mice gavaged three times with either OX or vehicle alone (olive oil). Mice pretreated with OX had suppressed PLN reactions following the footpad injection of OX (decrease in PLN size, weight, and cellularity), which was associated with an increase in the percentage of PLN CD8(+)T cells. In contrast, oral pretreatment with OX had no observable effect on the PLN reaction induced following footpad injection of the irrelevant hapten dinitrochlorobenzene (DNCB). Adoptive transfer studies were conducted to examine the mechanism of PLN hyporesponsiveness. It was found that either (1) unfractionated splenocytes or (2) purified CD8(+) splenocytes, but not (3) purified CD4(+) splenocytes isolated from mice gavaged with OX adoptively transferred PLN suppression to naive BALB/c mice. Because OX is not a pharmaceutical, we also examined the NSAID diclofenac (DF) (Voltaren). Like OX, DF caused dose-dependent increases in PLN size, weight, and cellularity in naive mice. Furthermore, like OX, the diclofenac-induced PLN reaction was attenuated in mice that had been orally pretreated three times with DF. However, splenocytes from mice orally treated with DF were not able to adoptively transfer PLN hyporesponsiveness. Collectively, these observations demonstrate that both OX and DF are potent immunostimulators in the PLNA. As importantly, these results demonstrate that the immunostimulating potential of OX and DF in the PLNA is significantly decreased in mice orally exposed to the respective drug, possibly due to the presence of a cellular mechanism of oral tolerance. For OX, the mechanism appears to involve, in part, CD8(+) T cells, whereas the mechanism(s) associated with PLN hyporesponsiveness using DF remain to be defined.     

Popliteal lymph node assay: facts and perspectives

The popliteal lymph node assay (PLNA) derives from the hypothesis that some supposedly immune-mediated adverse effects induced by certain pharmaceuticals involve a mechanism resembling a graft-versus-host reaction. The injection of many but not all of these compounds into the footpad of mice or rats produces an increase in the weight and/or cellularity of the popliteal lymph node in the treated limb (direct PLNA). Some of the compounds known to cause these adverse effects in humans, however, failed to induce a positive PLNA response, leading to refinements of the technique to include pretreatment with enzyme inducers, depletion of CD4(+) T cells or additional endpoints such as histological examination, lymphocyte subset analysis and cytokine fingerprinting. Alternative approaches have been used to improve further the predictability of the assay. In the secondary PLNA, the test compound is injected twice in order to illicit a greater secondary response, thus suggesting a memory-specific T cell response. In the adoptive PLNA, popliteal lymph node cells from treated mice are injected into the footpad of naive mice; a marked response to a subsequent footpad challenge demonstrates the involvement of T cells. Finally, the reporter antigens TNP-Ficoll and TNP-ovalbumin are used to differentiate compounds that induce responses involving neo-antigen help or co-stimulatory signals (modified PLNA). The PLNA is increasingly considered as a tool for detection of the potential to induce both sensitization and autoimmune reactions. A major current limitation is validation. A small inter-laboratory validation study of the direct PLNA found consistent results. No such study has been performed using an alternative protocol. Other issues include selection of the optimal protocol for an improved prediction of sensitization vs autoimmunity, and the elimination of false-positive responses due to primary irritation. Finally, a better understanding of underlying mechanisms is essential to determine the most relevant endpoints. The confusion resulting from use of the PLNA to predict autoimmune-like reactions as well as sensitization should be clarified. Interestingly, most drugs that were positive in the direct PLNA are also known to cause drug hypersensitivity syndrome in treated patients. This observation is expected to open new avenues of research.     

