Studies on in vitro IgE synthesis from lymph node cells of mice during infection with Nippostrongylus brasiliensis

The effect of serum factors on Ig synthesis (IgE, IgG) in vitro was analyzed. Spleen and mesenteric lymph node cells were obtained from Nippostrongylus brasiliensis-infected and non-infected mice. Sera and ammonium sulphate precipitated serum fractions from mice of different genetic origin (Balb/c - H-2^d, A.CA - H-2^f, B10.G - H-2^q) suppressed in vitro IgE synthesis whereas a pronounced enhancement of IgG antibody synthesis was obtained in several experiments. Our results obtained with sera from both high and low IgE responder strains demonstrated that no strain specificity exists as to the inhibitory efficacy of mouse sera for total IgE synthesis in vitro. The suppressive activity of the mouse sera was concentrated in a fraction precipitated with 20%–50% saturated ammonium sulphate. Amicon XM50 ultrafiltration suggested that this fraction had an apparent molecular weight >50,000 daltons. Suppressive activity was removed by immunoadsorption of the 20–50% fraction with anti-IgE Sepharose. After exogenous addition of monoclonal IgE to an inactive fraction in vitro neither the fraction enriched in IgE nor monoclonal IgE alone were able to suppress IgE synthesis in the culture. Our results suggest that one or more serum factors in the presence of IgE are responsible for the suppression of total IgE synthesis in vitro.Abbreviations BSA Bovine serum albumin - CFA Complete Freund's adjuvant - DNP Dinitrophenyl - EFA Enhancing factor of allergy - Fc R Fc-receptor for IgE - FCS Fetal calf serum - MLN Mesenteric lymph node - PBS Phosphate-buffered saline - PHA Phytohemagglutinin - PPD Purified protein derivative - SFA Suppressive factor of allergy     

T-cell-independent and T-cell-dependent IgE responses to the nematode Nippostrongylus brasiliensis: comparison of serum IgE and mast-cell-bound IgE.

The IgE immune response was studied in female athymic, nude (Lewis rnu/rnu) and euthymic (Lewis +/+) rats infected with the nematode Nippostrongylus brasiliensis. During the course of the infection, serum IgE levels were followed using an enzyme-linked immunosorbent assay technique (ELISA), while the surface expression and occupancy of IgE receptors on peritoneal mast cells were quantified using flow cytometry after immunolabelling with anti-IgE. The results show that the up-regulation of IgE receptors, which takes place on the mast cells of both athymic and normal rats during the early phase of the immune response, is more pronounced and longer-lasting in normal rats than in athymic ones, thereby suggesting that T cells are necessary for a full response to the parasite infection. The increased IgE occupancy observed on the mast cells during the early phase of the parasite immune response was not reflected in the serum IgE levels, which remained low during the entire infection period in athymic rats. In euthymic rats, on the other hand, there was a pronounced increase in serum IgE, as well as an increase in IgE occupancy on the mast cells, all reaching a peak level after 2 weeks of infection. However, there was no significant correlation between the serum IgE concentration and IgE occupancy or the density of IgE receptors on the mast cells of the individual euthymic rats. This indicates that the quantification of IgE occupancy on the mast cells may be a better way of detecting low-level IgE responses than the measurement of serum IgE. These findings, which were obtained in female Lewis rats, when compared with our previous findings in male rats of the same strain, suggest that sex differences may exist in terms of the intensity and duration of the IgE immune response to the parasite infection.     

IgE synthesis in vitro during infection of mice with the nematodeNippostrongylus brasiliensis: Effects of mitogens and antigens

In vitro IgE synthesis by lymphoid cells was studied during the course of infection of mice withNippostrongylus brasiliensis. The studies involved inbred strains of mice which had been shown to be high IgE responders (A.CA, B10.M), or non-responders (Balb/c, B10.D2) to parasite antigen. In addition, F₁ hybrids of low and high responders and irradiated non-responders were studied. Infection withN. brasiliensis led to an increase in IgE synthesis in vitro which was most pronounced during reinfection of mice. Addition of mitogens e.g. pokeweed mitogen (PWM), lipopolysaccharide (LPS), concanavalin A (ConA) to the cultures induced enhancement, suppression or had no effect on IgE synthesis. Addition ofN. brasiliensis homogenate or worm culture supernatant led to a fluctuating pattern of IgE synthesis. No correlation was found between lymphocyte proliferative response to mitogen and worm antigens and IgE synthesis. Our data suggest, that PWM is more likely to enhance IgE synthesis in vitro than LPS or ConA. An enhancement is more easily observed with the cells of non-infected animals or during the early phase of infection or reinfection. The mitogen-induced increase in IgE synthesis did not exceed the values obtained during infection or reinfection.     

