A subset of walnut allergic adults is sensitized to walnut 11S globulin Jug r 4.

BACKGROUND: The role of sensitization to commercially available allergens of English walnut (Juglans regia) Jug r 1, 2 and 3 in walnut allergy has been previously investigated in walnut allergic adults and was unable to explain all cases of walnut allergy. OBJECTIVES: Identify recognized walnut allergens, other than the ones previously investigated (Jug r 1-3), in walnut allergic adults and determine the sensitization frequency and diagnostic value. METHODS: Three different in-house walnut extracts were prepared and analysed on SDS-PAGE blots to identify allergenic walnut proteins. Immunoblots and immunoprecipitation, followed by LC-MS analysis, were performed to screen for, and confirm, IgE binding to walnut allergens in selected walnut allergic adults. In a cohort of 55 walnut challenged adults, including 33 allergic and 22 tolerant, sensitization to native and recombinant walnut allergen Jug r 4 was assessed using immunoblotting and immuno-line blot (EUROLINE), respectively. RESULTS: Screening of sera of 8 walnut allergic adults identified Jug r 4 as an allergen in our population. In the total cohort of 55 subjects, 5 were positive for Jug r 4 on immunoblot and 10 on EUROLINE. All but one EUROLINE positive subject had a positive food challenge (sensitivity 27%, specificity 95%, PPV 90%, NPV 47%). All 5 subjects positive on immunoblot were also positive on EUROLINE. LC-MS analysis showed a lack of Jug r 4 in the ImmunoCAP extract. Co-sensitization to other 11S albumins (eg hazelnut Cor a 9) was common in Jug r 4 sensitized subjects, potentially due to cross-reactivity. CONCLUSIONS: Walnut 11S globulin Jug r 4 is a relevant minor allergen, recognized by 27% of walnut allergic adults. It has a high positive predictive value of 90% for walnut allergy. Specific IgE against Jug r 4 occurred mostly with concomitant sensitization to other walnut components, mainly Jug r 1.

Component-resolved diagnostics demonstrates that most peanut-allergic individuals could potentially introduce tree nuts to their diet.

BACKGROUND: Nut allergy varies from pollen cross-allergy, to primary severe allergy with life-threatening symptoms. The screening of IgE antibodies to a wide spectrum of allergens, including species-specific and cross-reactive allergens, is made possible via microarray analysis. OBJECTIVE: We sought to study the association of variable IgE sensitization profiles to clinical response in peanut-challenged children and adolescents in a birch-endemic region. In addition, we studied the avoidance of tree nuts and species-specific sensitizations. METHODS: We studied 102 peanut-sensitized patients who underwent a double-blind placebo-controlled challenge to peanut. We analysed ISAC ImmunoCAP microarray to 112 allergens, singleplex ImmunoCAPs for hazelnut Cor a 14 and cashew Ana o 3, and performed skin prick tests to peanut, tree nuts and sesame seed. We surveyed avoidance diets with a questionnaire. RESULTS: Sensitization to PR-10 proteins was frequent (Bet v 1 90%), but equally high in the challenge negatives and positives. IgE to Ara h 2 and Ara h 6 discriminated peanut allergic (n = 69) and tolerant (n = 33) the best. Avoidance of tree nuts was common (52% to 96%), but only 6% to 44% presented species-specific sensitizations to tree nuts, so a great number could potentially introduce these species into their diet. CONCLUSIONS AND CLINICAL RELEVANCE: PR-10-sensitizations were frequent and strong regardless of peanut allergy status. Component-resolved diagnostics can be employed to demonstrate to patients that sensitization to seed storage proteins of tree nuts is uncommon. Several tree nuts could potentially be reintroduced to the diet.

Approaches to assess IgE mediated allergy risks (sensitization and cross-reactivity) from new or modified dietary proteins.

