Genetic attachment of undecane peptides to ovomucoid third domain can suppress the production of specific IgG and IgE antibodies

An undecane peptide (Gly-Ser-Pro-Gly-Ile-Pro-Gly-Ser-Thr-Gly-Met) was genetically attached to the N-terminus of ovomucoid third domain (DIII) to investigate structural characteristics of linear IgE and IgG (B cell) epitopes in DIII with respect to modulation of the immune response towards antigenicity and allergenicity. Balb/c mice were sensitized with native DIII, wild type recombinant DIII, and recombinant modified DIII containing the extra amino acid stretch. The immune responses to the antigens were compared using enzyme-linked immunosorbent assay. Interestingly, specific IgE and IgG levels were suppressed when the modified DIII was used as antigen. This was further confirmed by synthesizing immunodominant IgE and IgG epitopes of DIII on cellulose acetate membrane (SPOTs) and probing them with antibodies raised against DIII antigens. Anti-recombinant wild type DIII anti-serum showed strong binding activities to immunodominant IgE and IgG epitopes, while anti-modified DIII serum did not show any significant binding to the IgE and IgG epitopes. Thus, it is clearly demonstrated that the amino acid stretch in DIII is masking the immune reactive epitope. Genetical attachment of peptides into DIII was found to be effective in reducing the production of specific IgE and IgG antibodies in mice.     

Interleukin-18 enhances a Th2 biased response and susceptibility to Leishmania mexicana in BALB/c mice

Interleukin-18 deficient mice on a BALB/c background display increased resistance to cutaneous infection with Leishmania mexicana, with reduced lesion progression and reduced parasite burdens compared with wild-type mice. Infected IL-18-/- mice had lower antigen specific IgG1 levels and total IgE levels and conversely higher antigen specific IgG2a levels than similarly infected wild-type mice. Splenocytes isolated from infected IL-18-/- mice produced significantly lower levels of antigen induced IL-4 and higher levels of IFN-gamma than wild-type animals. Consequently IL-18 during L. mexicana infection of BALB/c mice promotes a Th2 biased response and thereby has a disease exacerbating role.     

T cell epitope-containing hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1, induce blocking antibodies

Allergen-specific immunotherapy represents one of the few curative approaches toward type I allergy. Up to 25% of allergic patients are sensitized against the major birch pollen allergen, Bet v 1. By genetic engineering we produced two recombinant (r) Bet v 1 fragments comprising aa 1-74 and aa 75-160 of Bet v 1, which, due to a loss of their native-like fold, failed to bind IgE Abs and had reduced allergenic activity. Here we show that both fragments covering the full Bet v 1 sequence induced human lymphoproliferative responses similar to rBet v 1 wild type. The C-terminal rBet v 1 fragment induced higher lymphoproliferative responses than the N-terminal fragment and represented a Th1-stimulating segment with high IFN-gamma production, whereas the N-terminal fragment induced higher IL-4, IL-5, and IL-13 secretion. Immunization of mice and rabbits with rBet v 1 fragments induced IgG Abs, which cross-reacted with complete Bet v 1 and Bet v 1-related plant allergens and strongly inhibited the IgE binding of allergic patients to these allergens. Thus, our results demonstrate that hypoallergenic T cell epitope-containing rBet v 1 fragments, despite lacking IgE epitopes, can induce Abs in vivo that prevent the IgE binding of allergic patients to the wild-type allergen. The overall demonstration of the immunogenic features of the hypoallergenic rBet v 1 fragments will now enable clinical studies for safer and more efficient specific immunotherapy.     

Engineered recombinant ovomucoid third domain can modulate allergenic response in Balb/c mice model