Carrier effect by Nippostrongylus brasiliensis infection on rat antihapten IgE antibody response.

Nippostrongylus brasiliensis infection was found to be highly effective in inducing a carrier-specific enhancing effect on primary and secondary antihapten IgE antibody response in the rat, when animals were immunized i.p. with 10 mug of dinitrophenylated N. brasiliensis protein (DNP-Nb) plus 10 mg Al(OH)3 2 weeks after the infection. The carrier effect by the infection was much greater than that obtained by any other supplementary immunization with the carrier (Nb) plus adjuvant, so far examined, in terms of the IgE antibody response. The results, together with our previous observation in the mouse, provide an explanation for the reason why antiworm IgE antibodies are so easily detectable in helminth infections.     

Potentiated IgE response in Nippostrongylus brasiliensis infested rats--sites of synthesis and traffic of cells secreting potentiated antibody

The distribution and the kinetics of potentiated antibody synthesis have been studied at the cellular level in rats infested with Nippostrongylus brasiliensis using the homologous adoptive cutaneous anaphylaxis technique. In animals immunized in the hind footpads with alum-absorbed ovalbumin 10 days prior to infestation with the parasite, the major sites of potentiated anti-ovalbumin homocytotropic antibody synthesis were the regional lymph nodes of the gut and the lungs. Peyer's patches were weakly active late in the response and the spleen produced considerable amounts of potentiated antibody. The regional lymph nodes of the ovalbumin immunization sites were the only organs in which specific homocytotropic antibody synthesis was detected in uninfested control rats. The kinetics of synthesis of the potentiated antibody by cells correlated well with the levels of anti-ovalbumin IgE antibodies in the sera of the infested rats. A traffic of cells secreting anti-ovalbumin homocytotropic antibody was detected in the thoracic duct lymph, but not the mesenteric lymph of immunized uninfested rats. After infestation, the mesenteric lymph also contained cells secreting potentiated antibody. The mesenteric lymph is a major route by which IgE and potentiated IgE antibodies reach the circulation in infested rats. The possible mechanisms responsible for the effects of the parasite on antibody secretion in distant lymphoid organs are discussed.     

Adoptive transfer of total and parasite-specific IgE responses in rats infected with Nippostrongylus brasiliensis.

Infection of rats with Nippostrongylus brasiliensis has both a parasite-specific and non-specific IgE stimulating effect. Both these responses can be adoptively transferred with thoracic duct lymphocytes (TDL) from infected rats. The character of the IgE response in the recipient rats was related to the stage after infection of the cell donors. TDL from hyperimmune rats adoptively transferred high serum titres of parasite-specific IgE to infected recipient rats and substantially increased the levels of total IgE. However, adoptive immunization with TDL from donors infected 10 days previously did not stimulate parasite-specific IgE and only slightly increased total IgE levels. After cell fractionation the sIg- cells from day 10 TDL increased the level of total IgE but not parasite-specific IgE whereas sIg- cells from hyperimmune TDL did not induce any IgE response unless given with sIg+ cells. The possible reasons for this are discussed.     

Immunmechanismus der Ratte gegen Nippostrongylus brasiliensis in vitro

Zusammenfassung Nippostrongylus brasiliensis wurden 6 Tage p.i. aus dem Darm von Ratten steril gewonnen und in Kulturen zusammen mit Serum, Lymphozyten und Peritonealzellen von immunen oder nicht infizierten Tieren in verschiedenen Kombinationen gehalten. Die Kulturmedien wurden in dem Versuchszeitraum von 10 Tagen 2–3mal gewechselt bzw. Serum und Zellen hinzugefügt.Die Lymphozyten stammten aus dem peripheren Blut oder aus den Mesenteriallymphknoten, während die monozytären Zellen aus der Bauchhöhle der Tiere gewonnen wurden. Serum und Lymphozyten aus dem peripheren Blut weder von immunen, noch von nicht infizierten Ratten zeigten einen Einfluß auf die Überlebensrate der in vitro gehaltenen Nippostrongylus.In Kulturen mit Zusätzen von Lymphozyten und Peritonealzellen sensibilisierter Ratten traten deutlich die höchsten Absterberaten der adulten Würmer auf, wobei es keine Rolle spielte, ob Serum infizierter oder nicht infizierter Tiere den Kulturen beigesetzt war. In den Kontrollchargen ohne Zellzusätze lag die Überlebensrate der Parasiten teilweise bei 88%.