Proteomic screening points to the potential importance of Ara h 3 basic subunit in allergenicity of peanut

Peanuts are complex storage proteins with high contents and have been identified as one of the most allergenic foods. In this review, we summarize some of the latest findings and the potential importance of the Ara h 3 basic subunit, which has been overlooked as an allergen in early literature. Some recent studies indicate that Ara h 3 basic subunit may be as significant as or even a more important allergen than the acidic subunit. For example, one clinical study found a group of children with peanut allergy who were specifically sensitized to the basic subunit of Ara h 3. Although, proteomic analysis of total peanut storage proteins has revealed limited polymorphic profiles of major proteins in diverse peanut germplasm accessions, a study reported a peanut breeding line 'GT-C9' lacking several seed protein peptides, in which the missed major proteins were basic subunits of Ara h 3. This breeding line was shown to exhibit significantly lower levels of advanced glycation end (AGE) products and IgE binding by the sera of peanut allergic patients, which implies a role for the basic subunit of Ara h 3 in the allergenicity of peanuts. Further studies are needed to investigate the contribution of Ara h 3 basic subunits to peanut allergenicity.     

Allergenicity attributes of different peanut market types

Four different market classes of peanut (Runner, Virginia Spanish, and Valencia) are commonly consumed in Western countries, but for some consumers peanuts are a main cause of food-induced anaphylaxis. Limited information is available on the comparative allergenicity of these distinct market classes. The aim of this study was to compare allergenicity attributes of different peanut cultivars.The protein content and protein profiles were highly comparable for all tested cultivars. All cultivar samples contained the major allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6, as assessed by SDS-PAGE and RP-HPLC, although some minor differences in major allergen content were found between samples. All samples were reactive in commercial ELISAs for detection and quantification of peanut protein. IgE-binding potency differed between samples with a maximum factor of 2, indicating a highly comparable allergenicity.Based on our observations, we conclude that peanuts from the main market types consumed in Western countries are highly comparable in their allergenicity attributes, indicating that safety considerations with regard to peanut allergy are not dependent on the peanut cultivar in question.     

Intradermal exposure of BALB/c strain mice to peanut protein elicits a type 2 cytokine response.

There is a growing need for the development of methods to characterize the allergenic properties of novel proteins, particularly those expressed by transgenic crop plants. Hence, there is considerable interest in the development of suitable animal models for this purpose. The production of specific IgE antibody has been reported following sensitization with food allergen via oral or systemic (intraperitoneal) routes of exposure. We have characterized cytokine profiles induced by intradermal treatment of BALB/c strain mice with a purified peanut allergen, Arachis hypogea lectin. Mice were exposed to peanut lectin by intradermal administration and the cytokine responses in the lymph node draining the site of exposure analyzed at the secreted protein level by enyzme-linked immunosorbent assay (ELISA) and cytokine mRNA level by ribonuclease protection assay (RPA). Exposure to peanut lectin, under conditions that induced robust IgE antibody titers, was found to be associated with a T helper 2 (Th2)-type cytokine expression profile at both the mRNA and secreted protein levels. Culture of na?ve lymph node cells with peanut lectin failed to stimulate marked proliferation or cytokine production, confirming this protein is not mitogenic for mouse lymphocytes. Furthermore, the expression of Th2 cytokines was associated with the effector/memory CD62L- cell population. Similar treatment with a non-allergenic protein, potato acid phosphatase, failed to induce Th2 cytokine expression. These data demonstrate that exposure of mice to peanut allergen results in the selective stimulation of a Th2-type response.     

Induction of IgE antibody responses by protein allergens: inter-laboratory comparisons.

