The impact of saliva collection methods on measured salivary biomarker levels.

Saliva diagnostics have become increasingly popular due to their non-invasive nature and patient-friendly collection process. Various collection methods are available, yet these are not always well standardized for either quantitative or qualitative analysis. In line, the objective of this study was to evaluate if measured levels of various biomarkers in the saliva of healthy individuals were affected by three distinct saliva collection methods: 1) unstimulated saliva, 2) chew stimulated saliva, and 3) oral rinse. Saliva samples from 30 healthy individuals were obtained by the three collection methods. Then, the levels of various salivary biomarkers such as proteins and ions were determined. It was found that levels of various biomarkers obtained from unstimulated saliva were comparable to those in chew stimulated saliva. The levels of potassium, sodium, and amylase activity differed significantly among the three collection methods. Levels of all biomarkers measured using the oral rinse method significantly differed from those obtained from unstimulated and chew-stimulated saliva. In conclusion, both unstimulated and chew-stimulated saliva provided comparable levels for a diverse group of biomarkers. However, the results obtained from the oral rinse method significantly differed from those of unstimulated and chew-stimulated saliva, due to the diluted nature of the saliva extract.

The role of intestinal dendritic cells subsets in the establishment of food allergy.

BACKGROUND: Food allergy affects approximately 6% of children and is the leading cause of hospitalization for anaphylactic reactions in westernized countries. Crucial in the establishment of allergy is the activation of dendritic cells (DC) leading to T helper 2-mediated responses. OBJECTIVE: We, therefore, investigated whether changes in DC subsets precede the establishment of food allergy, and which DC subsets have functional relevance during allergic sensitization in a mouse model. METHODS: Changes in DC populations in the intestine were analysed after exposure to cholera toxin alone and in combination with peanut extract (PE) as an allergen. To study the functional role of DC subsets in relation to food allergy, we used expansion of DC in vivo by treatment with Flt3L. RESULTS: Sensitization to PE in this mouse model was accompanied by a shift in DC subsets in intestinal tissues towards more CD11b(+) DC and less CD103(+) DC. No significant changes in the plasmacytoid DC (pDC) numbers were observed. Flt3L treatment, resulting in the expansion of all DC subtypes, inhibited allergic manifestations in our model, including Th2 cytokine production, PE-specific IgE and PE-induced mast cell degranulation. pDC depletion reversed Flt3L-induced inhibition of IgE responses and mast cell degranulation. conclusions and clinical relevance: The establishment of food allergy is accompanied by profound changes in DC subsets in the intestine towards more inflammatory CD11b(+) DC. In addition, expansion of DC numbers by Flt3L, in particular pDC, inhibits the establishment of allergic manifestations in the intestine. These findings are of relevance for understanding the role of DC subsets early during the process of allergic sensitization, and may lead to new therapeutic or prophylactic opportunities to prevent food allergy.

Diclofenac enhances allergic responses in a mouse peanut allergy model.

BACKGROUND: Diclofenac and other non-steroidal anti-inflammatory drugs (NSAIDs) interfere with cyclo-oxygenase-mediated synthesis of prostaglandins, resulting in the inhibition of inflammatory immune responses. In contrast, it is known that NSAIDs are able to induce gastrointestinal damage. OBJECTIVE: Our aim was to investigate whether NSAIDs are able to enhance sensitization or abrogate tolerance to food antigens. METHODS: Mice were exposed to diclofenac and sensitized to peanut using cholera toxin as a mucosal adjuvant. In a tolerance model, oral tolerance was induced via feeding of peanut 3 weeks before sensitization with peanut. Diclofenac was administered before peanut feeding. After 4 weeks, peanut-specific antibodies in the serum and cytokine production in the spleen were measured. Induction of intestinal damage after oral exposure with diclofenac and peanut + cholera toxin was examined microscopically. RESULTS: Diclofenac-exposed animals showed increased levels of peanut-specific IgG1, IgG2a and IgE in the serum compared with vehicle-treated animals. Furthermore, peanut-induced cytokine production in the spleen was elevated upon diclofenac treatment. Importantly, diclofenac did not induce peanut-allergic responses in the absence of the cholera toxin, although exposure to diclofenac and peanut + cholera toxin resulted in intestinal epithelial damage. Reduced peanut-specific antibody production in the case of oral tolerance was not reversed after diclofenac exposure. However, oral tolerance, as measured by inhibition of peanut-specific cytokine responses, was reverted by diclofenac. CONCLUSIONS: These data point towards an increased risk for induction of food-allergic responses by diclofenac, when other circumstances are also in favour of induction of allergy.