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Immune mechanism of rats on Nippostrongylus brasiliensis in vitroII. The influence of lymphocytes and peritoneal cells

Summary Nippostrongylus were collected from the intestines of rats 6 days p.i. and kept under sterile conditions in cultures. Serum, lymphocytes and peritoneal cells of immune or non-infected animals were added in various combinations to the culture media. The culture media were changed 2–3 times in an experimental period of 10 days, resp. serum and cells were added.The lymphocytes were isolated from the peripheral blood or from the mesenterial lymph nodes whereas the mononuclear cells were obtained from the peritoneal cavity. Serum and lymphocytes from the peripheral blood both from immune and non-infected rats, had no increased lethal effect on Nippostrongylus. The highest lethality rate of adults (65–68%) was achieved in cultures with peritoneal cells and lymphocytes from the lymph nodes of sensitized rats. Serum of infected or non-infected animals had no influence on adult Nippostrongylus in cultures with these cell combinations.In the controls without any cell-supplements the survival rate of the parasites was up to 88%.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Immunmechanismus der Ratte gegen Nippostrongylus brasiliensis in vitro

Zusammenfassung Für die Untersuchungen über den Einfluß von humoralen und sekretorischen Antikörpern sowie Zellzusätzen auf in vitro gehaltene Nippostrongylus brasiliensis verwendeten wir Sprague-Dawley-Ratten im Alter von mindestens 12 Wochen.Die für die in vitro-Haltung von N. brasiliensis verwendeten Nährmedien bestanden bei den Larvenkulturen aus je 25% Kükenembryoextrakt, Natriumcaseinat, Schweineleberextrakt und Rattenserum; bei den Adultenkulturen aus einem Gemisch von Yeast Extrakt (5%), Caseinat (15%), Krebs-Ringerlösung (30%) und Rattenserum (50%).Sekretorische Antikörper wurden aus der Spülflüssigkeit von Rattendärmen nach Überdruckfiltration (10–15 ), Reinigung über Sephadex G 15 und Einengung durch Kollodiumhülsen gewonnen. Die Mastzellen stammten aus dem Bauchhöhlenexsudat nach Separierung über Ficollzentrifugation.In den verschiedenen Versuchsserien mit Mediumzusätzen von Serum oder sekretorischen Antikörpern mehrmals infizierter immuner Ratten konnte weder bei den Larven noch Adulten eine unterschiedliche Wachstumsbeeinflussung oder erhöhte Absterberate gegenüber Kulturen mit Zusätzen nicht infizierter Tiere beobachtet werden.Mastzellen von sensibilisierten Ratten degranulieren innerhalb 14–22 Std zu 100% in Kulturen mit adulten N. brasiliensis. Eine unterschiedliche Beeinträchtigung der Überlebensrate von Nippostrongylus nach 10tägiger Bebrütung in Medien ohne oder mit Zellzusätzen sowie Seren immuner oder nicht infizierter Ratten war nicht zu beobachten.

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Immune mechanism of rats on Nippostrongylus brasiliensis in vitroI. The influence of humoral and secretory antibodies and of mast cells

Summary The influence of humoral and secretory antibodies as well as cell supplements on Nippostrongylus brasiliensis was tested in vitro. Adult Sprague-Dawley-rats approximately 12 weeks of age were used in these experiments.For the in vitro tests the following culture media were used: 25% chicken-embryo-extract, sodium casein, pig liver extract and rat serum for the larval stages. The medium for the cultures of adult N. brasiliensis consisted of 5% yeast extract, 15% casein, 30% Krebs-Ringer-solution and 50% rat serum.Secretory antibodies were isolated from the rinsing fluid of the rat intestines by high pressure filtration (10 to 15 micron), then cleaning of the fluid through a Sephadex G 15 column and finally narrowing down through collodene capsules. Mast cells were isolated from the peritoneal cavity by Ficoll-gradient-centrifugation.Various test series were conducted with the addition of serum or secretory antibodies of repeatedly infected and immune rats to the medium. In these tests there was never a difference in the influence on growth nor a higher mortality rate of larval or adult N. brasiliensis in contrast to cultures where serum and secretory antibodies of non-infected animals were used.A 100% degranulation of mast cells from infected rats occurred already within 14 to 22 hours in the cultures of adult N. brasiliensis. Variations were not noticed in the influence on the viability of N. brasiliensis kept in media for 10 days without or with cell supplements as well as sera of infected or not infected rats.The results from our experiments demonstrated that there was no variation in the influence on the development and a higher mortality rate of the larval stages and adult Nippostrongylus in media containing either sera and secretory antibodies of infected or not infected rats.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft     

