Butyl benzyl phthalate: effects on immune responses to ovalbumin in mice

During recent decades the prevalence of IgE-mediated (atopic) allergic diseases in Western Europe and the USA has been increasing dramatically. It has been suggested that one possible cause is the presence in the environment of chemicals that may act as adjuvants, enhancing immune and allergic responses. Certain commonly used phthalate plasticizers such as butyl benzyl phthalate (BBP) have been implicated in this way. In the current experiments, the impact of BBP, applied by a physiologically relevant exposure route, on the vigour of immune responses induced in BALB/c strain mice has been examined. Mice were immunized via subcutaneous injection with the reference allergen ovalbumin (OVA) and received concurrent topical treatment with doses of BBP that induced significant changes in liver weight. The generation of specific anti-OVA IgE and IgG1 antibodies was measured by passive cutaneous anaphylaxis and by enzyme-linked immunosorbant assays, respectively. Topical administration of BBP was without impact on anti-OVA IgE antibody responses, regardless of whether BBP was applied locally or distant to the site of OVA immunization. However, same-site treatment with high-dose BBP (100 mg) did result in a modest elevation in anti-OVA IgG1 antibody production, a subclass of antibody used as a surrogate marker of IgE responses. Taken together with human exposure data, these results suggest that the doses of phthalate encountered in the home environment are unlikely to be a major factor contributing to the increased incidence of asthma and allergy in the developed world.     

Effects of maternal exposure to di(2-ethylhexyl)phthalate (DEHP) during pregnancy on susceptibility to neonatal asthma

Di(2-ethylhexyl) phthalate (DEHP) is used as a plasticizer and is widely dispersed in the environment. In this study, we investigated the effects of maternal exposure to DEHP during pregnancy on neonatal asthma susceptibility using a murine model of asthma induced by ovalbumin (OVA). Pregnant BALB/c mice received DEHP from gestation day 13 to lactation day 21. Their offspring were sensitized on postnatal days (PNDs) 9 and 15 by intraperitoneal injection of 0.5 μg OVA with 200 μg aluminum hydroxide. On PNDs 22, 23 and 24, live pups received an airway challenge of OVA for 30 min. Offspring from pregnant mice that received DEHP showed reductions in inflammatory cell count, interleukin (IL)-4, IL-13, and eotaxin in their bronchoalveolar lavage fluid and in total immunoglobulin E and OVA-specific IgE in their plasma compared with offspring from pregnant mice that did not receive DEHP treatment. These results were consistent with histological analysis and immunoblotting. Maternal exposure to DEHP reduces airway inflammation and mucus production in offspring, with a decrease in inducible nitric oxide synthase (iNOS) in the lung tissue. This study suggests that maternal exposure to DEHP during pregnancy reduces asthmatic responses induced by OVA challenge in offspring. These effects were considered to be closely related to the suppression of Th2 immune responses and iNOS expression.     

Effect of a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin on immune function in male NC/Nga mice.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces immunosuppression in humans and animals. However, the effect of TCDD on Th2-type immune responses such as allergic reactions has been unclear. Using NC/Nga mice that developed atopic dermatitis-like skin lesions with marked elevation in plasma of total IgE when bred under conventional conditions, we investigated the effects of a single oral dose of TCDD on immune responses. NC/Nga mice received a single oral dose (0 or 20 microg/kg body weight) of TCDD. On day 7, treatment with TCDD alone decreased the cellularity of thymus. However, treatment with TCDD modified the cellularity of spleens and mesenteric lymph nodes (MLNs) but not of the thymus on day 28. When NC/Nga mice received ip immunization with OVA and alum on the same day as the TCDD treatment (0, 5, or 20 microg/kg body weight), TCDD markedly suppressed the concentrations of Th2-type cytokines (e.g., IL-4 and IL-5) in culture supernatants of spleen cells, whereas IFN-gamma production significantly increased. TCDD exposure reduced anti-OVA and total IgE antibody titers in plasma and did not induce the development of atopic dermatitis-like lesions in the pinnae or dorsal skin of NC/Nga mice. These results suggest that in NC/Nga mice, exposure to TCDD may impair the induction of Th2-type immune responses.     