IgE-mediated allergic reactions to egg white are a serious health problem and ovomucoid being the dominant egg white allergen has been on focus in the past decade. Engineered hypoallergens with reduced reactivity for IgE antibodies are being examined to modulate the allergic response and develop prophylactic allergen vaccines. In this study, we evaluated the immunomodulatory effect of a genetic variant of the third domain of ovomucoid (GMFA) which showed reduced IgE binding with egg allergic patient's sera in comparison to the native form of the third domain of ovomucoid (DIII) in a murine model system. Balb/c mice were injected intraperitoneally with DIII and GMFA antigens. Allergen-specific serum IgG, IgG1, IgG2a, and IgE responses were evaluated using enzyme-linked immunosorbent assay. Splenocyte cytokine levels in the medium of the cultured cells were examined by ELISA and levels of IL-4, INF-gamma, and IL-12 (p70) cytokines were quantified. Neutralization with anti-IL-12 monoclonal antibody was assayed and cytokine levels with respect to GMFA mutant antigen stimulation were measured. GMFA mutant form was found to have significantly reduced levels of specific IgE when compared to the DIII suggesting a mutation-induced abrogation of the IgE binding epitope in mice. The increase in IgG2a levels in GMFA together with the decline of IgE and IgG1 points to a shift from a Th2 response to a Th1 dominated response. The cytokine profile showed a modulation of anti-allergic Th1 phenotype in GMFA from a proallergic Th2 response observed with DIII. Low levels of IL-4 and increased levels of INF-gamma and IL-12 were observed and anti-IL-12 monoclonal antibody restored the levels of IL-4 and suppressed levels of INF-gamma and IL-12 in the GMFA sensitized group. These results indicate that GMFA has a marked suppressive effect on the allergic response of ovomucoid and caused a shift towards a Th1 pathway, thereby modulating the Th1/Th2 cytokine balance and could be used as a potential hypoallergenic candidate for allergen-immunotherapy in the treatment of egg white allergy.     

Effect of oral probiotics (Bifidobacterium lactis AD011 and Lactobacillus acidophilus AD031) administration on ovalbumin-induced food allergy mouse model

Recent study has demonstrated an increasing prevalence of food allergy in Korean children. Specific probiotic bacteria may promote potentially anti-allergenic processes through induction of Th1-type immunity and enhance the regulatory lymphocyte. This study investigated whether orally administrated probiotics could suppress allergic responses in an ovalbumin (OVA)-induced allergy mouse model. Thus, female C3H/HeJ mice were orally sensitized with OVA and cholera toxin for 4 weeks. Lactobacillus acidophilus AD031, Bifidobacterium lactis AD011, and L. acidophilus AD031 plus B. lactis AD011 were fed to mice from 2 weeks before the sensitization. The OVA-induced mice that were not treated with probiotics had significantly increased serum levels of OVA-specific IgE and IgG1, and OVAspecific IgA in feces. However, the mice treated with probiotics suppressed production of the OVA-specific IgE, IgG1, and IgA. The level of IL-4 was significantly lower, and the levels of INF-gamma and IL-10 were significantly higher in the mice treated with probiotics than that in the nontreated mice. The groups treated with probiotics had decreased levels of degranulated mast cells, eosinophil granules, and tail scabs. These results indicate that L. acidophilus AD031 and B. lactis AD011 might be useful for the prevention of allergy.     

IgE generation and mast cell effector function in mice deficient in IL-4 and IL-13

IL-4 and IL-13 are potent cytokines that drive production of IgE, which is critical to the development of atopic disease. In this study, we directly compared IgE generation and IgE-dependent mast cell effector function in mouse strains lacking IL-4, IL-13, IL-4 + IL-13, or their common receptor component, IL-4Ralpha. Although serum IgE was undetectable under resting conditions in most animals deficient in one or both cytokines, peritoneal mast cells from mice lacking IL-4 or IL-13 had only partial reductions in surface IgE level. In contrast, peritoneal mast cells from IL-4/13(-/-) and IL-4Ralpha(-/-) animals were severely deficient in surface IgE, and showed no detectable degranulation following treatment with anti-IgE in vitro. Surprisingly, however, intradermal challenge with high concentrations of anti-IgE Ab induced an ear-swelling response in these strains, implying some capacity for IgE-mediated effector function in tissue mast cells. Furthermore, upon specific immunization with OVA, both IL-4/IL-13(-/-) and IL-4Ralpha(-/-) mice produced detectable levels of serum IgE and Ag-specific IgG1, and generated strong ear-swelling responses to intradermal administration of anti-IgE. These findings suggest that a mechanism for IgE production exists in vivo that is independent of IL-4 or IL-13.     