Regulation by intestinal γδ T cells during establishment of food allergic sensitization in mice.

BACKGROUND: Food allergy affects approximately 5% of children and is the leading cause of hospitalization for anaphylactic reactions in westernized countries. The mucosal adjuvant cholera toxin induces allergic sensitization to co-administered proteins in mice, while feeding the protein alone induces oral tolerance. Intestinal γδ T cells could be of importance in the induction of oral tolerance. This study aims to investigate whether γδ T cells have functional relevance in food allergic sensitization. METHODS: Changes in γδ T cells on days 1, 2, 3, and 7 after initiation of food allergy were evaluated using flowcytometry. Furthermore, the anti-γδ T-cell receptor (TCR) antibody UC7 was used to block the γδ TCR in mice in vivo, followed by sensitization to peanut. After 4 weeks, peanut-specific antibodies in serum and cytokine production in spleen were measured. RESULTS: Induction of food allergy resulted in a profound decrease in the percentage of γδ T cells in intestinal tissues and Peyer's Patches, but not in mesenteric lymph nodes or spleen. This decrease could be detected from days 1 to 2 after the initiation of food allergy and the number of γδ T cells returned to normal on day 7. Blockade of the γδ TCR resulted in elevated food allergic responses upon sensitization with peanut characterized by increased IgE and Th2 cytokine production in splenocytes. CONCLUSION: These results demonstrate a unique regulatory role of γδ T cells, suggesting that targeting γδ T cells in the intestine may contribute to strategies to prevent and possibly treat food allergy.

A multi-center assessment to compare residual allergenicity of partial hydrolyzed whey proteins in a murine model for cow's milk allergy - Comparison to the single parameter guinea pig model.

INTRODUCTION: This 4-center study is part of a project to validate a food allergy murine model for safety testing of hydrolyzed infant formulas. AIM: The aim of the current multi-center experiment was to evaluate the residual allergenicity of three partial hydrolyzed whey proteins (pWH) in a multiple-parameter cow's milk allergy murine model and to compare to the classically used guinea pig model. Previous work showed differences in the magnitude of the allergic response to whey between centers. To get a first insight in the effect of housing on the robustness of the mouse model, microbiota composition of non-sensitized mice was analyzed and compared between centers. METHODS: Mice were sensitized intragastrically (i.g.) with whey, pWH or eWH using cholera toxin as an adjuvant. In mice, whey-IgE/IgG1, acute allergic symptoms were determined upon whey challenge. Guinea pigs were orally sensitized ad libitum via the drinking water (day 0-37) and challenged intravenously with whey on day 49. The microbial composition in fecal samples was determined in non-sensitized mice in all 4 research centers before and after conduct of the study. RESULTS: Elevated levels of whey-IgG1 were detected in whey-sensitized mice in all centers. Except for pWH-A in center 4, we observed elevated levels of whey-IgE in whey-sensitized mice and mice sensitized with pWH-A, -B, -C. Center 2 was excluded from further analysis because of non-significant IgE levels in the positive control. In contrast to whey-mice, pWH-A treated mice showed no acute skin response, mMCP-1 release or change in body temperature upon whey challenge in all centers, which corresponds with the absence of anaphylactic shock symptoms in both the mouse and guinea pig model. pWH-B and -C induced anaphylactic shock symptoms in the guinea-pig and mice whereas results on the remaining allergic outcomes in mice were inconclusive. No differences in microbiota composition were measured in response to the challenge and Microbiota composition depended on the location of the centers. CONCLUSIONS: Both animal models showed comparable results on the residual allergenicity of partial hydrolyzed whey proteins, but none of the centers was able to differentiate between the residual sensitizing capacities of the pWH-B and -C based on a single elicitation parameter in the murine model. Differences in microbiota composition might contribute to the robustness of the food allergy murine model. For a well-balanced prediction on the potential allergenicity of hydrolyzed infant formulas a multiple murine parameter model is suggested to decrease the risk of false positive or false negative results. A future challenge is to develop an overall scoring system for proper risk assessment, taking all parameters into account.