There is a growing interest in the development of methods for the evaluation of the allergenic potential of novel proteins. One approach is the measurement of specific IgE antibody production stimulated by systemic (intraperitoneal; i.p.) exposure of BALB/c strain mice. In the current investigations, inter-laboratory comparisons have been performed of IgE antibody production induced in mice by food proteins of differing sensitizing potential. Female BALB/c strain mice (n=5) were exposed to 0.1% peanut agglutinin, an allergenic constituent of peanuts, to 2% ovalbumin (OVA), a major allergenic constituent of hens' egg, or to a protein considered to lack significant allergenicity, potato agglutinin (5%). Specific IgE antibody was measured by homologous passive cutaneous anaphylaxis assay and IgG and IgG1 antibody production was analysed by enzyme-linked immunosorbent assay (ELISA). Two independent experiments were conducted in each laboratory, but with all serological analyses conducted in one of the laboratories. Each of the proteins induced vigorous IgG and IgG1 antibody responses, with no statistically significant differences in titres recorded between laboratories. Furthermore, OVA and potato agglutinin induced responses of equivalent immunogenicity with respect to both IgG and IgG1 antibody titres. Administration of peanut agglutinin and OVA each stimulated marked IgE antibody responses in every experiment. In the two laboratories, titres ranged from 1:32 and 1:64 for peanut agglutinin, and from 1:8 and 1:32 for OVA. In contrast, exposure to potato agglutinin failed to induce vigorous IgE production, with no detectable IgE (negative with neat serum), or titres of 1 (positive with neat serum only) recorded. These data demonstrate that the induction of IgE antibody by food proteins of differing allergenic potential is a relatively robust phenomenon and transferable between laboratories. Furthermore, these results provide additional evidence that the measurement of antibody (IgE) responses in BALB/c mice may allow discrimination between allergens and those materials that apparently lack allergenicity.     

Lactobacillus casei Shirota does not decrease the food allergic response to peanut extract in Brown Norway rats

Probiotics are claimed to beneficially affect the immune system and their involvement in allergy prevention is being investigated extensively. However, the efficacy of probiotics in allergy prevention remains controversial. We investigated whether the probiotic Lactobacillus casei Shirota (LcS) could modulate the food allergic response against peanut extract (PE) in Brown Norway (BN) rats. For this purpose BN rats were sensitized to PE (0, 1 and 10 mg/(rat d)) by daily oral gavage and the LcS-groups were additionally orally dosed with 1 x 10(9) colony forming units LcS/(rat d). LcS administration had minor effects in animals that were not sensitized. LcS increased Th1-(PE-specific IgG1), whereas the Th1/Th2 ratio based on PE-specific IgG1/PE-specific IgG2a shifted towards Th2 dominance in rats sensitized to PE in the presence of LcS as compared to rats that were sensitized to PE only. LcS stimulated PE-specific IgG2a; but for PE-specific IgE the effect was less clear; whereas there was no overall effect, two rats did not show detectable specific IgE antibodies, whereas the remainder showed significantly increased levels. LcS also resulted in increased numbers of basophilic granulocytes in blood. Furthermore, LcS increased levels of both Th1-(IFN-gamma) and Th2-(IL-4) related cytokines in PE stimulated spleen and mesenteric lymph node (MLN) cells, but predominantly IL-4 levels in the supernatants of both spleens and MLNs. Our study does not support the hypothesis that LcS down-regulates food allergic responses in a BN rat model for food allergy to peanut.     

BALB/c mice orally pretreated with diclofenac have augmented and accelerated PLNA responses to diclofenac