The development and introduction of new dietary protein sources has the potential to improve food supply sustainability. Understanding the potential allergenicity of these new or modified proteins is crucial to ensure protection of public health. Exposure to new proteins may result in de novo sensitization, with or without clinical allergy, or clinical reactions through cross-reactivity. In this paper we review the potential of current methodologies (in silico, in vitro degradation, in vitro IgE binding, animal models and clinical studies) to address these outcomes for risk assessment purposes for new proteins, and especially to identify and characterise the risk of sensitization for IgE mediated allergy from oral exposure. Existing tools and tests are capable of assessing potential crossreactivity. However, there are few possibilities to assess the hazard due to de novo sensitization. The only methods available are in vivo models, but many limitations exist to use them for assessing risk. We conclude that there is a need to understand which criteria adequately define allergenicity for risk assessment purposes, and from these criteria develop a more suitable battery of tests to distinguish between proteins of high and low allergenicity, which can then be applied to assess new proteins with unknown risks.

Unintended allergens in precautionary labelled and unlabelled products pose significant risks to UK allergic consumers.

BACKGROUND: Allergens in food may pose a risk to allergic consumers. While there is EU regulation for allergens present as an ingredient, this is not the case for unintended allergen presence (UAP). Food companies use precautionary allergen labels to inform allergic individuals of a potential risk from UAPs. This study investigates the risk of an allergic reaction within the milk-, wheat-, hazelnut- and peanut-allergic populations when ingesting UK foods across multiple product categories with and without precautionary allergen labelling. METHODS: Allergen risk assessment using probabilistic techniques enables the estimation of the residual risk after the consumption of a product that unintentionally contains an allergen. RESULTS: Within this selection of UK products, the majority that tested positive for an allergen contained a concentration of allergen predicted to cause a reaction in >1% of the allergic population. The concentrations of allergens measured were greater than the VITAL(®) 2.0 action levels and would trigger precautionary allergen labelling. This was found for products both with and without precautionary allergen labelling. CONCLUSIONS: The results highlight the need for the food industry and regulators to adopt a transparent, risk-based approach for the communication of the risk associated with potential cross-contact that could occur in the processing facility or production chain.

Objective eliciting doses of peanut-allergic adults and children can be combined for risk assessment purposes.

BACKGROUND: To improve food labelling strategies, information regarding eliciting doses (EDs) and the effect of patient characteristics on these EDs is necessary. OBJECTIVE: To establish EDs for objective and subjective symptoms and analyse the effect of sensitization levels and other patient characteristics on threshold distribution curves (TDCs). METHODS: Threshold data from 100 adults and 262 children with a positive food challenge were analysed with interval-censoring survival analysis (ICSA) and fitted to a TDC from which EDs could be extracted. Possible influencing factors were analysed as covariates by ICSA. A hazard ratio (HR) was calculated in case of a significant effect. RESULTS: TDCs for both objective and subjective symptoms were significantly different between adults and children (P < 0.001). Objective ED05 values, however, were comparable (2.86 mg peanut protein in adults and 6.38 mg in children). Higher levels of sIgE to Ara h 2 and peanut extract were associated with a larger proportion of patient groups reacting to a dose increase with objective symptoms (adults and children) or subjective symptoms (adults, in children a trend). Age had a similar effect in children (HR 1.05 for objective symptoms and 1.09 for subjective symptoms). Gender had no effect on TDCs. CONCLUSION AND CLINICAL RELEVANCE: Subjective and objective TDCs were different between adults and children, but objective ED05 values were comparable, meaning that threshold data from children and adults can be combined for elaboration of reference doses for risk assessment. Higher sIgE levels to Ara h 2 and peanut extract were associated with a larger proportion of both patient groups to react to a certain dose increase.

Remodeling of the actin cytoskeleton of target hepatocytes and NK cells during induction of apoptosis.