The mycotoxin deoxynivalenol facilitates allergic sensitization to whey in mice.

Intestinal epithelial stress or damage may contribute to allergic sensitization against certain food antigens. Hence, the present study investigated whether impairment of intestinal barrier integrity by the mycotoxin deoxynivalenol (DON) contributes to the development of whey-induced food allergy in a murine model. C3H/HeOuJ mice, orally exposed to DON plus whey once a week for 5 consecutive weeks, showed whey-specific IgG1 and IgE in serum and an acute allergic skin response upon intradermal whey challenge, although early initiating mechanisms of sensitization in the intestine appeared to be different compared with the widely used mucosal adjuvant cholera toxin (CT). Notably, DON exposure modulated tight-junction mRNA and protein levels, and caused an early increase in IL-33, whereas CT exposure affected intestinal γδ T cells. On the other hand, both DON- and CT-sensitized mice induced a time-dependent increase in the soluble IL-33 receptor ST2 (IL-1R1) in serum, and enhanced local innate lymphoid cells type 2 cell numbers. Together, these results demonstrate that DON facilitates allergic sensitization to food proteins and that development of sensitization can be induced by different molecular mechanisms and local immune responses. Our data illustrate the possible contribution of food contaminants in allergic sensitization in humans.

Is Candida albicans a trigger in the onset of coeliac disease?

Coeliac disease is a T-cell-mediated autoimmune disease of the small intestine that is induced by ingestion of gluten proteins from wheat, barley, or rye. We postulate that Candida albicans is a trigger in the onset of coeliac disease. The virulence factor of C albicans-hyphal wall protein 1 (HWP1)-contains aminoacid sequences that are identical or highly homologous to known coeliac disease-related alpha-gliadin and gamma-gliadin T-cell epitopes. HWP1 is a transglutaminase substrate, and is used by C albicans to adhere to the intestinal epithelium. Furthermore, tissue transglutaminase and endomysium components could become covalently linked to the yeast. Subsequently, C albicans might function as an adjuvant that stimulates antibody formation against HWP1 and gluten, and formation of autoreactive antibodies against tissue transglutaminase and endomysium.

The vehicle modulates cellular and humoral responses in contact hypersensitivity to oxazolone.

The development of contact hypersensitivity (CHS) greatly depends on the allergenicity of the inducing agent. However, various cofactors are known to influence the outcome of the response as well. From this perspective, we have compared the effects of five different vehicles: acetone, ethanol, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), and a 4 to 1 mixture of acetone and olive oil (AOO) on the cellular and humoral immune responses to epicutaneously applied oxazolone in female BALB/c mice. A single application of 0.2% oxazolone dissolved in acetone or ethanol induced stronger proliferative responses and higher lymph node cell numbers than the other three vehicles. Moreover, both vehicles led to higher numbers of oxazolone-specific Ab forming cells in the draining lymph nodes of sensitized animals. When the IgG2a/IgG1 ratios were determined to indicate the type of T helper cell involved, the highest values were obtained with AOO and lowest with DMF and DMSO, while acetone and ethanol were in between. Moreover, no correlation was found between oxazolone-specific antibody production and cellular responses, measured as [3H]thymidine incorporation of draining lymph node cells after sensitization and increased ear thickness after challenge. From this study it can be concluded that cellular and humoral responses in CHS to oxazolone are dissimilarly affected by the vehicles used.

Immunotoxic organotins as possible model compounds in studying apoptosis and thymocyte differentiation.

In the mid-seventies it appeared that some organotin compounds selectively caused thymus atrophy. From that time onward efforts were made to reveal molecular and cellular mechanisms involved. In this review recent studies into organotin-sensitive stages and processes of thymocyte maturation are discussed. Together these studies resulted in the recognition of organotin compounds as possible model compounds in studying immature thymocyte differentiation and protein synthesis-independent apoptotic cell death of thymocytes.

The SCID-hu mouse as a tool in immunotoxicological risk assessment: effects of 2-acetyl-4(5)-tetrahydroxybutyl-imidazole (THI) and di-n-butyltin dichloride (DBTC) on the human thymus in SCID-hu mice.