The nonsteroidal anti-inflammatory drug (NSAID) diclofenac (DF) is associated with idiosyncratic hepatotoxicity and several other distinct hypersensitivity reactions. The mechanism(s) are unknown but evidence suggests both cell-mediated and antibody-mediated immune effector systems may be involved. In the present studies, the immunostimulating potential of DF was evaluated using the direct and TNP-Ficoll (trinitrophenyl (TNP)-Ficoll) popliteal lymph node assays (PLNA). These assays were conducted in naive mice, T-cell-deficient mice, or in mice that had been pretreated with a single oral dose of DF. In naive mice, DF induced a dose-, and time-dependent reaction in the direct PLNA. A significant increase in popliteal lymph node (PLN) weight and PLN cellularity was detected 7 days after the injection of 0.50 and 0.75 mg DF, whereas 0.25 mg DF produced no observable effect. With 0.75 mg, there was a rapid accumulation of cells in the PLN between days 5 and 6, with maximum PLN cellularity observed between days 7 and 10. The immunostimulating effects of DF were significantly attenuated in T-cell-deficient mice. In the TNP-Ficoll PLNA conducted in naive mice, DF caused a dose-dependent increase in PLN cellularity on day 7 with a time-dependent increase in anti-TNP antibody forming cells (AFCs) in the PLN; the reaction was dominated by IgM anti-TNP AFCs from day 4 through day 7, but IgG1 anti-TNP AFCs and IgG3 anti-TNP AFCs were detected beginning on day 5 and day 6, respectively. Relative to mice pretreated with vehicle (ddH2O), mice orally pretreated with DF had a significantly greater increase in PLN weight 5 days following the injection of 0.25 mg DF and a significantly greater increase in PLN weight and cellularity 4 days following the injection of 0.50 mg DF. Oral pretreatment with DF had no observable effect on the direct PLN reaction induced following the footpad injection of the irrelevant drugs, D-penicillamine (D-PEN) or streptozotocin. When 0.50 mg DF was co-injected with TNP-Ficoll, mice orally pretreated with DF, compared to vehicle-pretreated mice, and had a significantly greater increase in IgM anti-TNP AFCs on day 4, and a significant increase in both IgG1 and IgG3 anti-TNP AFCs on day 7. Additionally, IgG1 anti-TNP AFCs were detected in the PLN of DF-pretreated mice as early as day 4. No differences in anti-TNP AFCs were detected when orally pretreated mice were injected with 0.50 mg D-PEN. Collectively, these results demonstrated that DF (i) is an immunostimulating drug that induced a dose-, time- and T-cell-dependent PLN reaction in naive mice, (ii) provided non-cognate help that produced antibody against co-injected TNP-Ficoll, and (iii) mice orally pretreated with DF had DF-specific increased responsiveness in the direct PLNA, which (iv) resulted in accelerated and augmented AFC production against co-injected TNP-Ficoll. These novel findings suggest that oral administration of DF may result in primed T cells that respond with footpad injection. Thus, the oral pretreatment modification of the PLNA should be further explored as a possible alternative to hypersensitivity testing with drugs administered via the oral route. Additional studies with other compounds known to produce hypersensitivity reactions are needed.     

Partial characterization of red gram (Cajanus cajan L. Millsp) polypeptides recognized by patients exhibiting rhinitis and bronchial asthma

We sought to assess the allergenic potential of red gram by identifying and characterizing the responsible proteins. Immunoblotting was performed to detect IgE binding proteins. Identities of these proteins were confirmed by mass spectrometry. To evaluate allergenic potential, BALB/c mice were sensitized with red gram proteins and levels of specific immunoglobulins, histamine, Th2 cytokines were measured. Allergenic response was evident by significant increase in specific IgE, IgG1, histamine and Th2 cytokine levels. Prominent anaphylactic symptoms, discernible histopathological responses and down regulation of IFN-γ levels give strong support towards allergenicity of red gram proteins. IgE immunoblot detected five proteins; one of 66 kDa, three of 45 kDa (pI of ∼5.3, 5.9 and 6.6) and one of 30 kDa. All these proteins showed homology to known allergens of soybean (different subunits of β-conglycinin), lentil (Len c1 and Len c2), peanut (Ara h1) and pea (vicilin). In conclusion, five novel IgE binding proteins (namely Caj c1, Caj c2, Caj c3, Caj c4 and Caj c5) were identified as putative clinically relevant allergens.     

Immunogenic properties of rapidly digested food proteins following gavage exposure of mice: a comparison of ovalbumin with a potato acid phosphatase preparation.