Natural Killer cells are immune cells that recognize and eliminate altered and non-self cells from the circulation. To study the interaction between NK cells and target cells, we set up an experimental system consisting of rat Interleukin-2 activated Natural Killer cells (A-NK cells) and rat hepatocytes with a masked Major Histocompatibility Complex (MHC). The masking of the MHC induces recognition of the hepatocytes by the NK cells as non-self. We showed that in vitro apoptosis is rapidly induced in the hepatocytes [Blom et al., 1999] after co-incubation with A-NK cells. Now we describe the morphological changes that occur during and after interaction of A-NK cells with hepatocytes. Confocal laser scanning microscopy showed that the actin cytoskeleton of the NK cells was remodeled during attack of hepatocytes. Some NK cells were in close contact with the hepatocytes while others had formed actin-containing dendrites of varying length that made contact with the hepatocytes. However, dendrite formation is not obligatory for induction of apoptosis because cells that were unable to form these did induce FAS-dependent apoptosis in hepatocytes. Apparently both direct as well as distant contact resulted in apoptosis. Formation of the dendrites was calcium-dependent as EGTA largely prevented it. Importantly, chelation of the calcium also suppressed killing of the hepatocytes. Within 1 h after addition of the A-NK cells, morphological changes in hepatocytes that are characteristic of apoptosis, such as the formation of apoptotic bodies and fragmented nuclei, became apparent. Specifically, the actin cytoskeleton of the hepatocytes was remodeled resulting in the formation of the apoptotic bodies. Inhibition of caspase activity by z-Val-Ala-DL-Asp-fluoromethylketone (100 microM) partly protected against the rearrangement of the actin filaments in the hepatocytes.

Prevention of cycloheximide-induced apoptosis in hepatocytes by adenosine and by caspase inhibitors.

The mechanism by which cycloheximide induces apoptosis in isolated rat hepatocytes was studied. Cycloheximide (1-300 microM) induced apoptosis within 3-4 hr in the hepatocytes. Specific apoptotic characteristics such as blebbing, phosphatidyl serine (PS) exposure, chromatin condensation, and nuclear fragmentation were induced. Cycloheximide (CHX) dose dependently activated the caspase-3-like proteases, but not the caspase-1-like proteases. Pretreatment of the hepatocytes with 100 microM of the caspase inhibitors z-Val-Ala-DL-Asp-fluoromethylketone or Ac-Asp-Glu-Val-Asp-aldehyde completely abrogated the caspase activation and the apoptosis. Addition of adenosine (100 microM) reduced phosphatidyl serine exposure and other morphological characteristics of apoptosis by 50%; however, it did not prevent the activation of the caspases, suggesting that adenosine inhibited downstream of caspase activation. The adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine abolished the capacity of adenosine to prevent apoptosis, indicating that prevention was receptor-mediated. During apoptosis, the mitochondrial membrane potential in apoptotic cells (cells with PS exposition) was decreased to 50-60% of the control value; in the population viable cells, however, the mitochondrial membrane potential remained stable. Prevention of apoptosis by the caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone or adenosine prevented the decrease in mitochondrial membrane potential. In conclusion, CHX rapidly induces apoptosis in isolated rat hepatocytes, which is inhibited by adenosine at a relatively late step.

Changes of G-actin localisation in the mitotic spindle region or nucleus during mitosis and after heat shock: a histochemical study of G-actin in various cell lines with fluorescent labelled vitamin D-binding protein.

The presence and localisation of G-actin in various cell lines was studied using the highly G-actin specific, fluorescence-labelled vitamin D-binding protein. In various cell-types, pig kidney-derived cells (LLC-PK1), Chinese hamster ovary (CHO) cells, SV-40 transformed African green monkey kidney (COS) cells and human hepatoma (HepG2) cells, G-actin was only visible in the cytoplasm of interphase cells. However, in mitotic cells, depending on the mitotic phase, intense G-actin staining was found associated with the mitotic spindle (early mitosis) or overlapping the DNA-staining pattern (late mitosis). Also after heat shock (60-180 min at 43 degrees C), an intense nuclear staining of G-actin was observed. In LLC-PK1 cells, the increase of nuclear G-actin staining disappeared again after 24 h at 37 degrees C, but in COS, CHO and HepG2 cells, it was still present in the nucleus after 24 h at 37 degrees C, indicating that the process was not rapidly reversible in these cells; the increased nuclear G-actin was not associated with cell division. Comparison of the amount of G-actin present in the nucleus and in the cytosol before and after heat shock using Western blotting demonstrated that the total amount of G-actin in both nucleus and cytosol was unchanged after heat shock. This indicates that the increased G-actin staining is not a result of import of G-actin into the nucleus. These observations suggest a rearrangement of G-actin in the nucleus during both mitosis and heat shock, which may be due to changes in interaction of G-actin with chromosomes.