SCID mice engrafted with human fetal thymus and liver tissue fragments (SCID-hu mice) are currently considered as a new tool in human immunotoxicological risk assessment. Testing of various immunotoxicants exerting thymotoxicity via different intrathymic target cell types is necessary for validation of this model. Therefore, SCID-hu mice were exposed to 2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)-imidazole (THI), the immunotoxic component in the food additive, Caramel Colour III, or the organotin compound, di-n-butyltin dichloride (DBTC). Histopathological examination of the human thymus grafts of SCID-hu mice either exposed to THI or to DBTC showed a reduction in the relative size of the thymus cortex, an effect also described in rodents. These results indicate that the human thymus is a target for the immunotoxic action of both THI and DBTC. In addition, they indicate the promising potential of the SCID-hu mouse model as a tool for human immunotoxicological risk assessment.

AHTN and HHCB show weak estrogenic--but no uterotrophic activity.

The ubiquitous presence of the polycyclic musks AHTN (6-acetyl-1,1,2,4,4,7-hexamethyltetraline) and HHCB (1,2,4,6,7,8-hexahydro-4,6,6,7,8-hexamethylcyclopenta-gamma-2-b enzopyreen) in surface waters and their identification in human milk fat together with their polycyclic nature, which makes them potential candidates for interference with estrogen receptors, prompted us to assess these compounds for their potential estrogenic effects. We therefore investigated the effects of AHTN and HHCB in ERalpha- and ERbeta-dependent gene transcription assays with Human Embryonal Kidney 293 (HEK293) cells, which have proven to be very suitable to estimate the estrogenic activity of compounds with low binding activity (Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., Van der Saag, P.T., Van der Burg, B., Gustafsson, J.A., 1998. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139, 4252-4264). Both AHTN and HHCB were found to induce a slight but dose-dependent stimulation of transcriptional activity in the transiently ERalpha transfected HEK293 cells. This weak estrogenic response was not observed in the ERbeta transiently transfected cells. However, these cells were less responsive to estradiol than the ERalpha transfected HEK293 cells. Also, no significant increase in transcriptional activity was observed in HEK293 cell lines, permanently expressing the same estrogen-responsive reporter gene construct and either ERalpha or ERbeta. In the classical uterine weight assay performed in juvenile Balb/c mice, no uterotrophic activity of AHTN and HHCB was noted at relatively high dietary exposure levels up to 50 and 300 ppm, respectively, at which levels an increase in liver weight was evident. Also the vitellogenin production by carp hepatocytes, a sensitive marker of estrogenic activity, was not affected by these two fragrance materials (Smeets, J.M.W., Rouhani Rankouhi, T., Nichols, K.M., Komen, H., Kaminsky, N.E., Giesy, J.P., Van den Berg, M., 1999. In vitro vitellogenin production by carp (Cyprimus carpio) hepatocytes as a screening method for determining (anti-) estrogenic activity of xenobiotics. Toxicol. Appl. Pharmacol., 157, 68-76). Therefore it is concluded that these compounds have very weak estrogenic potency, too weak to induce estrogenic effects in wildlife species or humans at the current levels of exposure. These results give further support to the promiscuity of estrogen receptors.

Cellular and molecular aspects of organotin-induced thymus atrophy.

1. Organotin compounds, di-n-butyltin dichloride (DBTC) in particular, have been shown to cause thymus atrophy in the rat. 2. DBTC-induced thymus atrophy results from a depletion of small CD4+CD8+ thymocytes which is caused by a diminished production of immature CD4-CD8+ and CD4+CD8+ thymoblasts. 3. DBTC inhibits the activation, but not the differentiation of immature CD4-CD8+ thymocytes in vitro and in vivo suggesting a selective antiproliferative activity of DBTC. 4. DBTC inhibits the adhesion molecule-mediated binding of thymocytes to thymic epithelial cells. 5. DBTC enhances the Ca2+ release elicited by cross-linking of the T cell receptor complex (TcR alpha beta-CD3) on thymocytes and moreover delays cap formation of the TcR alpha beta-CD3 receptor. 6. It is concluded that DBTC possibly interferes with the functioning of the cytoskeleton. The relation of the in vitro findings to the inhibition of immature CD4-CD8+ thymocyte activation and the induction of thymus atrophy is unknown as yet.

