Marty Blom

To improve food labeling strategies, information regarding eliciting doses (ED) and the effect of patient characteristics on these EDs is necessary. Establish EDs for objective and subjective symptoms and analyze the effect of sensitization levels and other patient characteristics on threshold distribution curves (TDC). Threshold data from 100 adults and 262 children with a positive food challenge were analyzed with Interval-Censoring Survival Analysis (ICSA) and fitted to a TDC from which EDs could be extracted. Possible influencing factors were analyzed as covariates by ICSA. A hazard ratio (HR) was calculated in case of a significant effect. TDCs for both objective and subjective symptoms were significantly different between adults and children (P<0.001). Objective ED05 values however were comparable (2.86mg peanut protein in adults and 6.38mg in children, respectively). Higher levels of sIgE to Ara h 2 and peanut extract were associated with a larger proportion of patient gro...

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. The current 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. Allergen risk assessment using probabilistic techniques enables estimation of the residual risk after the consumption of a product that unintentionally contains an allergen. 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 level...

The spatio-temporal relationship between a decrease in the mitochondrial membrane potential (MMP) and externalization of phosphatidylserines (PS) during induction of apoptosis was investigated in single freshly isolated hepatocytes. Apoptosis was induced in the hepatocytes in three different ways: attack by activated Natural Killer cells, exposure to ATP, or exposure to the inhibitor of protein synthesis cycloheximide. Fluorescence microscopy showed staining of externalized PS at those areas where the staining for MMP was lost whereas in other areas the mitochondria remained intact for longer periods of time, indicating coupling between local loss of MMP and local PS exposure. To discriminate whether the decrease in MMP itself or a decrease in ATP induced PS externalization, hepatocytes were treated with rotenone, which resulted in a rapid collapse of cellular ATP but left the MMP intact for a much longer period. Addition of fructose prevented the decrease of ATP to approximately 30...

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.

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.

Natural Killer (NK) cells are important in the first response against viruses and tumours. Compounds that modulate human NK cell activity offer interesting prophylactic and therapeutic options, however, a systematic screening tool is lacking. Development of suitable NK cell lines or receptor-based assays is hindered by the highly complicated regulation of the different NK cell subsets by multiple receptors. Here, we describe a cell-based flowcytometric activity assay adapted to identify NK cell modulating compounds. Fresh human peripheral blood mononuclear cells (PBMC) were incubated with NK-sensitive K562 target cells labelled with 5-(6)-carboxyfluorescein succinimidyl ester, followed by DNA-labelling with propidium iodide to identify dead cells. The assay demonstrated a good performance with an average Z'-factor of 0.6 and over 95% of the assays fulfilled the quality criteria, suggesting that it is possible to use a complex system with two different cell types to screen compounds. A large number of (natural) compounds and extracts were tested and normalized to the positive control, Interleukin-2. Promising and less promising compounds were distinguished. Effectiveness of compounds was based on the augmentation of NK cell activity as well as the number of responding subjects. To conclude the assay is robust, reliable and can be used for functional screening of natural compounds modulating NK cell activity.

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.

Previously, we showed that interleukin-2 activated Natural Killer cells (A-NK cells) in vitro rapidly induced apoptosis in freshly isolated rat hepatocytes (Blom et al., 1999. Hepatology 29 (3): 785-792) which was caused by a rapid decrease in the mitochondrial membrane potential and activation of caspases. In the present study we investigated the involvement of calpains in A-NK cell-induced apoptosis in isolated hepatocytes. When NK cells and hepatocytes were incubated in the presence of a calpain inhibitor the number of apoptotic cells decreased from 46 to 36%. However, more hepatocytes became necrotic (48 vs. 30%) as compared to the uninhibited situation. Inhibition of the calpains alone could not prevent the induction of the nuclear and cytoskeletal disruptions occurring in the hepatocytes. Inhibition of both calpains and caspases increased the number of necrotic cells as compared to incubation with a single inhibitor. However, the damage to the cytoskeleton of the surviving cells was completely inhibited. We conclude that calpains play a role in induction of apoptosis by NK cells. However, their role is limited as compared to caspases.

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.