The adjuvant effect of di-(2-ethylhexyl) phthalate is mediated through a PPARalpha-independent mechanism

Previous studies in BALB/c mice revealed an adjuvant effect of di-(2-ethylhexyl) phthalate (DEHP) to simultaneously administered ovalbumin. DEHP is the most commonly used phthalate plasticizer. In vivo formed metabolites of DEHP are peroxisome-proliferator-activated receptor (PPAR) ligands, a group of chemicals that may have immunomodulatory properties. To study whether the PPARalpha receptor was involved in the adjuvant effect of DEHP, PPARalpha-deficient 129/Sv mice were exposed intraperitoneally to a mixture of OVA and DEHP, and the OVA-specific IgE, IgG1 and IgG2a responses were compared to the corresponding responses in the wild-type strain. The study showed that the adjuvant mechanism of DEHP is mediated through a PPARalpha-independent mechanism. Compared to mice only given OVA, DEHP induced highly increased levels of OVA-specific IgG1 and IgG2a, both in the wild-type and in the PPARalpha knock-out strains, indicating that DEHP is a mixed Th1/Th2 adjuvant.     

Characteristics of antibody responses induced in mice by protein allergens

Whereas many foreign proteins are immunogenic, only a proportion is also allergenic, having the capacity to induce the quality of immune response necessary to support the production of IgE antibody. We have demonstrated previously that intraperitoneal administration to mice of proteins such as ovalbumin (OVA) or the industrial enzyme A. oryzae lipase, which possess significant allergenic potential, stimulates the production of both IgG and IgE antibody. Identical exposure to bovine serum albumin (BSA), a protein with limited potential to cause immediate respiratory or gastrointestinal hypersensitivity reactions, induced IgG responses only. In the current investigations, the quality of immune responses induced following exposure to these proteins via mucosal tissue (intranasal) has been compared with those provoked following administration via a non-mucosal (intraperitoneal) route of exposure. Intranasal or intraperitoneal administration of BSA, OVA or A. oryzae lipase elicited in each case vigorous IgG and IgG1 antibody responses. For all three proteins, at every concentration tested, and via both routes of exposure, IgG1 antibody titres paralleled closely IgG titres. However, the three materials displayed a differential potential to provoke IgE responses and this correlated with their known allergenic potential in humans. Thus, OVA and A. oryzae lipase stimulated strong IgE antibody responses, whereas BSA provoked low titre IgE only at the highest concentration tested (5% administered intraperitoneally). The quality of induced responses was not affected by the route of exposure. It would appear, therefore, that the stimulation of IgG and IgG1 antibody responses is a reflection of protein immunogenicity whereas protein allergenicity is associated with the induction of strong IgE responses.     

Covalent linkage of IL-12 and ovalbumin confines the effects of IL-12 to ovalbumin-specific immune responses

In order to direct the form of the immune response in an antigen-specific manner, we constructed a fusion protein (OVA/IL12) that contained the T cell-dependent antigen, ovalbumin (OVA), covalently linked to murine interleukin-12 (IL-12). The OVA/IL12 protein was produced in a baculovirus expression system and was purified by anti-OVA immunoaffinity chromatography. The purified OVA/IL12 protein displayed potent IL-12 bioactivity in an IL-12 proliferation assay. BALB/c mice immunized with the OVA/IL12 protein produced increased quantities of anti-OVA IgG2a antibody compared with mice immunized with recombinant OVA alone. Lymph node cells from the immunized mice with the OVA/IL12 protein produced large amounts of IFN-gamma when restimulatedin vitro with OVA, while those from mice immunized with the OVA protein produced little or no IFN-gamma. In contrast, immunization with a mixture of OVA and free recombinant IL-12 also induced IFN-gamma production, which was not OVA-specific. These studies indicate that the OVA/IL12 fusion protein can induce OVA-specific, Th1-dominated imnune responses, and that the covalent linkage of OVA and IL-12 confines the effect of IL-12 to OVA-specific cells.     

