Immune responses induced by diclofenac or carbamazepine in an oral exposure model using TNP-Ficoll as reporter antigen.

Immune-mediated drug hypersensitivity reactions (IDHR) may result from immuno-sensitization to a drug-induced neo-antigen. They rarely occur in patients and are usually not predicted preclinically using standard toxicity studies. To assess the potential of a drug to induce T-cell sensitization, trinitrophenyl (TNP)-Ficoll was used here as a bystander antigen in animal experiments. TNP-Ficoll will only elicit TNP-specific IgG antibodies in the presence of non-cognate T-cell help. Therefore, the presence of TNP-specific IgG antibodies after co-injection of drug and TNP-Ficoll was indicative of T-cell sensitization potential. This TNP-Ficoll-approach was used here to characterize T-cell help induced by oral exposure to diclofenac (DF) or carbamazepine (CMZ). DF or CMZ was administered orally to BALB/c mice and after 3 w, the mice were challenged in a hind paw with TNP-Ficoll and a dose of the drug that by itself does only elicit a sub-optimal popliteal lymph node assay (PLNA) response. T-cell-dependent responses were then evaluated in paw-draining popliteal lymph nodes (PLN). Also, shortly after oral exposure, mesenteric lymph nodes (MLN) were excised for evaluation of local responses. Both drugs were able to increase PLN cellularity and TNP-specific IgG1 production after challenge. Both DF and CMZ stimulated CD4+ and CD8+ T-cells and caused shifts of the subsets toward an effector phenotype. DF, but not CMZ, appeared to stimulate interferon (IFN)-γ production. Remarkably, depletion of CD8+, but not CD4+, T-cells reduced TNP-specific IgG1 production, and was more pronounced in CMZ- than in DF-exposed animals. Local responses in the MLN caused by DF or CMZ also showed shifts of CD4+ and CD8+-cells toward a memory phenotype. Together, the data indicate that oral exposure to CMZ and DF differentially induced neo-antigen-specific T-cell reactions in the PLNA.

Oral exposure to immunostimulating drugs results in early changes in innate immune parameters in the spleen.

The development of immune-dependent drug hypersensitivity reactions (IDHR) is likely to involve activation of the innate immune system to stimulate neo-antigen specific T-cells. Previously it has been shown that, upon oral exposure to several drugs with immune-adjuvant capacity, mice developed T-cell-dependent responses to TNP-OVA. These results were indicative of the adjuvant potential of these drugs. The present study set out to evaluate the nature of this adjuvant potential by focusing on early immune changes in the spleen, by testing several drugs in the same experimental model. Mice were exposed to one or multiple oral doses of previously-tested drugs: the non-steroidal-anti-inflammatory drug (NSAID) diclofenac (DF), the analgesic acetaminophen (APAP), the anti-epileptic drug carbamazepine (CMZ) or the antibiotic ofloxacin (OFLX). Within 24 h after the final dosing, early innate and also adaptive immune parameters in the spleen were examined. In addition, liver tissue was also evaluated for damage. Exposure to APAP resulted in severe liver damage, increased levels of serum alanine aminotransferase (ALT) and local MIP-2 expression. DF exposure did not cause visible liver damage, but did increase liver weight. DF also elicited clear effects on splenic innate and adaptive immune cells, i.e. increased levels of NK cells and memory T-cells. Furthermore, an increase in plasma MIP-2 levels combined with an influx of neutrophils into the spleen was observed. OFLX and CMZ exposure resulted in increased liver weights, MIP-2 expression and up-regulation of co-stimulatory molecules on antigen-presenting cells (APC). The data suggested that multiple immune parameters were altered upon exposure to drugs known to elicit immunosensitization and that broad evaluation of immune changes in straightforward short-term animal models is needed to determine whether a drug may harbor the hazard to induce IDHR. The oral exposure approach as used here may be applied in the future as an immunotoxicological research tool in this type of evaluation.