The ability of food proteins to resist digestion in simulated gastric fluid (SGF) correlates with allergenic potential. The purpose of the current investigations was to determine whether this association is due solely to the failure of unstable proteins to elicit an immune response when administered orally. We have examined immune responses induced in BALB/c mice by gavage administration of ovalbumin (OVA) and a crude potato protein extract (PPE) containing acid phosphatase activity. The stability of OVA and PPE in SGF was measured using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The ability of these proteins to stimulate specific IgG and IgE antibody production in mice following parenteral (intraperitoneal; ip) or oral (gavage) exposure was compared using enzyme-linked immunosorbent and homologous passive cutaneous anaphylaxis assays, respectively. Both OVA and PPE induced specific IgG antibody responses when administered either by gavage or by ip injection. Parenteral, but not gavage, exposure to OVA was associated with robust IgE antibody responses. Administration of PPE failed to stimulate strong IgE production via either route of exposure. Differential stability in SGF was observed, with PPE being digested extremely rapidly (within 1 min), whereas OVA was more resistant. The strong association reported by others between stability in SGF and allergenic potential is unlikely to be solely due to orally-ingested labile proteins failing to provoke immune responses due to degradation in the stomach.     

Evaluating the effect of food processing on the potential human allergenicity of novel proteins: international workshop report.

The ILSI Health and Environmental Sciences Institute Protein Allergenicity Technical Committee organized an international workshop in June 2006 in Estoril, Portugal, co-sponsored by the ILSI Research Foundation, ILSI International Food Biotechnology Committee and ILSI Europe. The objective was to discuss the effects of food processing on the allergenic potential of proteins and foods. The impact of food processing on the sensitization/induction phases of food allergy, and the bioavailability of allergens to the immune system were presented. Studies evaluating the stability, digestibility, and allergenicity of processed food allergens were identified, and their complexity and limitations discussed. Participants agreed that investigating food allergy mechanisms, validating appropriate methods for identifying allergenic proteins, and refining strategies to assess and manage the risks from food allergy were important before processing considerations are integrated into public-health decision-making for novel proteins. Other factors may also play a role in food allergy and include: food matrix; multiplicity of epitopes; geographic variation in patterns/prevalence of food allergies; and genetic factors, but required further exploration. Food processing may increase or decrease the intrinsic allergenicity of a protein, but current data do not facilitate the identification of specific variables that could be used to reliably determine how processing will influence protein allergenicity.     

Assessment of sensitization potential of monoterpenes using the rat popliteal lymph node assay.

The popliteal lymph node assay (PLNA) has been proposed as a screening test for detecting chemicals with potential of inducing allergic and auto-immune-like reactions in humans. In the present study, we used the rat PLNA to evaluate the immuno-sensitizing potential of 10 monoterpenes found in the essential oils of a variety of aromatic, edible and medicinal plants. The primary or direct PLNA was performed with the monoterpenes, and chlorpromazine (CPZ) and barbital were used as positive and negative controls, respectively. Female, 7-8 week-old Wistar rats were injected subcutaneously (50 microL) with the test substance (0.5, 2.5 or 5mg) into the right hind footpad while the contralateral footpad was injected with the vehicle (DMSO) alone. Weight (WI) and cellularity (CI) indices for draining PLNs were determined 7 days after treatment. PLNA was positive (WI >or= 2 and CI >or= 5) for CPZ, citral, alpha-terpinene, beta-myrcene and (-)-alpha-pinene, and negative for barbital, DMSO, (-)-menthol, 1,8-cineole, (+/-) citronellal, (+)-limonene, (+/-) camphor and terpineol. A secondary PLNA, a T-cell priming test, was carried out with the four substances that had been positive in the primary assay. Six weeks after being locally primed with 5 mg/paw, rats were sc injected into the same footpad with a dose (0.5 mg/paw) of the substance that had been previously found to be insufficient to cause a positive response. WI and CI were then calculated 4 and 7 days after the second injection. CPZ was also positive in the secondary assay thereby confirming that it is a sensitizing agent. Citral, alpha-terpinene, beta-myrcene and (-)-alpha-pinene, however, were negative in the secondary assay. In summary, citral, alpha-terpinene, beta-myrcene and (-)-alpha-pinene induced a clear immuno-stimulatory response due to their irritant properties but no monoterpene proved to be a sensitizing agent in the PLNA.     