Induction of apoptosis and changes in nuclear G-actin are mediated by different pathways: the effect of inhibitors of protein and RNA synthesis in isolated rat hepatocytes.

Stressor-induced changes in the cytoskeleton, of which actin is a major component, may lead to apoptosis. The role of drug-induced changes in nuclear G-actin and apoptosis was studied in freshly isolated hepatocytes. Several protein synthesis inhibitors, cycloheximide, puromycin, and emetine, induced 10 to 15% apoptosis in hepatocytes after 4 h, as was determined by changes in nuclear morphology and flow cytometric analysis of Annexin V-positive cells. Apoptosis induced by protein synthesis inhibition could be prevented by the caspase inhibitors Z-Val-Ala-DL-Asp fluormethylketone (zVAD-fmk) and Ac-Asp-Glu-Val-Asp-aldehyde (DEVD-cho). Several (chemical) stressors cause a rapid increase in nuclear G-actin staining in hepatocytes or cell lines (Meijerman et al., Biochem. Biophys. Res. Commun. 240, 697-700, 1997). The protein synthesis inhibitors also increased G-actin staining in nuclei after 2 h; this could not be inhibited by zVAD-fmk or DEVD-cho. Changes in the cytosolic F-actin pattern did not occur until nuclear G-actin staining had already increased. The mRNA synthesis inhibitor actinomycin D, also increased nuclear G-actin staining, but did not induce apoptosis within the studied time frame. The results suggest that the induction of apoptosis and the increased nuclear staining of G-actin by protein synthesis inhibition are differently controlled.

Interleukin-2-activated natural killer cells can induce both apoptosis and necrosis in rat hepatocytes.

Natural killer (NK) cells play a crucial role in the elimination of virus-infected or transformed cells in the liver. In this article, we describe the mechanism by which liver cells are killed by NK cells. Interleukin-2-activated natural killer (A-NK) cells from the rat induced apoptotic cell death in 30% of freshly isolated rat hepatocytes within 60 minutes. Recognition by the A-NK cells of the hepatocytes as nonself was established by masking the major histocompatibility complex (MHC) class I molecules on the hepatocytes with the OX18 antibody. During the killing process, a decrease of the mitochondrial membrane potential (MMP), formation of blebs, phosphatidyl serine (PS) externalization, chromatin condensation, and nuclear fragmentation were observed. The hepatocytes became apoptotic before permeabilization of the plasma membrane occurred, suggesting that the observed cytolysis was caused by secondary necrosis. The apoptotic process was completely abolished by the caspase inhibitors, Z-Val-Ala-DL-Asp fluormethylketone (zVAD-fmk) and Ac-Asp-Glu-Val-aldehyde (DEVD-cho). However, under these conditions, A-NK cells killed a smaller fraction of the hepatocytes by (primary) necrosis. These results indicate that apoptosis is the major cytotoxic process induced by A-NK cells in hepatocytes. If apoptosis is prevented, a more limited necrotic effect is induced. Therefore, this study shows that NK cells are fully equipped to induce both apoptosis and necrosis in hepatocytes, but appear to prefer the apoptotic route.

Nuclear accumulation of G-actin in isolated rat hepatocytes by adenine nucleotides.

Extracellular ATP induces bleb formation in isolated rat hepatocytes. We examined the effect of extracellular ATP on the actin cytoskeleton of these hepatocytes. Exposure to 100 microM ATP caused pronounced nuclear accumulation of G-actin. ADP, AMP, adenosine, and dibutyryl-cAMP induced the same effect. Adenosine deaminase could inhibit both ATP- and adenosine-induced nuclear accumulation. The P2-receptor agonists, UTP and 2' & 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate, did not induce this redistribution of G-actin. Phalloidin, which prevents depolymerisation of F-actin filaments to G-actin monomers, inhibited adenosine-induced nuclear accumulation of G-actin. These observations suggest that nuclear accumulation of G-actin is mediated by adenosine receptors.