The aryl hydrocarbon receptor and food allergy.

The immune system is important for protection against pathogens and malignant cells. However, malfunction of the immune system can also result in detrimental auto-immune diseases, inflammatory diseases, cancers and allergies. The aryl hydrocarbon receptor (AhR), present in numerous tissues and cell subsets, including cells of the immune system, plays an important role in the functioning of the immune system. Activation of the AhR is for example associated with various effects on dendritic cells (DCs), regulatory T cells and the Th1/Th2 cell balance. These cells play a major role in the development of food allergy. Food allergy is an increasing health problem in both humans and animals. Despite the knowledge in risk factors and cellular mechanisms for food allergy, no approved treatments are available yet. Recently, it has been shown that activation of the AhR by dioxin-like compounds suppresses allergic sensitization by suppressing the absolute number of precursor and effector T cells, by preserving CD4(+)CD25(+)Foxp3(+) Treg cells and by affecting DCs and their interaction with effector T cells. Future research should elucidate whether and how AhR activation can be used to interfere in food allergic responses in humans and in animals. This may lead to new prevention strategies and therapeutic possibilities for food allergy.

Identification and quantification of drug-albumin adducts in serum samples from a drug exposure study in mice.

The formation of drug-protein adducts following the bioactivation of drugs to reactive metabolites has been linked to adverse drug reactions (ADRs) and is a major complication in drug discovery and development. Identification and quantification of drug-protein adducts in vivo may lead to a better understanding of drug toxicity, but is challenging due to their low abundance in the complex biological samples. Human serum albumin (HSA) is a well-known target of reactive drug metabolites due to the free cysteine on position 34 and is often the first target to be investigated in covalent drug binding studies. Presented here is an optimized strategy for targeted analysis of low-level drug-albumin adducts in serum. This strategy is based on selective extraction of albumin from serum through affinity chromatography, efficient sample treatment and clean-up using gel filtration chromatography followed by tryptic digestion and LC-MS analysis. Quantification of the level of albumin modification was performed through a comparison of non-modified and drug-modified protein based on the relative peak area of the tryptic peptide containing the free cysteine residue. The analysis strategy was applied to serum samples resulting from a drug exposure experiment in mice, which was designed to study the effects of different acetaminophen (APAP) treatments on drug toxicity. APAP is bioactivated to N-acetyl-p-benzoquinoneimine (NAPQI) in both humans and mice and is known to bind to cysteine 34 (cys34) of HSA. Analysis of the mouse serum samples revealed the presence of extremely low-level NAPQI-albumin adducts of approximately 0.2% of the total mouse serum albumin (MSA), regardless of the length of drug exposure. Due to the targeted nature of the strategy, the NAPQI-adduct formation on cys34 could be confirmed while adducts to the second free cysteine on position 579 of MSA were not detected.

Aryl hydrocarbon receptor activation affects the dendritic cell phenotype and function during allergic sensitization.

Aryl hydrocarbon receptor (AhR) activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses peanut sensitization by affecting T cell subsets. However, effects of AhR activation on dendritic cells (DC) in an allergic setting were not investigated yet. Therefore, we analysed the effects of AhR activation on DC phenotype in vivo, as well as their ex vivo potency to stimulate allergen-specific splenic T cells and to induce CD4+CD25+Foxp3+ regulatory (T(reg)) cells. C3H/HeOuJ mice were treated with TCDD by gavage and subsequently sensitized to peanut extract (PE). After eight days, mice were sacrificed and DC in spleen and mesenteric lymph nodes (MLN) were characterized or cocultured with PE-specific CD4+ T cells. AhR activation almost doubled the absolute number of CD11c+CD103+ DC, while not affecting CD11b+ DC, the absolute number of DC, the expression of the activation makers MHCII, CD86, CD80, CD40, CD54 and CD8 on CD11c+ and the activation status of CD11c+CD103+ DC in the spleen. In the MLN, TCDD decreased the absolute number of DC and CD103+ DC, while not affecting CD11b+ DC and the expression of activation markers on DC. PE-pulsed splenic DC from TCDD-treated mice suppressed IL-5, IL-13 and IFN-γ production by PE-specific T cells, but did not induce CD4+CD25+Foxp3+ T(reg) cells. This suppression of cytokine production was not mediated by the TCDD-induced increase in CD103+ DC in the spleen. Combined, these results indicate that AhR activation suppresses the initiation of food allergic responses by affecting DC and their interaction with effector T cells.

