Direct effects of IL-4/IL-13 and the nematode Nippostrongylus brasiliensis on intestinal epithelial cells in vitro.

Nematode infections induce the upregulation of mucin- and glycosylation-related genes in intestinal epithelial cells in vivo. However, the factor(s) that induce these changes in epithelial cells have not been fully elucidated. In the present study, we analysed the effects of the Th2 cytokines IL-4 and IL-13 and the excretory-secretory (ES) product of the nematode Nippostrongylus brasiliensis on the gene expression of the major mucin core peptide MUC2, the sialyltransferase ST3GalIV (Siat4c) and the sulphotransferase HS3ST1 in intestinal epithelium-derived IEC-6 cells by quantitative reverse transcription (RT)-PCR. The administration of IL-4 and IL-13 resulted in a significant upregulation of ST3GalIV and HS3ST1 gene transcription, but had no effect on MUC2, in IEC-6 cells. RT-PCR studies also demonstrated the constitutive expression of IL-13Ralpha1 and IL-4R in IEC-6 cells. On the other hand, the ES product induced upregulation of ST3GalIV, but not HS3ST1 or MUC2, while coadministration of IL-13 and the ES product induced a slight but significant upregulation of MUC2. Co-incubation of live N. brasiliensis adult worms with IEC-6 cells resulted in the upregulation of ST3GalIV and MUC2. These results suggested that HS3ST1 gene expression is strictly regulated by IL-4/IL-13, while ST3GalIV and MUC2 gene expressions are regulated by redundant mechanisms.

Immunity-mediated regulation of fecundity in the nematode Heligmosomoides polygyrus--the potential role of mast cells.

Previous studies have shown that host immunity regulates the fecundity of nematodes. The present study was aimed at clarifying the reversible nature of fecundity in response to changes of immunological status and to determine which effector cells are responsible for compromising fecundity in Heligmosomoides polygyrus. Enhanced fecundity was observed in immunocompromised SCID and nu/nu mice compared to those in the corresponding wild-type mice, with significantly fewer numbers of intrauterine eggs produced in the wild-type than in the immunodeficient mice. When 14-day-old adult worms from BALB/c mice were transplanted into naïve BALB/c mice, their fecundity increased significantly as early as 24 h post-transplantation, but not when they were transferred into immune mice, suggesting the plastic and reversible nature of fecundity in response to changes in host immunological status. In mast cell-deficient W/W(v) mice, nematode fecundity was significantly higher than in mast cell-reconstituted W/W(v) or +/+ mice. The serum levels of the mast-cell protease mMCP1 were markedly increased in the wild-type as well as the mast cell-reconstituted W/W(v), but not in the W/W(v), SCID, or nu/nu mice during infection. These findings raise the interesting possibility that certain activities of mast cells, either directly or indirectly, regulate parasite fecundity during infection.

Depleted intestinal goblet cells and severe pathological changes in SCID mice infected with Heligmosomoides polygyrus.

To determine the role of T cells and mast cells in intestinal pathology and immune expulsion of intestinal nematodes, worm burdens, goblet cell responses and villus structures were analysed in T- and B-cell-deficient severe combined immunodeficiency (SCID) mice, athymic nu/nu mice and mast cell deficient W/W(v) mice after infection with the nematode Heligmosomoides polygyrus. SCID and nu/nu mice showed significantly higher worm burdens at week 9 post-infection compared with the wild-type controls. SCID and nu/nu mice showed compromised goblet cell hyperplasia and/or Muc 2 expression, indicating that both events are T-cell dependant. On the other hand, the SCID mice showed increased pathology (villus atrophy and crypt hyperplasia) and increased numbers of proliferating cell nuclear antigen positive cells compared to the wild-type controls. W/W(v) mice, conversely, were able to expel the worms normally, had normal goblet cell hyperplasia, and did not demonstrate the changes in mucosal architecture seen in SCID mice, confirming that a normal mast cell response is not necessarily required for these changes. These results suggest that a functional T-cell response, but not a mast cell response, is necessary for anti-parasite responses, goblet cell function, and maintaining normal mucosal architecture.

Mucosal mast cell proliferation following normal and heterotopic infections of the nematode Nippostrongylus brasiliensis in rats.