The Effect of IFN-gamma, Alum and Complete Freund Adjuvant on TNP-KLH Induced Ig.G(1), IgE and IgG(2a) Responses in Mice

Adjuvants are considered to play an important role in directing the isotype and amount of antibodies produced upon immunization by conducting the development of either Th-1 or Th-2 cells upon T-cell stimulation. This is based on the different cytokine production patterns that were observed after in vitro resttmulation of T cells isolated from mice immunized with antigen either adsorbed on alum or emulsified in complete Freund adjuvant (CFA). However, other studies suggest that primarily the type of antigen determines which isotypes are produced and to what extent. In these studies, however, IgE was not determined. Therefore, this study examined whether alum and CFA influenced the amount and/or ratio of IgG(1), IgE and IgG(2a) produced after TNP-KLH immunization. Similar levels of IgG(1), IgE and IgG(2a) antibodies were found upon immunization with TNP-KLH either adsorbed on alum or emulsified in CFA. Moreover, administration of IFN-gamma in combination with TNP-KLH adsorbed on alum did not increase the amount of IgG(2a) produced. IFN-gamma treatment resulted in an increased IL-6 and decreased IFN-gamma production by spleen cells upon Con A stimulation, whereas it did not change the IL-4 production in similar conditions. The presented results suggest that upon immunization with TNP-KLH high IL-4 levels are produced, resulting in an antibody response that is dominated by IgG(1), independent of the adjuvant employed. The IL-4 inducing property of TNP-KLH is substantiated by the finding that repeated immunization of mice with TNP-KI, without adjuvant, increases the serum total IgE level. The presented data suggest that the carrier part of TNP-KLH preferentially results in Th-2 cell activity after which the adjuvant merely enhances the antibody responses generated.     

Induction of Th2 responses and IgE is largely due to carbohydrates functioning as adjuvants on Schistosoma mansoni egg antigens

Infection with the helminth parasite Schistosoma mansoni induces a pronounced Th2-type response that is associated with significant IgE production. To better understand how the parasite drives these responses, we investigated the relative roles of proteins and carbohydrates in driving Th2-type and/or IgE responses using a murine model of intranasal sensitization with soluble egg Ags (SEA) of Schistosoma mansoni. We found that repeated intranasal sensitization with soluble egg Ags led to the induction of both total and specific IgE production and nasal eosinophilia. By comparing the responses of mice sensitized with SEA or metaperiodate-treated SEA we were able to demonstrate that carbohydrates on SEA are the major inducers of IgE production and nasal recruitment of eosinophils. Mice sensitized with periodate-treated SEA displayed a significant decrease in both total and specific IgE levels in comparison to mice sensitized with native SEA. Furthermore, sensitization of mice with periodate-treated SEA significantly reduced levels of Ag-specific IgG1, but had no effect on IgG2a production. Nasal lymphocytes from mice sensitized with native SEA, but not with periodate-treated SEA, produced IL-4, IL-5, and IL-10 when restimulated with native SEA in vitro. On the other hand, lymphocytes from mice sensitized with periodate-treated SEA did not produce any of these same cytokines following in vitro restimulation, suggesting that carbohydrates were required for in vivo induction of Th2 response and for that of associated cytokine responses in this model. Lastly, competitive inhibition ELISA showed that although carbohydrates are required for SEA-specific IgE induction, they are not targets of the induced IgE response.     

Induction of systemic Th1 and Th2 immune responses by oral administration of soluble antigen and diesel exhaust particles

The present study was undertaken to examine whether oral administration of soluble antigen together with diesel exhaust particles (DEP) induced the systemic immune response in mice. Mice were orally given 1 mg of hen egg lysozyme (HEL) with varying doses of DEP every 3 days over a period of 15 days. The results showed that oral administration of HEL plus DEP produced anti-HEL IgG antibodies in serum in a dose-related fashion, while either HEL or DEP alone failed to show the antigen-specific IgG antibody production. Production of anti-HEL IgG2a and IgG1 antibodies, which are dependent on Th1 and Th2 CD4(+) T cells, respectively, was seen in mice fed with combined HEL and DEP, although anti-HEL IgG1 antibodies appeared to be more efficiently produced by lower doses of DEP than anti-HEL IgG2a antibodies. There was marked antigen-specific proliferation of spleen cells in mice treated with HEL and DEP. The anti-HEL antibody production and lymphoid cell proliferation to the antigen were associated with marked secretion of the Th1 cytokine IFN-gamma as well as the Th2 cytokine IL-4. These results suggest that DEP may act as a mucosal adjuvant in the gut enhancing systemic Th1 and Th2 immune responses and might play a role in oral immunization and food allergy.     