Effects of the phosphodiesterase IV inhibitor rolipram on Th1 and Th2 immune responses in mice

The present study was designed to investigate the effect of the phosphodiesterase IV inhibitor rolipram on Th1 and Th2 immune responses in mice. Mice were immunized subcutaneously at the base of the tail with ovalbumin (OVA) emulsified with complete Freund's adjuvant (day 0) and were treated daily with oral administration of various doses of rolipram from days 0 to 20. On day 21, production of anti-OVA IgG and proliferative responses to the antigen were determined. Anti-OVA IgG2a and interferon-gamma (IFN-gamma), as indicators of Th1 responses, and anti-OVA IgG1 and interleukin-10 (IL-10), as indicators of Th2 responses, were also measured. The results showed that treatment with rolipram failed to affect the production of OVA-specific IgG but decreased the proliferation of spleen cells to the antigen. Its inhibitory effect on these immune responses was correlated with a marked decrease in IFN-gamma but not IL-10 production, although neither anti-OVA IgG2a nor IgG1 production was affected by rolipram. These results suggest that rolipram may preferentially inhibit Th1 responses more effectively than Th2 responses. Administration of rolipram resulted in suppression of antigen (OVA)-induced arthritis in mice. The suppression of joint inflammation by rolipram was associated with the inhibition of the OVA-specific proliferative responses of spleen cells and IFN-gamma secretion. These results indicate that rolipram may be effective in regulating Th1-mediated diseases such as rheumatoid arthritis.     

Airway inflammation and adjuvant effect after repeated airborne exposures to di-(2-ethylhexyl)phthalate and ovalbumin in BALB/c mice

BACKGROUND: Epidemiological studies have suggested an association between exposure to phthalate plasticizers, including di-(2-ethylhexyl)phthalate (DEHP), and increased prevalence of asthma, rhinitis or wheezing. Furthermore, studies in mice have demonstrated an adjuvant effect from DEHP after parenteral administration with the model allergen ovalbumin (OVA). OBJECTIVE: Exposures to DEHP were investigated for adjuvant effects and airway inflammation in a mouse inhalation model. METHODS: BALB/cJ mice were exposed to aerosols of 0.022-13 mg/m(3) DEHP and 0.14 mg/m(3) OVA 5 days/week for 2 weeks and thereafter weekly for 12 weeks. Mice exposed to OVA alone or OVA+Al(OH)(3) served as control groups. Finally, all groups were exposed to a nebulized 1% OVA solution on three consecutive days. Serum, bronchoalveolar lavage (BAL) fluid, and draining lymph nodes were collected 24h later. RESULTS: In the OVA+Al(OH)(3) group, significantly increased levels of OVA-specific IgE and IgG1 in serum as well as of eosinophils in BAL fluid were observed. DEHP affected OVA-specific IgG1 production in a concentration-dependent manner, whereas little effect was seen on IgE and IgG2a. Dose-dependent increases in inflammatory cells were observed in BAL fluids, leading to significantly higher lymphocyte, neutrophil and eosinophil numbers in the OVA+13 mg/m(3) DEHP group. Ex vivo cytokine secretion by cultures of draining lymph nodes suggested that DEHP has a mixed Th1/Th2 cytokine profile. CONCLUSION: Airborne DEHP is able to increase serum IgG1 and lung inflammatory cell levels, but only at very high concentrations. Realistic DEHP levels do not have an adjuvant effect or induce allergic lung inflammation in the present mouse model.     

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.     