Infections of intestinal nematodes induce the T cell-dependent proliferation of intestinal mucosal mast cells (MMC). To examine whether nematode-induced MMC proliferation is affected by the site of infestation, adult-stage nematode Nippostrongylus brasiliensis (NB) was transplanted into the normal infection site, the duodenum, or into heterotopic sites, the peritoneal cavity (i.p.) or subcutaneous tissue (s.c.), of rats. Two weeks after duodenal inoculation, MMC numbers in the small intestine had increased 6.5-fold. In contrast, i.p. and s.c. inoculation induced only slight increases of intestinal MMC. After i.p. inoculation, worm granulomas developed in the connective tissues adhering to stomach and duodenum, and large numbers of mast cells appeared around the granulomas. The majority of the latter mast cells showed histochemical features similar to MMC: they were formalin sensitive, berberine sulfate-, alcian blue+/safranine-, and rat mast cell protease (RMCP) II+. After s.c. inoculation, worm granulomas developed at the inoculation site, but the number of mast cells around the granulomas was not significantly increased. These results suggest that intense proliferation of MMC or MMC-like cells is induced only by the infections on mucosa or in mucosa-associated tissues.

Lack of active lung anaphylaxis in congenitally mast cell-deficient Ws/Ws rats sensitized with the nematode Nippostrongylus brasiliensis.

Ws/Ws rats are deficient in both mucosal- and connective tissue-type mast cells. To study the role of mast cells in active anaphylaxis, changes in vascular permeability in the trachea upon intravenous antigen challenge with Evans blue dye were examined in Ws/Ws, heterogenic Ws/+, and normal +/ + rats sensitized with the nematode Nippostrongylus brasiliensis. Antigen challenge resulted in fatal anaphylactic shock in some +/+ and Ws/+ rats, but not in Ws/Ws rats. Marked dye leakage developed within 30 min in the trachea of +/+ and Ws/+ rats, while Ws/Ws rats showed no substantial increases in the levels of vascular permeability. Ex vivo stimulation of sensitized lung fragments from +/+ animals with specific antigen induced significant releases of histamine and leukotriene (LT) C4, while sensitized Ws/Ws rat-lung fragments did not. In Ws/Ws rats, levels of nematode-specific IgE, IgG1 and IgG2a antibodies as well as levels of lung eosinophilia were not significantly different from those in +/+ rats. These results show that mast cell-deficient Ws/Ws rats fail to develop active anaphylaxis, and this is mediated probably by the lack of mast cell-derived mediators required for initiation of the reaction.

Migration of neutrophils is dependent on mast cells in nonspecific pleurisy in rats.

To determine the role of mast cells in the recruitment of neutrophils and eosinophils, acute nonspecific pleurisy was induced by injecting isologous serum into normal +/+ and mast cell-deficient Ws/Ws rats. In +/+ rats, neutrophil infiltration peaked 4 h after serum administration, followed by influx of eosinophils after 24-48 h. The levels of neutrophil influx after 4 h as well as the activity of myeloperoxidase (MPO) in pleural lavage-cell extract were significantly lower in Ws/Ws rats than in +/+ rats. In contrast, numbers of eosinophils as well as activity of eosinophil peroxidase (EPO) did not differ significantly between Ws/Ws and +/+ rats. For local reconstitution of mast cells, +/+ rat peritoneal mast cells (PMC) or mesenteric lymph node cells (MLNC) as a control were transferred into the Ws/ Ws pleural cavity. Serum injection into animals with PMC transfer 7 days previously triggered augmented neutrophil influx by approximately 4.7-fold as compared to that in MLNC-transferred animals. Mast cells recovered from the pleural cavity of PMC-transferred rats showed histamine contents equivalent to 20% of that of freshly isolated PMC and retained the reactivity to compound 48/80. These results indicated that dependency of neutrophil recruitment on resident mast cells is greater than that of eosinophils in isologous serum-induced pleurisy.

Acanthocephalan, probably Bolbosoma, from the peritoneal cavity of man in Japan.

An immature female acanthocephalan in a tumor on the serosa over the ileum of a 16-year-old boy in Kagoshima, Japan, was identified as probably a species of Bolbosoma. This is the second such case to be reported.

Antibody responses and protective immunity in rats receiving repeated inoculations of Strongyloides ratti.