Interlaboratory evaluation of a cow's milk allergy mouse model to assess the allergenicity of hydrolysed cow's milk based infant formulas.

This study describes two phases of a multi-phase project aiming to validate a mouse model for cow's milk allergy to assess the potential allergenicity of hydrolysed cow's milk based infant formulas (claim support EC-directive 2006/141/E). The transferability and the discriminatory power of this model was evaluated in 4 research centers. Mice were sensitized by oral gavage with whey or extensively hydrolysed whey (eWH) using cholera toxin as an adjuvant. Whey-specific antibodies, mMCP-1 levels, anaphylactic shock symptoms, body temperature and the acute allergic skin response were determined upon whey challenge. In phases I and II, all 4 centers detected elevated levels of whey-specific IgE/IgG1 in whey sensitized animals. Elevated levels of mMCP-1, anaphylactic symptoms, body temperature drop and acute allergic skin response were scored upon whey challenge in 3 out of 4 research centers. In contrast, none of the evaluated parameters were elevated in eWH orally exposed groups. The cow's milk allergy mouse model is capable to distinguish the sensitizing capacity of complete or hydrolysed cow's milk protein. The model uses straightforward parameters relevant to food allergic responses and can be effectively transferred between different laboratories. We propose this mouse model as a new strategy for the screening of new hypoallergenic cow's milk formulas.

Activation of the aryl hydrocarbon receptor reduces the number of precursor and effector T cells, but preserves thymic CD4+CD25+Foxp3+ regulatory T cells.

Aryl hydrocarbon receptor (AhR) activation suppresses immune responses, including allergic sensitization, by increasing the percentage of regulatory (Treg) cells. Furthermore, AhR activation is known to affect thymic precursor T cells. However, the effect of AhR activation on intrathymic CD4+CD25+Foxp3+ Treg cells is unknown. Therefore, we investigated the effect of AhR activation on the percentage and number of CD4+CD25+Foxp3+ Treg cells during allergic sensitization in relevant immunological organs. C3H/HeOuJ mice were treated on day 0 with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and subsequently sensitized to peanut. On day 8, mice were sacrificed and thymus, spleen and mesenteric lymph nodes (MLN) were isolated. TCDD treatment decreased the number of CD4-CD8-, CD4+CD8+, CD4+CD8- and CD4-CD8+ precursor T cells, but not the number of thymic CD4+CD25+Foxp3+ Treg cells. TCDD treatment increased the number of splenic CD4+CD25+Foxp3+ Treg cells and decreased Th1, Th2 and cytotoxic T cells in the spleen. This appeared to be independent of allergic sensitization. In MLN, TCDD treatment suppressed the increase of the number of CD4+CD25+Foxp3+ Treg cells, Th1, Th2 and cytotoxic T cells induced by peanut sensitization. Together, TCDD treatment preserves thymic CD4+CD25+Foxp3+ Treg cells and decreases peripheral T helper and cytotoxic T cells. This effect of TCDD may contribute to the increased influence of CD4+CD25+Foxp3+ Treg cells on immune mediated responses and to the understanding of how AhR activation modulates immune mediated diseases, including food allergy.

Activation of the aryl hydrocarbon receptor suppresses sensitization in a mouse peanut allergy model.