In vivo manipulation of endogenous metallothionein with a monoclonal antibody enhances a T-dependent humoral immune response.

Metallothionein (MT) is a small stress response protein that can be induced by exposure to heavy metal cations, oxidative stressors, and acute phase cytokines that mediate inflammation. In previous experiments, we have shown that exogenous MT can affect cell proliferation, macrophage and cytotoxic T lymphocyte function, and humoral immunity to T-dependent antigens. In the studies described here, we have explored the effect of a monoclonal anti-MT antibody (clone UC1MT) on the role that endogenous MT plays in the humoral immune response. In vivo injection of UC1MT significantly increased the humoral response to simultaneous challenge with ovalbumin (OVA). In contrast, mice immunized with OVA in the presence of an isotype-matched antibody control (MOPC 21) showed no change in the anti-OVA humoral response. The predominant anti-OVA response that was enhanced by UC1MT treatment was the IgG(1) response; the IgG(2a) anti-OVA response was not altered by UC1MT treatment. UC1MT treatment increased the numbers of IgG anti-OVA secreting cells as measured by ELISPOT assay, suggesting that blocking the effects of MT synthesized during the immune response augments the differentiation of antigen-specific plasma cells. The percentages of T and B cells in the spleens of animals from each treatment group were not significantly different, suggesting that this regimen of UC1MT treatment does not significantly affect hematopoiesis, but rather alters antigen-induced differentiation of lymphocytes. These observations are compatible with previous results from our laboratory that suggest that endogenous MT synthesized during the normal immune response or as a consequence of toxicant exposure suppresses in vivo immune function. In light of the fact that significant amounts of MT can be synthesized during toxicant exposure, manipulation of MT levels with an anti-MT antibody may ultimately represent an important therapeutic approach to the treatment of immune dysfunctions that result from toxicant exposure.     

Divergent antibody isotype responses induced in mice by systemic exposure to proteins: a comparison of ovalbumin with bovine serum albumin.

Whereas many foreign proteins are immunogenic, only a proportion is associated commonly with allergy, having the potential to induce the quality of immune response necessary for IgE antibody production and the development of immediate type hypersensitivity reactions in the gastrointestinal and/or respiratory tracts. In the context of toxicological evaluations there is a need to identify those properties that confer on proteins the ability to provoke allergic reactions. The characteristics of antibody responses induced in BALB/c strain mice following administration of ovalbumin (OVA), a significant human allergen, have been compared with those provoked by bovine serum albumin (BSA), a protein considered to have more limited allergenic potential. Intranasal or intraperitoneal (ip) administration of BSA or OVA elicited vigorous IgG and IgG1 antibody responses. Differential IgE antibody production was observed, however, with OVA stimulating relatively high IgE antibody titres at all doses tested whereas no or low titre IgE antibody was detected following exposure to BSA. Furthermore, a differential capacity for IgG2a antibody responses was observed, with only BSA provoking high titres of this IgG subclass. The relative quality of induced responses was equivalent following administration of these proteins via mucosal (in) tissue or via a non-mucosal (ip) route of exposure. IgG2a antibody production is promoted by the type 1 cytokine interferon gamma (IFN-gamma), whereas IFN-gamma and the type 2 cell product interleukin 4 exert reciprocal antagonistic effects on IgE antibody responses. Although cytokine expression patterns were not analysed in this series of experiments, the differential IgE and IgG subclass antibody responses induced by BSA and OVA are consistent with the preferential activation of T helper (Th) 1- and Th2-type cells, respectively. These data indicate that proteins can provoke in mice characteristic antibody (IgE and IgG) isotype profiles suggestive of discrete T lymphocyte responses and that such differences may be associated with variable allergenic activity.     

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.     