The relationship between specific antibody responses and protective immunity against Strongyloides ratti was examined in rats receiving 10, 50, or 500 infective larvae (L3) at weekly intervals. No specific IgG response was detected in rats receiving 10-L3 inoculations for 7 wk. Fifty- and 500-L3 inoculations induced an IgG response by weeks 2 and 3, respectively, and a higher IgG response was induced in rats receiving the higher doses. All 3 inoculation doses induced high IgE responses, but the kinetics were different. IgE in the 10-L3 group continued to rise from weeks 4 to 7. In the 50- and 500-L3 groups, IgE was detected first at week 3 and increased until week 5. It then declined in the 500-L3 group and the titer at week 7 was significantly lower than that at week 5, whereas it remained the same in the 50-L3 group. The number of larvae recovered from the head 40 hr after a challenge inoculation (1,000 L3) significantly declined by weeks 7, 3, and 2 in rats receiving 10, 50-, and 500-L3 inoculations, respectively. Intestinal worm burdens increased for 7 wk in the 10-L3 group, 5 wk for the 50-L3 group, and 2 wk for the 500-L3 group. These findings indicate that repeated inoculations of low doses of L3 induce delayed and limited protective immunity to a heavy challenge and worm expulsion from the intestine. There was a temporal correlation between the levels of protection and serum IgG, whereas circulating IgE level did not seem to affect directly either the level of the resistance or expulsion of intestinal worms.

Lectin-binding properties of the surfaces of in vitro-transformed Schistosoma mansoni and Echinostoma paraensei sporocysts.

As carbohydrates on the surfaces of sporocysts of digenetic trematodes may be targets of attack by the molluscan internal defense system, the lectin-binding patterns of living, in vitro-transformed sporocysts of Schistosoma mansoni and Echinostoma paraensei were characterized. Schistosoma mansoni sporocysts specifically bound 8 and E. paraensei 6 of 11 lectins examined. Sporocysts of the 2 species responded differently to 7 of the 11 lectins. Lectins inhibitable by mannose, galactose, and N-acetylgalactosamine were bound by both species. Lectins inhibited by fucose and N-acetylglucosamine bound uniquely to S. mansoni, and an N-acetylneuraminic acid (NeuNAc)-inhibitable lectin bound only to E. paraensei. Preincubation of sporocysts of either species in the plasma of the host snail Biomphalaria glabrata for as long as 24 hr only marginally altered the subsequent binding of lectins. Pretreatment of S. mansoni sporocysts with pronase E and trypsin substantially altered subsequent lectin binding, but similar treatment of E. paraensei sporocysts had little effect. A neuraminidase enzyme derived from Clostridium perfringens diminished binding of the NeuNAc-inhibitable lectin to E. paraensei sporocysts. This study indicates that lectin-binding monosaccharides are expressed abundantly on sporocyst surfaces, they vary considerably between 2 species parasitizing the same host, and they are not obscured readily or altered by exposure to host plasma.

The effects of single and repeated inoculations of various larval doses on Strongyloides ratti burden and distribution in rats.

The changes in worm burden, distribution, length, and fecundity after and during single and repeated inoculations of 10, 50, or 500 larvae of Strongyloides ratti were examined in rats. Worm burden after a single inoculation of a higher larval dose reduced rapidly. Repeated inoculations of lower larval doses at weekly intervals led to a delayed peak and slower reduction of worm burden; the repeated inoculations of 10 larvae did not induce worm expulsion for at least 7 wk. In repeated inoculations at 3-wk intervals, a primary inoculation of 500 larvae induced strong resistance to reinfection at week 3, whereas no resistance was induced until week 6 in rats receiving repeated inoculations of 10 or 50 larvae. Similar dose-dependent reductions in worm length and fecundity were observed in single and repeated inoculations, and the reductions began earlier than worm expulsion. Intestinal migration of worms from the upper small intestine to the large intestine was observed during the course of single and repeated inoculations. Earlier and clearer migration was observed in rats receiving higher doses. These findings indicate that in S. ratti infection, the changes of worm burden, distribution, length, and fecundity are dependent on the inoculated larval dose.

Immunological characteristics of patients infected with common intestinal helminths: results of a study based on reverse-transcriptase PCR.