Comparison of six commercial ELISA kits for their specificity and sensitivity in detecting different major peanut allergens.

Six commercial peanut enzyme-linked immunosorbent assay kits were assessed for their ability to recover peanut from the standard reference material 2387 peanut butter and also for their specificity in detecting four major peanut allergens, Ara h 1, Ara h 2, Ara h 3, and Ara h 6. The percentage recovery of peanut from peanut butter differed across different kits as well as at different sample concentrations. The highest recovery was observed with the Romer and R-Biopharm kits, while four other kits were found to underestimate the protein content of the reference peanut butter samples. Five of the kits were most sensitive in detecting Ara h 3 followed by Ara h 1, while hardly recognizing Ara h 2 and Ara h 6. The other kit showed the highest sensitivity to Ara h 2 and Ara h 6, while Ara h 1 and Ara h 3 were poorly recognized. Although Ara h 2 and Ara h 6 are known to be heat stable and more potent allergens, antisera specific to any of these four peanut proteins/allergens may serve as good markers for the detection of peanut residues.

Non-dioxin-like AhR ligands in a mouse peanut allergy model.

Recently, we have shown that AhR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses sensitization to peanut at least in part by inducing a functional shift toward CD4(+)CD25(+)Foxp3(+) T cells. Next to TCDD, numerous other AhR ligands have been described. In this study, we investigated the effect of three structurally different non-dioxin-like AhR ligands, e.g., 6-formylindolo[3,2-b]carbazole (FICZ), ß-naphthoflavone (ß-NF), and 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF), on peanut sensitization. Female C57BL/6 mice were sensitized by administering peanut extract (PE) by gavage in the presence of cholera toxin. Before and during peanut sensitization, mice were treated with FICZ, ß-NF, or 6-MCDF. AhR gene transcription in duodenum and liver was investigated on day 5, even as the effect of these AhR ligands on CD4(+)CD25(+)Foxp3(+) T(reg) cells in spleen and mesenteric lymph nodes (MLNs). Mice treated with TCDD were included as a positive control. Furthermore, the murine reporter cell line H1G1.1c3 (CAFLUX) was used to investigate the possible role of metabolism of TCDD, FICZ, ß-NF, and 6-MCDF on AhR activation in vitro. TCDD, but not FICZ, ß-NF, and 6-MCDF, suppressed sensitization to peanut (measured by PE-specific IgE, IgG1, IgG2a and PE-induced interleukin (IL)-5, IL-10, IL-13, IL-17a, IL-22, and interferon-γ). In addition, FICZ, ß-NF, and 6-MCDF treatments less effectively induced AhR gene transcription (measured by gene expression of AhR, AhRR, CYP1A1, CYP1A2, CYP1B1) compared with TCDD-treated mice. Furthermore, FICZ, ß-NF and 6-MCDF did not increase the percentage of CD4(+)CD25(+)Foxp3(+) T(reg) cells in spleen and mesenteric lymph nodes compared with PE-sensitized mice, in contrast to TCDD. Inhibition of metabolism in vitro increased AhR activation. Together, these data shows that TCDD, but not FICZ, ß-NF, and 6-MCDF suppresses sensitization to peanut. Differences in metabolism, AhR binding and subsequent gene transcription might explain these findings and warrant further studies to investigate the role of the AhR in food allergic responses.

Contribution of classic and alternative effector pathways in peanut-induced anaphylactic responses.