Identification and characterization of a novel antigen from the nematode Nippostrongylus brasiliensis recognized by specific IgE

Identification and characterization of IgE-inducing antigens are important for elucidating the mechanisms involved in IgE-mediated immune responses in allergic diseases and parasite infections. While many allergens have been characterized, little is known about parasite antigens inducing specific IgE following infection. In order to identify antigens from the nematode Nippostrongylus brasiliensis, we generated an IgE-producing B cell hybridoma from N. brasiliensis-infected C57BL/6 mice and constructed a cDNA phage display library from N. brasiliensis. We successfully cloned and expressed an N. brasiliensis antigen (Nb-Ag1) that showed specific binding to anti-N. brasiliensis IgE. Nb-Ag1 localized to the pharynx of adult N. brasiliensis, suggesting that Nb-Ag1 is a potential pharyngeal gland antigen. Nb-Ag1-specific IgE could be detected in the serum of N. brasiliensis-infected mice, but only for a short time and only following a challenge infection. In contrast, local administration of Nb-Ag1 during primary, secondary and tertiary infections induced Nb-Ag1-specific IgE-mediated active cutaneous anaphylaxis. Therefore, amongst the high amounts of polyclonal total IgE, low levels of parasite-specific IgE responses are induced during primary helminth infections. Here, we show that even such low levels of parasite-specific IgE are sufficient to prime mast cells in vivo and mediate degranulation.     

Release of cholinesterase by adult Nippostrongylus brasiliensis in vitro

Summary Adult N. brasiliensis incubated in Tyrode's medium released between 3% and 11% of their initial total cholinesterase into the medium per hour; release of enzyme continued for at least 4 hours. Immune-damaged worms, which have much higher cholinesterase levels than normal adults, released 2 to 3 times more enzyme per unit wet weight than did the normal worms. There was no significant difference between the amounts of cholinesterase released per unit wet weight by male and female worms. Adult Nematospiroides dubius released about 1% of their total cholinesterase per hour; i. e. about 1/20 as much enzyme as that released by normal adult N. brasiliensis. These results are discussed with reference to possible functions of cholinesterase in the host/parasite relationship.     

Characterisation of the secretory acetylcholinesterases from adult Nippostrongylus brasiliensis.

The biochemical nature and relationship between the different isoforms of acetylcholinesterase (AChEs) secreted by adult Nippostrongylus brasiliensis was investigated, primarily via staining for enzyme activity and active-site labelling with [3H]-diisopropylfluorophosphate (DFP). Analysis by 1-dimensional SDS-PAGE under non-reducing conditions revealed the existence of 2 proteins of 74-kDa and 39-kDa, and each protein resolved as 2 species by isoelectric focusing. Both AChEs were co-purified via affinity chromatography on 9-[N beta-(epsilon-aminocaproyl)-beta-aminopropylamino]-acridine-coupled Sepharose 6B, and utilised to raise a polyclonal rabbit antiserum. Examination of the expression of secretory AChEs by adult worms during their residence in the gastrointestinal tract showed that the initial secretion of both forms on day 4 post-infection switched to predominant secretion of the 39-kDa protein by day 8. Immunoprecipitation of 35S-labelled products of in vitro translation via RNA from day 4 and day 8 worms predicted a single primary translation product of 59 kDa. These data suggested that the 'switching' event seen in vivo most likely corresponded to processing of the 74-kDa molecule. This interpretation was supported by limited digestion with V8 protease and chymotrypsin, which showed that the 74-kDa and the 39-kDa proteins possessed structural similarities.     

IgE antibody production in rats against multiple components of excretory-secretory products of the nematode Nippostrongylus brasiliensis.

Infection with the nematode Nippostrongylus brasiliensis (NB) induces the intense production of specific and non-specific immunoglobulin E (IgE) in rats. In the present study, we analysed NB-derived allergenic substances and the variability of IgE antibody production in response to these allergens in outbred Sprague-Dawley rats. Two kinds of crude allergens were used: the excretory-secretory products (ES) of adult NB, and an extract of homogenized adult worms (AW). ELISA showed that IgE antibody titres to ES were more than five times higher than the titres to AW. In the homologous passive cutaneous anaphylaxis (PCA) reaction using serum from infected rats, as little as 50 micrograms of ES had a maximal PCA activity, while even 1 mg of AW still gave a slightly lower PCA titre. Thus, it appeared that ES contained more allergen than AW at the same amount of total proteins. By immunoblot analysis, at least six components were recognized by IgE antibodies from infected animals, and these components were exactly the same in both ES and AW. The results indicated that the allergenic components in ES and AW were the same molecules, and that only those molecules which could be excreted or secreted from living worms were allergenic. Among the array of allergens, 130,000 and 70,000 molecular weight (mw) molecules were commonly recognized by IgE from all serum samples examined, while other components of the allergens were recognized variably by IgE antibodies from individual animals. These findings suggested that individual animals varied considerably in their IgE antibody production to the different constituents of the nematode allergens.     