Food allergy is an increasing health problem in Western countries. Previously, it has been shown that the intensity of food allergic reactions can be regulated by regulatory T (T(reg)) cells. In addition, it has been shown that activation of the aryl hydrocarbon receptor (AhR) regulates T-cell responses by induction of T(reg) cells. Therefore, we hypothesized that activation of the AhR pathway can suppress development of food allergic responses through the induction of T(reg) cells. This was investigated by using a mouse model for peanut allergy. C3H/HeOuJ mice (AhR(b)(-2)) were sensitized to peanut by administering peanut extract (PE) by gavage in the presence of cholera toxin and were treated with the prototypical AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0.6, 1.7, 5, and 15 µg/kg body weight) on days 3 and 11 orally. The functional role of CD4(+)CD25(+)Foxp3(+) T(reg) cells was investigated by depleting these cells with anti-CD25 mAb during sensitization to PE. TCDD treatment dose dependently suppressed sensitization to peanut (PE-specific IgE, IgG1, and IgG2a and PE-induced IL-5, IL-10, and IL-13, respectively). The percentage, but not the number, of CD4(+)CD25(+)Foxp3(+) T(reg) cells dose dependently increased by AhR activation in both spleen and mesenteric lymph nodes. Depletion of CD4(+)CD25(+)Foxp3(+) T(reg) cells markedly reversed the suppressive effect of TCDD on PE-specific antibody levels and PE-induced IL-5, IL-10, and IL-13 cytokine production. Present data demonstrate for the first time that activation of the AhR by TCDD suppressed the development of Th2-mediated food allergic responses. A functional shift within the CD4(+) cell population toward CD4(+)CD25(+)Foxp3(+) T(reg) cells appeared to underlie this effect. This suggests that the AhR pathway might provide potential therapeutic targets to treat food allergic diseases.

Hexachlorobenzene treatment increases the number of splenic B-1-like cells and serum autoantibody levels in the rat.

In the present study, the role of B-1 cells in hexachlorobenzene (HCB)-induced autoimmune aberrations in the Wistar rat was investigated. To that end, male and female rats were exposed to a semi-synthetic diet containing 0 or 1000 mg HCB/kg food for 3 weeks. After dissection, serum was prepared form coagulated blood to determine (auto)antibody levels, and spleens and lymph nodes were isolated and weighed. Cell suspensions were prepared, counted and analysed for B- and T-cell subsets by flow cytometry. Quantification of antibody-secreting cells (ASC) in spleen cell suspensions was done with an ELISPOT assay. Previous findings that HCB treatment induced an increase of relative lymph node and spleen weights and serum (auto)antibody levels were confirmed, while it appeared that numbers of some lymph nodal, and of the splenic large cell populations, were elevated as well. HCB treatment did not change subsets of lymph nodal T and B cells, but elevated the absolute numbers of large splenic CD4+ T cells by about 70%, IgMdull/IgDbright B cells by about 60%, and IgMbright/IgDdull B cells by about 200% cells of control numbers, and the absolute numbers of splenic IgM and IgG (auto) ASC by 300-400% of the control numbers. As splenic IgMbright/IgDdull numbers and ASC numbers correlated with statistical significance, the results indicate that HCB treatment selectively activates rat splenic B-1 cells, which may underlie the elevation of serum autoantibody levels.

The organotin-induced thymus atrophy, characterized by depletion of CD4+ CD8+ thymocytes, is preceded by a reduction of the immature CD4- CD8+ TcR alpha beta-/low CD2high thymoblast subset.

Thymic changes in the rat induced by the thymus atrophy-inducing organotin compound di-n-butyltin dichloride (DBTC) were examined using FACS analyses. The number of CD4+CD8+ thymocytes was reduced by DBTC treatment from Day 2 onwards and reached minimum level on Days 4 and 5 after dosing. On these days the CD4-CD8- and both the CD4-CD8+ and CD4+CD8- subsets were not affected. On Day 2 we observed a reduced proportion of transferrin receptor (CD71)-positive CD4-OX44- cells, representing the cycling immature CD4-CD8+ cells, and of CD71+OX44- cells, representing the cycling CD4+CD8+ cells, but not of CD71+CD4-CD8- cells. When compared to controls, the FSChigh cell population of DBTC-treated rats contained less CD4-OX44- and OX44- cells, which were further characterized as CD2high and T-cell receptor (TcR)alpha beta- low. Moreover, fewer TcR alpha beta high cells were detected in the OX44- thymoblast subset of DBTC-treated rats. The number of CD4-CD8- thymoblasts appeared marginally decreased while the numbers of CD4+OX44+ cells, representing mature CD4+ cells, were not affected. These data indicate that DBTC causes a preferential initial depletion of immature CD4-CD8+CD2high TcR alpha beta-low thymoblasts. This initial event may result in a decreased formation of CD4+CD8+ thymoblasts and of small CD4+CD8+ thymocytes. These characteristics of the initially depleted subset indicate a specific anti-proliferative effect of DBTC and may give clues for the mechanism involved in the induction of thymus atrophy.