To determine whether common helminth infections could modify the intestinal immunopathological status of the host, the expression in the human duodenal mucosa of cytokines, eosinophil- and mast-cell-specific molecules and monosaccharide transporters of the glucose-transporter (GLUT) family was explored. The 31 subjects were all patients at the gastro-intestinal disease unit of Nongkhai Hospital, Thailand. Four of the 10 patients who presented with eosinophilia (> or = 6.0% of their leucocytes were eosinophils), and five of the other 21 patients, had intestinal infections with helminths when they presented or within the previous 3 months. Studies based on semi-quantitative, reverse-transcriptase PCR revealed that the interleukin-5/interferon-gamma ratio was significantly higher in the noneosinophilic, helminth-infected patients than in the non-eosinophilic, uninfected patients, whereas the IgE receptor type I (Fc epsilon RI)/mast-cell tryptase ratio was significantly higher in the eosinophilic, helminth-infected patients than in the eosinophilic, uninfected patients. Expression of Charcot-Leyden-crystal protein, GLUT-1 and GLUT-5, however, showed no significant inter-group differences. Principal-components analysis of the data on eosinophils, interleukin-5, interferon-gamma, Fc epsilon RI and mast-cell tryptase revealed that one principal component could discriminate the patients who had helminth infection from the non-eosinophilic, uninfected patients, but not from the eosinophilic, uninfected patients. These results indicate that, whatever the intestinal pathology, patients infected with common intestinal helminths tend to develop a mucosal immunological response of the Th2 type.

Immunoglobulin E and mast cell responses are related to worm biomass but not expulsion of Hymenolepis diminuta during low dose infection in rats.

It is well known that the destrobilation and later expulsion are characteristics of multiple Hymenolepis diminuta infections in rats. This process is suggested to be mediated by a variety of host cellular responses. It has also been suggested that immunoglobulin (Ig) E may have a beneficial role for some cestodes including H. diminuta. We examined the intestinal mast cell and serum IgE responses to a 10-H. diminuta infection in three different rat strains. Tapeworm infection induced no increased mast cell and IgE responses in F344 rats in which neither worm biomass nor worm burden decreased during 6 weeks of observation. The number of mast cells and amounts of serum rat mast cell protease (RMCP) II and IgE markedly increased from 3 weeks postinfection (p.i.) in BN rats. The worm biomass in BN rats was significantly lower than that in F344 rats, but worm burden was not different from that in F344 rats at 3 or 6 weeks p.i. In DA rats, the number of mast cells and levels of serum RMCP II and IgE increased at 6 weeks but not at 3 weeks p.i. Although numbers of mast cells and serum RMCP II and IgE levels were lower in DA rats than in BN rats, smaller and fewer worms were recovered in DA rats than in F344 and BN rats at from 3 and 6 weeks p.i. Worms were recovered from all of F344 and BN rats, while only 40% of DA rats harboured worms at 6 weeks p.i. These results suggested that the worm biomass was related to mast cell and IgE responses, but these responses were not required for worm expulsion during low dose H. diminuta infection in rats.

Transmammary transmission of Strongyloides ratti.

The rate of transmammary transmission of Stronglyloides ratti was examined in albino rats in terms of the route of subcutaneous (s.c.) migration from the infection site (the skin) to the cranium. Inoculation sites nearer the cranium resulted in less frequent transmammary infection. The maximum number of adult worms was recovered from the sucklings when the mother was inoculated in her hindquarter and sucklings were allowed to feed for 30-36 h after inoculation (AI). Few worms were recovered from sucklings when they were allowed to nurse during periods of less than 24 h AI or greater than 42 h AI. In lactating mothers, larval infection of the mammary glands was commonly observed, and these larvae showed an increased esophagus length. In nonlactating mothers, most larvae completed their migration to the cranium within 36 h AI.

Echinostoma paraensei and Schistosoma mansoni: adherence of unaltered or modified latex beads to hemocytes of the host snail Biomphalaria glabrata.