Food allergy affects approximately 5% of children and is the leading cause of hospitalization for anaphylactic reactions in westernized countries. However, the pathways of anaphylaxis in food allergy are still relatively unknown. We investigated the effector pathways of allergic and anaphylactic responses of different strains of mice in a clinical relevant model of peanut allergy. C3H/HeOuJ, C57BL/6 and BALB/c mice were sensitized by intragastric peanut extract and challenged by intragastric or intraperitoneal injection of peanut. Peanut-specific T cell responses, IgE, IgG1 and IgG2a and mucosal mast cell degranulation were induced to different extent in C3H/HeOuJ, C57BL/6 and BALB/c mice. Interestingly, anaphylactic symptoms after systemic challenge were highest in C3H/HeOuJ followed by C57BL/6 but were absent in BALB/c mice. Mechanistic studies showed that the food allergic systemic anaphylaxis was dependent on platelets, FcRγ and mast cells, and partially dependent on platelet activating factor and monocytes/macrophages, depending on mouse strain. These data demonstrate that in three mouse strains, components of the classic and alternative anaphylactic cascade are differently expressed, leading to differential outcomes in parameters of allergic disease and food induced systemic anaphylaxis.

Oral exposure to drugs with immune-adjuvant potential induces hypersensitivity responses to the reporter antigen TNP-OVA.

Immune-mediated drug hypersensitivity reactions are important causes of black box warnings and drug withdrawals. Despite the high demand for preclinical screening tools, no validated in vitro or in vivo models are available. In the current study, we used a previously described oral administration model using trinitrophenyl-ovalbumin (TNP-OVA) as an antigen to report immuno-adjuvating effects of the analgesic drug acetaminophen (APAP) and its nonhepatotoxic regioisomer 3'-hydroxyacetanilide (AMAP), the antibiotic ofloxacin (OFLX), the antiepileptic drug carbamazepine (CMZ), and the antidiabetic drug metformin (MET). Furthermore, APAP and AMAP were tested in a popliteal lymph node assay (PLNA) combined with TNP-OVA as reporter antigen (RA). C3H/HeOuJ mice were dosed by oral gavage with diclofenac (DF), APAP, AMAP, OFLX, MET, or CMZ. On the first exposure day, the mice received an ip injection with TNP-OVA. Fifteen days later, they were ear challenged with TNP-OVA and delayed-type hypersensitivity (DTH) responses were assessed 24 h later. One week after challenge, the ear-draining lymph node was removed and TNP-specific antibody-secreting cells were determined. DF, APAP, CMZ, and OFLX showed a significant increase in DTH responses to ear injection with TNP-OVA, whereas AMAP and MET did not. C57BL/6 mice were slightly less responsive to APAP and DF after oral gavage, and importantly both AMAP and APAP were negative in the RA-PLNA. The present work shows that the oral exposure model using RA and the RA-PLNA may serve to screen the immune-adjuvant potential of new chemical entities during preclinical drug development.

Bovine Intestinal Alkaline Phosphatase Reduces Inflammation After Induction of Acute Myocardial Infarction in Mice.

BACKGROUND: There has been increasing evidence suggesting that lipopolysaccharide or endotoxin may be an important activator of the innate immune system after acute myocardial infarction. Bovine intestinal alkaline phosphatase reduces inflammation in several endotoxin mediated diseases by dephosphorylation of the lipid A moiety of lipopolysaccharide. The aim of this study was to investigate the effect of bovine intestinal alkaline phosphatase on reducing inflammation after acute myocardial infarction. METHODS: Just before permanent ligation of the left anterior descending coronary (LAD) artery to induce acute myocardial infarction in Balb/c mice, bovine intestinal alkaline phosphatase (bIAP) was administrated intravenously. After 4 hours, mice were sacrificed and the inflammatory response was assessed. Acute myocardial infarction induced the production of different cytokines, which were measured in blood. RESULTS: Treatment with bovine intestinal alkaline phosphatase resulted in a significant reduction of the pro-inflammatory cytokines IL-6, IL-1ß and the chymase mouse mast cell protease-1. No difference in the production of the anti-inflammatory cytokine IL-10 was observed between the control group and the bovine intestinal alkaline phosphatase treated group. CONCLUSION: In a mouse model of permanent LAD coronary artery ligation, bIAP diminishes the pro-inflammatory responses but does not have an effect on the anti-inflammatory response in the acute phase after acute myocardial infarction.