Differential immunogenic and neurogenic inflammatory responses in an allergic mouse model exposed to low levels of formaldehyde

It is suspected that exposure to low levels of formaldehyde induces or aggravates airway inflammation mediated by immunological and neurological reactions. To clarify the effect of this exposure on allergic inflammatory responses, we exposed female C3H/He mice to 0, 80, 400, or 2000ppb formaldehyde for 12 weeks. When mice were immunized with ovalbumin (OVA) and then exposed to formaldehyde, the numbers of total bronchoalveolar lavage cells, macrophages, and eosinophils in the mice exposed to 2000ppb formaldehyde were significantly increased compared to 0ppb controls. However, the production of interleukin-1beta from bronchoalveolar lavage fluid of these mice decreased significantly. Immunization with OVA significantly increased the production of nerve growth factor, but exposure to 80 and 400ppb formaldehyde significantly reduced the nerve growth factor levels in bronchoalveolar lavage fluid of the immunized mice. In in vitro study, markedly increased lipopolysaccharide-stimulated interferon-gamma production in culture supernatants of spleen cells from 2000ppb formaldehyde-exposed, nonimmunized mice, and significantly increased OVA-stimulated monocyte chemoattractant protein-1 production in culture supernatants of spleen cells from 400 and 2000ppb formaldehyde-exposed, immunized mice were observed. Exposure to 400ppb formaldehyde induced significant decreases in anti-OVA IgG1 and IgG3 antibody productions in plasma, whereas anti-OVA IgE antibody production was not affected. In addition, the levels of nerve growth factor in plasma of 80 and 400ppb formaldehyde-exposed, immunized mice significantly decreased compared to 0ppb control, immunized mice. These results provide the first experimental evidence that low levels of long-term formaldehyde inhalation can induce differential immunogenic and neurogenic responses in allergic mice.     

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.     

Effects of kampo-hozais on the induction of oral tolerance to ovalbumin in mice.

We studied here the effect of 3 kinds of kampo-hozais (Shofu-san, Ji-zuso-ippo and Unsei-in) on the induction of oral tolerance to ovalbumin (OVA) in C3H/HeN mice by measuring serum levels of OVA-specific antibodies. Oral tolerance was induced by a single administration of OVA (2 or 20 mg, p.o.) 7 d before the immunization. Among these kampo-hozais, mice administered Ji-zuso-ippo for 7 d before feeding of 2 mg OVA had significantly lower levels of anti-OVA IgG and IgG1, and further, Ji-zuso-ippo tended to reduce anti-OVA Igs in 20 mg OVA-fed mice, whereas Ji-zuso-ippo did not alter anti-OVA Igs in non-OVA-fed mice. Unsei-in also slightly augmented the suppression of anti-OVA IgG and IgG1; however, this lowering action of Unsi-in should be due to its immunosuppressive effect. Shofu-san did not alter these anti-OVA Ig levels in serum. These results suggested that Ji-zuso-ippo could augment the induction of oral tolerance.     

Dose and Load Studies for Subcutaneous and Oral Delivery of Poly(lactide-co-glycolide) Microspheres Containing Ovalbumin

Poly(lactide-co-glycolide) microspheres containing different loads of OVA (0.05, 0.1, 0.5 and 1.0% w/w) were manufactured by a w/o/w emulsion/solvent evaporation method. Low load efficiencies of less than 20% were observed. Normal size distributions with mean volume diameters ranging from 3.7 to 4.7 µm were obtained for different batches. The in vitro release of OVA from different loaded microspheres showed an expected burst release with all batches. The in vivo dose study (1, 10, 25, 50 µg of OVA) was performed by subcutaneous and oral inoculation in mice by single (0 week) or double (0 and 3 weeks) administration of PLGA 50/50 microspheres containing 0.1% OVA. Subcutaneous administration showed an immune response (serum Ig levels by ELISA) statistically (Fishers paired t-test; P < 0.05) above OVA saline negative controls at 3, 6 and 12 weeks after administration. Oral administration of microspheres produced statistically higher systemic immune responses at the higher doses. Single and double inoculation orally and subcutaneously produced similar serum antibody levels. The in vivo load study was performed by subcutaneous and oral administration to mice of 25 µg OVA contained in various loaded (0.05, 0.1, 0.5 and 1.0% w/w) microspheres. Serum immune responses at 3, 6, and 12 weeks after inoculation were statistically above OVA saline controls and were inversely proportional to the OVA load using either route. This observation suggested a relationship between the number of microspheres delivered and the in vivo serum response. Single subcutaneous administration of 0.05 or 0.1% OVA loaded PLGA 50/50 microspheres induced larger immune responses compared with complete Freunds adjuvant.     