The reactive D-glucopyranose moiety of streptozotocin is responsible for activation of macrophages and subsequent stimulation of CD8+ T cells

The antitumor drug streptozotocin (STZ) is commonly used as a diabetogenic compound in animal models. At relatively low doses, STZ-induced beta cell destruction is associated with Th1-driven type 1 immune reactions, including macrophages (MPhi) and IFN-gamma-producing CD8(+) T cells. STZ induces similar Th1-dependent effects in the popliteal lymph node assay (PLNA), and because this assay allows straightforward examination of early immunostimulating processes, the PLNA was used to further examine the importance of MPhi and structural properties of STZ in relation to the induction of type 1 immune responses. Results show that elimination of MPhi with clodronate-containing liposomes prior to exposure to STZ prevents the occurrence of some (CD8(+) T cell activation, IFN-gamma production, and tissue destruction) but not all (IgG2a formation) type 1 immune responses. It appeared that stimulation of MPhi depends on the d-glucopyranose moiety of STZ, as well as on the intact reactive N-methyl-N-nitrosourea (MNU) moiety. However, the MNU moiety suffices to induce IgG2a formation. In addition, STZ-derived nitric oxide may have modulating effects on the elicitation of STZ-induced immune responses. Present results support the idea that MPhi activation is indispensable for the STZ-induced tissue destructive type 1 responses and that various STZ-induced type 1 immune responses are differently regulated.     

Consideration of interaction between nanoparticles and food components for the safety assessment of nanoparticles following oral exposure: A review

Nanoparticles (NPs) are increasingly used in food, and the toxicity of NPs following oral exposure should be carefully assessed to ensure the safety. Indeed, a number of studies have shown that oral exposure to NPs, especially solid NPs, may induce toxicological responses both in vivo and in vitro. However, most of the toxicological studies only used NPs for oral exposure, and the potential interaction between NPs and food components in real life was ignored. In this review, we summarized the relevant studies and suggested that the interaction between NPs and food components may exist by that 1) NPs directly affect nutrients absorption through disruption of microvilli or alteration in expression of nutrient transporter genes; 2) food components directly affect NP absorption through physico-chemical modification; 3) the presence of food components affect oxidative stress induced by NPs. All of these interactions may eventually enhance or reduce the toxicological responses induced by NPs following oral exposure. Studies only using NPs for oral exposure may therefore lead to misinterpretation and underestimation/overestimation of toxicity of NPs, and it is necessary to assess the synergistic effects of NPs in a complex system when considering the safety of NPs used in food.     

Why are some proteins allergens?

The ability of certain proteins to induce an allergic response in susceptible individuals is well established. Symptoms can range from mild erythema or rhinitis, to acute, and possibly fatal, anaphylactic shock. Because such allergic responses require complex interactions between the protein and the immune system, they are notoriously difficult to predict. Nevertheless, it is clear that some proteins are intrinsically more allergenic than others. The challenge for toxicologists is to identify those characteristics that confer on proteins the potential to induce allergic sensitization and allergic disease. Here, we first consider the potential contribution that individual epitopes may make to the allergenicity of a protein. These are the minimal peptide units within proteins that can be recognized by the immune system and are a fundamental requirement for all immune responses, including those resulting in allergic sensitization. It appears that allergens must necessarily contain B-cell epitopes to which immunoglobulin E (IgE) can bind, and T-cell epitopes capable of inducing a type 2 T-lymphocyte response. Nevertheless, it appears doubtful that the presence of appropriate epitopes alone is sufficient to endow a protein with allergenic potential. We therefore consider also the contribution that other features and characteristics of proteins may make to their overall allergenicity. In particular, we consider the effects that resistance to proteolysis, post-translational glycosylation, and enzymatic activity may have. It appears that relative stability in simulated gastric fluid (SGF) sometimes correlates with allergenic activity. However, this is not universally true, and it is known that there are protein allergens, such as some of those associated with oral allergy syndrome, that are unstable. Nevertheless, if stability in SGF is associated with the intrinsic allergenicity of many proteins irrespective of the route of exposure, then this may reflect some more fundamental property of proteins, and possibly their stability in other biologic matrices and/or to intracellular proteases. Post-translational modification appears generally to enhance allergenicity, perhaps by increasing uptake and detection of the protein by the immune system. Some enzymatic activities also enhance allergenicity through what appear to be several different mechanisms, including nonspecific activation of cells participating in the immunologic response. Overall, it appears likely that many factors can contribute to the overall allergenicity of any given protein. Some, such as the presence of epitopes with allergenic potential, may be essential. Others, such as the glycosylation status, resistance to proteolysis, and enzymatic activity, may play a subsidiary but nevertheless critically important role. By better defining the limits within which these factors operate, we can hope to gain a better understanding of the fundamental origins of protein allergenicity, and therefore be in a position to identify and characterize the hazards and risks of allergic disease associated with novel proteins.     