Hemocytes derived from a strain (13-16-R1) of Biomphalaria glabrata resistant to Schistosoma mansoni were significantly more likely to bind untreated latex beads than hemocytes from the schistosome-susceptible M line strain. Beads preincubated in 13-16-R1 plasma were more readily bound by both 13-16-R1 and M line hemocytes than beads preincubated in M line plasma. Beads preincubated in plasma derived from snails of either strain infected with the trematode Echinostoma paraensei were more readily bound by hemocytes than beads preincubated in plasma from control snails of the corresponding strain. Plasma from snails exposed to S. mansoni did not have a similar effect. Throughout these experiments, beads receiving a particular treatment were consistently bound at higher rates by 13-16-R1 than M line hemocytes. SDS-PAGE of plasma components eluted from beads revealed differences between treatments, particularly in diffuse bands falling into two groups, of 75-130 and 150-220 kDa. The results indicate that both hemocytes and plasma components from the two host strains differ and identify plasma molecules deserving of additional study as possible modulators of hemocyte effector functions. Also, S. mansoni and E. paraensei provoked different responses in the same host snail.

Suppression of gamma interferon transcription and production by nematode excretory-secretory antigen during polyclonal stimulation of rat lymph node T cells.

Although certain helminth infections preferentially induce type 2 T-cell responses, the immunological mechanisms responsible for type 2 T-cell polarization remain unclear. In the present study, we investigated the effects of excretory-secretory (ES) antigen from the nematode Nippostrongylus brasiliensis on cytokine production by mesenteric lymph node (MLN) cells isolated from naive rats. MLN cells produced considerable levels of gamma interferon (IFN-gamma) during a 72-h stimulation with concanavalin A (ConA) or with immobilized anti-CD3 plus soluble anti-CD28 antibodies (anti-CD3/CD28). With either stimulation, 10 microg of ES antigen per ml significantly suppressed IFN-gamma and interleukin-2 (IL-2) production without cytotoxic activity. The copresence of anti-IL-4, anti-IL-10, or transforming growth factor beta (TGF-beta) blocking antibodies did not alter the suppressive effect of ES antigen on IFN-gamma production. ES antigen did not affect IL-10 production. Kinetic studies of the effect of ES antigen indicated that the antigen suppressed even ongoing IFN-gamma production. Reverse transcription-PCR study showed that in the presence of ES antigen, IFN-gamma mRNA expression by MLN cells was suppressed 6 and 12 h after ConA or anti-CD3/CD28 stimulation. ES antigen also significantly suppressed IFN-gamma production by purified CD4(+) or CD8(+) T cells during anti-CD3/CD28 stimulation but did not affect IL-4 production by CD4(+) T cells. These findings suggested that the nematode antigen suppressed production of IFN-gamma and IL-2 but not IL-4 or IL-10 production. ES antigen-mediated suppression of IFN-gamma during the initiation of the immune response may provide a microenvironment that helps generation of type 2 T cells.

The presence of Angiostrongylus cantonensis in Viti Levu, Fiji.

Wild rats and molluscs were examined for Angiostrongylus cantonensis infection on Viti Levu, Fiji. A. cantonensis were recovered from 29.6% (16/54) of Rattus rattus and 59.5% (25/42) of R. exulans. A. cantonensis-like larval nematodes were found in all of four slugs, Laevicaulis alte, and ten of 20 unidentified land snails. The larvae developed to adult A. cantonensis in the pulmonary arteries of laboratory rats 40 to 42 days after ingestion. This is the first record of A. cantonensis in Fiji.

Immunocytochemical localization of secretory acetylcholinesterase of the parasitic nematode Nippostrongylus brasiliensis.

Various parasitic nematodes secrete acetylcholinesterase (AChE). In this study, the localization of AChE in the nematode Nippostrongylus brasiliensis and the secretory forms of AChE in culture fluid were examined. A thiocholine method revealed that AChE activity was localized in the subventral glands, which have a secretory and excretory function via a duct connected to the excretory pore. By electron microscopy, AChE activity was found mainly in the matrix of secretory granules, and sometimes in the Golgi apparatus in the subventral gland cells. These results show that nematode AChE is produced and stored in the subventral glands. Monoclonal antibodies against AChE of human erythrocytes or electric rays also bound to the nematode subventral gland, suggesting immuno-cross-reactivity of AChE among these species. When AChE activity in the nematode excretory-secretory product was examined by SDS polyacrylamide gel electrophoresis combined with the thiocholine method, intense activity was demonstrated as a single band at 74 kDa. Immunoblot analysis showed specific recognition of this molecule by IgE and IgG1 antibodies, but not by IgG2a antibody, in nematode-infected rat sera. These results indicate that the nematode AChE molecule produced in and secreted from the subventral glands is antigenic for the production of IgE/IgG1 in host animals.