An assessment of the ability of phthalates to influence immune and allergic responses

It has been suggested that one possible contributor to the increasing prevalence of atopic (IgE-mediated) allergic diseases and asthma in Europe and the US is exposure to chemicals that may act as adjuvants. Certain commonly used phthalate plasticisers, such as di-(2-ethylhexyl) phthalate, have been implicated in this regard. The evidence for the ability of phthalates to impact on immune and allergic responses has been examined, encompassing epidemiological investigations and results deriving from studies using experimental animals and from analyses in vitro. The epidemiological data provide some evidence that exposure to phthalates may be associated with increased risk of development of allergies and asthma, however, the lack of objective exposure information limits the interpretation. A variety of studies have been performed in mice to examine the influence of phthalate (delivered via various routes of exposure) to impact on immune responses. Measurement of antibody responses is the commonest read out, although other parameters of inflammation such as eosinophil infiltration and cytokine production have been used also. Although certain phthalates, when delivered at appropriate doses, and via an appropriate route, have been reported to impact on immune and inflammatory function in rodents, as yet no consistent pattern has emerged. Results ranged from potentiation of immune or inflammatory responses, to the absence of any effect, to inhibitory or immunosuppressive activity. In addition, comparatively low doses of phthalates have been associated with immune effects only when routes of administration (subcutaneous or intraperitoneal) are used that do not reflect, and are much less relevant for, opportunities for human contact with phthalates. There is clearly a case to be made for the design of more definitive animal studies that will allow development of a more detailed understanding of whether and to what extent, and under what conditions, phthalates are able to effect meaningful changes in immune function that may in turn impact on human health.     

Effect of methotrexate on Th1 and Th2 immune responses in mice

We investigated the effect of the anti-rheumatic drug methotrexate (MTX) on Th1 and Th2 immune responses in mice. For this investigation, mice were immunized subcutaneously at the base of the tail with ovalbumin (OVA) emulsified with complete Freund's adjuvant (day 0). Varying doses of MTX were orally administered daily from days 0 to 20. On day 21, anti-OVA IgG2a and interferon-gamma (IFN-gamma) as indicators of Th1 responses and anti-OVA IgG1 and interleukin-10 (IL-10) as those of Th2 responses were measured. The results showed that treatment with MTX was followed by decreases in OVA-specific IgG and proliferation of spleen cells to the antigen. The anti-rheumatic drug inhibited both anti-OVA IgG2a and IgG1 production, although the inhibitory effect of MTX on the antigen-specific IgG2a production appeared to be greater than that on IgG1 production. IFN-gamma, but not IL-10, secretion was markedly downregulated by MTX. Administration of MTX resulted in suppression of antigen (OVA)-induced arthritis in mice. The suppression of the joint inflammation by MTX was associated with inhibition of OVA-specific proliferative responses of spleen cells, anti-OVA IgG, IgG2a and IgG1 production, and IFN-gamma and IL-10 secretion, although more pronounced decreases in IgG2a and IFN-gamma were observed compared with those in IgG1 and IL-10 in MTX-treated mice. These results indicate that MTX appears to suppress Th1 and, to a lesser extent, Th2 immune responses and its anti-arthritic effect on human rheumatoid arthritis might be at least in part explained by down-regulation of Th1 responses involved in the disease.