IgE production after four routes of injections of Japanese cedar pollen allergen without adjuvant: crucial role of resident cells at intraperitoneal or intranasal injection site in the production of specific IgE toward the allergen

The production of specific IgE antibodies directed toward cedar pollen correlates well with the onset of allergic rhinitis; but the mechanisms of allergen recognition as nonself and Ig class switch to IgE by the immune system are still not fully understood. In the present study, we injected cedar pollen into mice through 4 different routes (intranasal (i.n.), intraperitoneal (i.p.), intravenous (i.v.), and subcutaneous (s.c.)) without adjuvant 1 to 3 times, and determined time-dependent changes in the total and specific serum IgE levels compared with those in the serum levels of other isotype Igs. After an i.p. or i.n. injection of allergen into the mice, they produced a 1.5-to 1.7-fold increase in total IgE, but none in IgG, IgM, or IgA antibodies in their serum, whereas an i.v. or s.c. injection of allergen was inactive as an inducer of total IgE antibodies. Upon a 2nd (s.c.) injection of the allergen into the i.p. or i.n. sensitized mice, a large amount of allergen-specific IgE antibodies was found in the serum. In the case of i.v. or s.c. sensitized mice, however, they produced total, but not specific, IgE antibodies; and a 3rd (s.c.) injection of the allergen resulted in a large amount of specific IgE antibodies in the serum. These results imply that resident cells at the i.p. or i.n. injection site may play a crucial role in the efficient production of total and specific IgE antibodies toward the allergen.     

Oral administration of ovalbumin after sensitization attenuates symptoms in a mouse model of food allergic enteropathy

Oral immunotherapy (OIT) is a promising treatment of food allergy. To administer an appropriate oral dose of an allergenic component as OIT to individuals sensitized with a food allergen may prevent inducing food allergic inflammation in them. So we attempted to establish a mouse model to evaluate efficacy for oral administration of food allergen after sensitization. In BALB/c mice sensitized by injecting ovalbumin (OVA) with alum twice, OVA was administered before inducing inflammation by feeding the mice with egg white (EW) diet. Severe inflammatory responses, such as enteropathy, weight loss, IL-4 production, and increase of IgE antibody levels, were suppressed by administration with 4 mg of OVA 7 times before feeding EW diet. OVA administration alone induced a slight Th2 response, but no symptoms. The current study demonstrated that severe food allergic enteropathy could be prevented by pre-administration with appropriate dose of OVA to sensitized mice.     

Immune response induced by airway sensitization after influenza A virus infection depends on timing of antigen exposure in mice

To study which phase of viral infection promotes antigen sensitization via the airway and which type of antigen-presenting cells contributes to antigen sensitization, BALB/c mice were sensitized by inhalation of ovalbumin (OA) during the acute phase or the recovery phase of influenza A virus infection, and then 3 weeks later animals were challenged with OA. The numbers of eosinophils and lymphocytes, the amounts of interleukin-4 (IL-4) and IL-5 in the bronchoalveolar lavage fluid, and the serum levels of OA-specific immunoglobulin G1 (IgG1) and IgE increased in mice sensitized during the acute phase (acute phase group), while a high level of gamma interferon production was detected in those sensitized during the recovery phase (recovery phase group). In the acute phase group, both major histocompatibility complex class II molecules and CD11c were strongly stained on the bronchial epithelium; in the recovery phase group, however, neither molecule was detected. OA-capturing dendritic cells (DCs) migrated to the regional lymph nodes, and a small number of OA-capturing macrophages were also observed in the lymph nodes of the acute phase group. In the recovery group, however, no OA-capturing DCs were detected in either the lungs or the lymph nodes, while OA-capturing macrophages were observed in the lymph nodes. These results indicate that the timing of antigen sensitization after viral infection determines the type of immune response.     

The polyclonal and antigen-specific IgE and IgG subclass response of mice injected with ovalbumin in alum or complete Freund's adjuvant