Differentially enhanced cytokine gene expression in CD4+ and CD8+ T cells in mesenteric lymph nodes in rats infected with Nippostrongylus brasiliensis.

Autoinfective strongyloidiasis is potentially fatal, yet the majority of infected individuals harbour asymptomatic and chronic infections. The role of humoral responses in modulating autoinfection was assessed by examining antibody isotype responses to filariform larval antigens amongst chronically infected ex-Far East Prisoners of War (exFEPOWs) with longstanding (> 30 years) infection. Serum immunoglobulin (Ig)G1, IgG4, IgE and IgA responses to whole Strongyloides stercoralis L3 extracts and their constituent antigenic components were characterized by ELISA and quantitative immunoblotting. Comparison of two groups of S. stercoralis infected exFEPOWs with and without detectable larvae in stool demonstrated novel trends. Significantly enhanced recognition of six immunodominant antigenic components by IgA was associated with undetectable larval output, as was enhanced IgE recognition of several components. Additionally, IgE and IgG4 exhibited parallel antigen recognition patterns. These findings are consistent with roles for IgA in modulating larval output, for IgE in regulating autoinfection, and for IgG4 in blocking IgE-mediated responses in human strongyloidiasis.

Cytokine mRNA expression profiles in rats infected with the intestinal nematode Nippostrongylus brasiliensis.

Although the immune responses to intestinal nematode infection have been well studied and have been shown to be strongly driven by Th2-associated cytokines in mice, such information has been limited with respect to rats. We investigated changes in levels of the mRNAs encoding interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-10, and gamma interferon in the mesenteric lymph nodes of rats infected with Nippostrongylus brasiliensis by reverse transcription-PCR in comparison with immunoglobulin E (IgE)/IgG2a antibody, eosinophil, basophil, and mucosal mast cell responses. In the two rat strains used, Brown Norway and Fischer-344, which show different responses to allergens, serum IgE increased to much higher levels in the former than in the latter 2 weeks after infection. Intestinal mastocytosis was observed much earlier and more intensely in Brown Norway rats than in Fischer-344 rats, but the degrees of peripheral eosinophilia and basophilia did not differ between the two strains. In both strains, IL-3, IL-4, and IL-5 mRNA expression increased and peaked around 7 to 14 days after infection, while expression of IL-2, IL-10, and gamma interferon mRNAs did not change notably throughout the experimental period. The highest IL-4 mRNA expression was observed slightly earlier in Brown Norway than in Fischer-344 rats, but levels of IL-3 and IL-5 mRNAs peaked synchronously in both strains. The amounts of mRNAs encoding these three cytokines were always higher in Brown Norway than in Fischer-344 rats. It is suggested that in rats, Th2 or Th2-like cells are also induced after nematode infection, and IgE elevation is mainly related to increased IL-4 gene expression.

IgE antibody responses induced by transplantation of the nematode Nippostrongylus brasiliensis in rats: a possible role of nematode excretory-secretory product in IgE production.

In order to examine the effective site of sensitization for IgE responses, we transplanted 2000 adult-stage worms of the nematode Nippostrongylus brasiliensis into the duodenum or the peritoneal cavity of naive rats. Total serum IgE began to increase 1 week after the nematode inoculations and reached a peak at week 2. Living worms inoculated into the duodenum induced the highest serum IgE, this being 800 times the level in control animals. Intraperitoneal inoculations of living and dead worms resulted in increases of the serum IgE levels to 120 and 13 times the control level, respectively. The intraduodenal inoculation of living adult worms also induced a significant increase in specific IgE against the excretory-secretory (ES) product of adult N. brasiliensis 1 week later than the rise in total IgE, whereas intraperitoneal inoculations did not induce such an increase. These results suggest that sensitization through the intestinal mucosa with adult N. brasiliensis might be important for the effective induction of both specific and non-specific IgE responses. Since these findings also indicated that factors secreted by living worms play an important role in the induction of total IgE response, the ES product was injected to naive rats for 6 consecutive days (total 2.7-4.4 mg). Intraperitoneal injection of the ES product alone induced a 14.7-fold increase in total IgE without any specific IgE response. This indicates that some constituents of the ES product have the potential to trigger a non-specific IgE response.