Assessment of three human FcepsilonRI-transfected RBL cell-lines for identifying IgE induced degranulation utilizing peanut-allergic patient sera and peanut protein extract

Specific IgE sera screening studies are employed to investigate protein cross-reactivity. Such nonfunctional immunochemical methods cannot measure the biological activity of proteins. Therefore, an assay using RBL cells transfected with human FcepsilonRI was developed. Our objective was to evaluate the degranulation of three cell-lines expressing either the alpha-(RBL-hEI(a)-2B12 and RBL-30/25cells) or alpha-, beta-, and gamma-subunits (RBL SX-38) of the human FcepsilonRI by beta-hexosaminidase release. Purified human IgE and serum-derived polyclonal IgE from peanut-allergic subjects following challenge with anti-IgE or peanut protein extract, respectively, were utilized. Robust degranulation was induced in all three: RBL-30/25 (84%), -hEI(a)-2B12 (54%), SX-38 (94%), respectively, using purified IgE+anti-human IgE. Good release (18%, 40-45%, and 65%, respectively) occurred for one peanut-allergic subject+peanut extract with all cell-lines. With serum from three other peanut-allergic subjects, no beta-hexosaminidase release occurred with RBL-hEI(a)-2B12 cells+peanut extract, while only serum from one subject induced good degranulation, 30% and 60%, respectively, with RBL-30/25 and RBL SX 38 cells. Consistent degranulation with a potent food allergen (peanuts) was not observed. The assay's utility in safety assessment, predictive value and reproducibility for evaluating the cross-reactivity of proteins with allergens needs further investigation with additional proteins and well-characterized sera.     

Oral immunogenicity of the plant proteinase bromelain

Bromelain is a natural mixture of proteolytic enzymes derived from pineapple stem that has been shown to have anti-inflammatory activity when administered orally. Although most proteins given orally without adjuvant (e.g., food) result in tolerance, we previously reported that long-term oral exposure to bromelain stimulated the development of high serum anti-bromelain antibody titers. The purpose of these studies was to further investigate the mechanisms responsible for the immunogenicity of oral bromelain. Results showed that repeated exposure was required for development of anti-bromelain antibodies, with strong antibody responses in all mice that received at least 12 doses of bromelain either orally or intragastrically over 3-6 weeks. Proteolytic activity was required for strong oral immunogenicity in the absence of conventional adjuvant, with strong serum antibody responses generated against proteolytically active bromelain and trypsin, but not against ovalbumin, lysozyme, or inactivated bromelain. Significantly higher anti-bromelain antibody titers were seen in IL-10-deficient versus wild-type mice, suggesting that simultaneous treatments that decrease IL-10 activity may further enhance systemic antibody responses following oral exposure. The antibodies generated did not affect the proteolytic activity of bromelain. The data demonstrate that proteolytically active antigens such as bromelain can stimulate both systemic and mucosal immune responses following repeated oral exposure. Further studies of the mechanisms involved in generation of immune responses following oral exposure to proteolytically active antigens can lead to a better understanding of mechanisms of oral tolerance and to the development of novel adjuvants for oral vaccines.