Intestinal alkaline phosphatase contributes to the reduction of severe intestinal epithelial damage.

Inflammatory bowel disease is characterized by chronic inflammation of the intestine and is accompanied by damage of the epithelial lining and by undesired immune responses towards enteric bacteria. It has been demonstrated that intestinal alkaline phosphatase (iAP) protects against the induction of inflammation, possibly due to dephosphorylation of lipopolysaccharide (LPS). The present study investigated the therapeutic potential of iAP in intestinal inflammation and epithelial damage. Intestinal epithelial damage was induced in C57BL/6 mice using detran sulfate sodium (DSS) and iAP was administered 4days after initial DSS exposure. Loss in body weight was significantly less in iAP-treated mice and accompanied with reduced colon damage (determined by combination of crypt loss, loss of goblet cells, oedema and infiltrations of neutrophils). Treatment with iAP was more effective in case of severe inflammation compared to situations of mild to moderate inflammation. Rectal administration of LPS into a moderate inflamed colon did not aggravate inflammation. Furthermore, soluble iAP did not lower LPS-induced nuclear factor-kappaB activation in epithelial cells in vitro but induction of cellular AP expression by butyrate resulted in decreased LPS response. In conclusion, the present study shows that oral iAP administration has beneficial effects in situations of severe intestinal epithelial damage, whereas in moderate inflammation endogenous iAP may be sufficient to counteract disease-aggravating effects of LPS. An approach including iAP treatment holds a therapeutic promise in case of severe inflammatory bowel disease.

An in vivo tiered approach to test immunosensitization by low molecular weight compounds.

New chemical entities are tested in general toxicity assays during development before entering clinical trials. However, immunosensitization of these entities is not tested on a standard basis. There are no in vitro or in vivo standardized methods available for testing immunosensitization or immunostimulation. In this chapter, we describe a tiered strategy oral exposure model for assessing immunosensitization or immunostimulation capacity of low molecular weight compounds. The strategy starts from a set of data that may provide information on bioactivation, conjugation (hapten-protein conjugate formation), cytotoxicity and signs of inflammation in any of the animals in a 28 day-toxicity study. In case of concern, a reporter antigen-popliteal lymph node assay (RA-PLNA) and, subsequently, an oral exposure experiment with the reporter antigen can be performed. Based on the presence of RA-specific immune responses an indication for immunosensitization can be found.

Calf intestinal alkaline phosphatase, a novel therapeutic drug for lipopolysaccharide (LPS)-mediated diseases, attenuates LPS toxicity in mice and piglets.

It has been demonstrated that human placental alkaline phosphatase (HPLAP) attenuates the lipopolysaccharide (LPS)-mediated inflammatory response, likely through dephosphorylation of the lipid A moiety of LPS. In this study, it is demonstrated that also alkaline phosphatase derived from calf intestine (CIAP) is able to detoxify LPS. In mice administered CIAP, 80% of the animals survived a lethal Escherichia coli infection. In piglets, previous to LPS detoxification, the pharmacokinetic behavior of CIAP was studied. CIAP clearance was shown to be dose-independent and showed a biphasic pattern with an initial t1/2 of 3 to 5 min and a second phase t1/2 of 2 to 3 h. Although CIAP is cleared much faster than HPLAP, it attenuates LPS-mediated effects on hematology and tumor necrosis factor-alpha responses at doses up to 10 microg/kg in piglets. LPS-induced hematological changes were antagonized, and the tumor necrosis factor-alpha response was reduced up to 98%. Daily i.v. bolus administration of 4000 units CIAP, the highest dose used in the LPS intervention studies, in piglets for 28 days was tolerated without any sign of toxicity. Therefore, CIAP potentially encompasses a novel therapeutic agent in the treatment of LPS-mediated diseases. Based on the data mentioned above, human clinical trials have been initiated.