BALB/c mice were injected ip with 1 microgram ovalbumin (OVA) in alum or complete Freund's adjuvant (cFA) and the changes of the IgE and IgG subclass serum levels and isotypes of the anti-OVA specific antibodies determined by radioimmunoassays. By Day 10, OVA in alum had induced a 5- to 10-fold increase of the IgE serum level and an initial decrease of the IgG subclass levels which subsequently increased to two to threefold over the preinjection level. OVA in cFA induced a gradual twofold increase of the IgE serum level, a rapid fourfold increase of the IgG2a level occurring by Day 7, and a gradual two to threefold increase of the other IgG subclasses. Over 90% of the anti-OVA antibodies were of the IgGl isotype with both adjuvants; OVA in alum induced slightly more IgGl anti-OVA antibodies than cFA. In contrast, the OVA in alum injected mice formed significantly more (5- to 10-fold) IgE anti-OVA antibodies than the cFA-injected mice. OVA in alum also induced a large nonspecific increase of the IgE serum level because only approximately 40% of the increase observed on Day 14 was absorbable with OVA, whereas approximately 90% the IgE increase in cFA injected mice was absorbable with OVA. The data demonstrate that mice form mainly IgGl and IgE antibodies to OVA irrespective of the adjuvant. The low specific and lack of nonspecific IgE formation by mice injected with OVA in cFA may be the result of cFA-induced interferon-gamma (IFN-gamma) production because IFN-gamma has been shown to stimulate IgG2a and inhibit IgE secretion in vitro.     

IgE isotype switch and IgE production are enhanced in IL-21-deficient but not IFN-gamma-deficient mice in a Th2-biased response

IgE plays a critical role in the pathogenesis of allergy and asthma. Therefore, suppression of IgE production would provide therapeutic benefits to patients suffering from these diseases. We have reported that the production of IgE is regulated differently in the spleen vs. the draining lymph nodes (LN). IgE isotype switch and IgE producing B cell expansion occur in the draining LN after antigen (Ag) immunization, but do not happen in the spleen. In addition, a population of pre-existing IgE+ cells is observed in the spleen of normal or sham immunized mice, but is not present in the draining LN. To further understand the regulation of IgE production in different lymphoid organs, and the potential inhibitory factors of IgE isotype switch in the spleen, the involvement of IL-21 and IFN-gamma in regulating IgE production was investigated by using the IL-21 and the IFN-gamma deficient mice. We found that in the absence of IL-21 IgE isotype switch and IgE+ cell clonal expansion were dramatically enhanced in the spleen and IgE isotype switch was partially increased in the draining LN. In addition, IgE production of the pre-existing CD19-CD5+B220(low) IgE+ cells in the spleen was also increased in the absence of IL-21 under physiological conditions. In contrast, using the IFN-gamma deficient mice, we did not observe a negative impact of IFN-gamma on either IgE isotype switch or IgE production. Our data suggest that IL-21 appears to be a critical cytokine to keep low IgE levels under physiological and pathological conditions.     

Unlipidated Outer Membrane Protein Omp16 (U-Omp16) from Brucella spp. as Nasal Adjuvant Induces a Th1 Immune Response and Modulates the Th2 Allergic Response to Cow’s Milk Proteins

The discovery of novel mucosal adjuvants will help to develop new formulations to control infectious and allergic diseases. In this work we demonstrate that U-Omp16 from Brucella spp. delivered by the nasal route (i.n.) induced an inflammatory immune response in bronchoalveolar lavage (BAL) and lung tissues. Nasal co-administration of U-Omp16 with the model antigen (Ag) ovalbumin (OVA) increased the amount of Ag in lung tissues and induced OVA-specific systemic IgG and T helper (Th) 1 immune responses. The usefulness of U-Omp16 was also assessed in a mouse model of food allergy. U-Omp16 i.n. administration during sensitization ameliorated the hypersensitivity responses of sensitized mice upon oral exposure to Cow’s Milk Protein (CMP), decreased clinical signs, reduced anti-CMP IgE serum antibodies and modulated the Th2 response in favor of Th1 immunity. Thus, U-Omp16 could be used as a broad Th1 mucosal adjuvant for different Ag formulations.     

Establishment and characterization of a novel murine model for pollen allergy

Although there have been many studies revealing the mechanism and establishing the therapeutical method for allergic rhinitis, no suitable animal models for allergic rhinitis, especially for pollen allergy, are currently available. We therefore aimed in this study to develop a murine model producing IgE in response to an inhaled antigen without using any adjuvants. Ovalbumin (OVA)-specific T cell receptor transgenic mice (DO11.10) inhaled an OVA solution for one h, twice a week, for six weeks. The resulting increase of OVA-specific IgE in the serum was observed depending on the times of inhalation. Spleen cells from mice that had inhaled the antigen produced more IL-4 and less IFN-γ than those from the control mice in vitro. These results indicate that inhaled antigen enhanced the Th2-type responses and induced IgE production in a T cell-mediated manner. Our findings would contribute to studies on prevention and treatment of pollen allergy.     

Primary anti-trinitrophenyl memory cells investigated by plaque-forming cells and ELISA.

Primary anti-trinitrophenyl antibody production was investigated from spleen cells of mice immunized with trinitrophenylated-keyhole limpet hemocyanin, using the plaque-forming cell method and ELISA. Cells taken 5 days after antigen injection do not produce IgE, but do produce IgM and IgG1 anti-trinitrophenyl antibodies as demonstrated by plaque-forming cells. Substantial increase of IgM, IgG1, and IgE antibody production was seen from cells taken 7 days after immunization, followed by a rapid decline. By ELISA it was seen that cells taken 3 days after immunization already produce small amounts of anti-trinitrophenyl antibodies. Presence of antigen from the start of the cultures did not increase antibody production from cells taken 3 days after immunization, but potentiated antibody secretions from cells taken 5 days or later after immunization. This potentiation was interpreted as recruitment of antibody-forming cells from early memory B cells. The presence of IL-4 from the start of the cultures had no appreciable effect. Cell sorting with specific antibody-coated magnetic beads showed that plaque-forming cells from nonsorted cells, membrane IgE+ or membrane IgE- cells secreted similar amounts of anti-trinitrophenyl IgG1 and IgE antibodies. No difference in anti-trinitrophenyl IgM, IgG1, or IgE production was found in controls; cells sorted negatively or positively for CD23. The data show that memory B cells can be demonstrated already on Day 5 after immunization, and their antigen-induced antibody secretion is IL-4 dependent.     

Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4- or IL-13-mediated signaling

Previous studies demonstrate that aluminium hydroxide adjuvant (alum) produces increased Th1 responses in IL-4-deficient mice compared with wild-type animals, although the continued production of IL-5 by spleen cells from these mice also indicates that Th2 responses are induced. In the present study, we demonstrate that alum can induce Th2-associated IL-4 and IL-5 production in the absence of IL-4 signaling in mice deficient in either IL-4Ralpha or Stat6. The Th2 responses observed could not be due to IL-13 as IL-13 responses are also impaired in IL-4Ralpha- and Stat6-deficient mice. We also detected higher levels of IL-4 in IL-4Ralpha gene-deficient, though not Stat6-deficient, mice compared with their wild-type counterparts. The increased levels of IL-4 could be explained by the IL-4R being unavailable to neutralize this cytokine in IL-4Ralpha-deficient mice. While levels of IL-5 production in IL-4Ralpha- or Stat6-deficient mice were similar to IL-4-deficient and wild-type mice, other type 2-associated responses, which are largely or wholly IL-4 dependent, such as the production of IgG1 or IgE Abs, were either reduced or absent. We conclude that alum adjuvants can induce IL-4 production and Th2 responses independently of IL-4 or IL-13, negating the requirement for an early source of IL-4 in the Th2 response induced by this adjuvant.     

The IgE and IgG subclass responses of mice to four helminth parasites

To investigate whether the formation of IgE is linked in vivo to an IgG subclass, mice were infected with four helminth parasites, Nippostrongylus brasiliensis (Nbr), Mesocestoides corti, Taenia crassiceps and Trichinella spiralis, and the changes in the serum levels of the different Ig isotypes as well as the antibody response to M. corti and T. crassiceps antigen extracts were determined by radioimmunoassays. All four parasites induced a concomitant increase of the IgE and IgG1 serum levels and usually a decrease of the IgG2a level. They also induced an increase of the IgM level but had little effect on the IgG2b, IgG3, and IgA serum levels. The specific antibodies to an M. corti antigen extract were mainly of the IgG1 subclass, whereas it was of both IgG1 and IgG2a subclasses to T. crassiceps. Injections of dead M. corti induced an increase of all IgG subclasses and similar levels of IgG1 and IgG2a anti-parasite antibodies. Subcutaneous instead of intraperitoneal infection with T. crassiceps induced higher IgG2a than IgG1 levels and 10-fold lower IgE levels than the natural ip infection; however, despite the greater IgG2a polyclonal response, anti-parasite antibodies were predominantly of the IgG1 subclass. The data demonstrate that natural infection with four different helminth parasites induces a concomitant polyclonal IgG1 and IgE response. These in vivo observations corroborate the recent in vitro findings demonstrating that interleukin-4 induces lipopolysaccharide-activated murine B cells to secrete both IgG1 and IgE, suggesting that the regulation of these two isotypes is linked.