Restricted sets of parasite antigens from the surface of different stages and sexes of the nematode parasite Nippostrongylus brasiliensis.

Surface molecules of parasitic stages of the nematode Nippostrongylus brasiliensis can be readily iodinated by the chloramine T technique, and assessed for antigenic reactivity with humoral antibody from infected animals. Free-living infective larvae are less amenable to analysis by this, or similar methods, but within 18 hr of larvae entering the host, new macromolecular surface antigens can be detected. The parasites change their surface antigens twice more in the course of the maturation to the adult stage. Surface antigens are stage-specific: lung larvae (L3), intestinal larvae (L4) and gut-living adults each possess characteristic sets of cuticular molecules. Single stage infections result in antibody reactive only to the antigens from the homologous stage. The adult surface appears to bear the greatest number of antigens, one of which is found only on the male worm. The composition of these antigens does not differ grossly between adult worms from a naive or immune host, or worms established after the adaptation of a 'trickle' (multiple low dose) infection. There appears to be an interesting contrast between the rapidity and extent of changes in surface antigens in the early phases of infection, and the stability of adult antigens analysed at different points in the host immune response.     

The production of IgE and IgGa antibodies in normal rats and rats infected with Nippostrongylus brasiliensis.

The time courses of production of IgE and IgGa homocytotropic antibodies were measured in Wistar rats during a primary and secondary response to egg albumin with pertussis or Freund's adjuvants. An anamnestic IgE antibody response occurred in animals previously sensitized to antigen with killed Bordetella pertussis as adjuvant. IgGa antibodies were formed in the primary response with Freund's complete adjuvant only, but were found during the secondary response with all adjuvants used. The time courses of formation of IgE and IgGa antibodies were very different during the secondary response. The production of both classes of antibody to egg albumin was studed in Wistar and Hooded Lister rats infected with Nippostrongylus brasiliensis. IgGa antibody formation was not potentiated by the infection. However, increased levels of IgE antibody, formed during a secondary response to antigen in infected animals, were consistently higher in both strains than during a primary response.     

Dissociation of specific and total IgE antibody responses following repeated low-level infections with Nippostrongylus brasiliensis in rats.

IgE, IgG and mast cell responses were studied in rats infected weekly with 10 larvae of Nippostrongylus brasiliensis (NB). Worm recovery at 8 weeks of repeated infections was six-fold greater than that of a single infection with 10 larvae, suggesting the accumulation of worms during the repeated infections. Total serum IgE was increased after 2 weeks of infection, and further increased after repeated infections: at 6 weeks of infection the level was four to six times higher than that after a single infection. Anti-NB IgG1 levels were also significantly higher after repeated infections than after a single infection. On the other hand, there was no significant difference in the level of anti-NB IgE between single and repeated infections, as determined by ELISA, as well as by passive cutaneous anaphylaxis (PCA) reaction. Mastocytosis was induced in the small intestine after both single and repeated infections, but the levels did not differ between the two. These results indicate that total IgE and specific IgG1 production are augmented by repeated helminth infections, but specific IgE and mast cell responses are not. This pattern of response may minimize the development of IgE-dependent hypersensitivity reactions with repeated helminth infections.     

Use of secondarily revised VH genes in IgE antibodies produced in mice infected with the nematode Nippostrongylus brasiliensis.

Although a high level of IgE is produced after primary infection with Nippostrongylus brasiliensis (Nb), most of the IgE antibodies (Abs) are not specific to the worm. Analyses with Western blotting and enzyme-linked immunosorbent assay (ELISA) revealed that the IgE Abs from Nb-infected BALB/c mice did not show reactivity with Nb-derived excretory-secretory proteins (NES) and antigens present in the cell-free extracts of the worm. Monoclonal IgE Abs obtained from the Nb-infected mice were not reactive with these Nb antigen either. To characterize Nb-induced IgE response, we used (QM x C57BL/6)F1 (QBF1) mice that bear the knock-in 17.2.25 VHDJH segment (VHT) encoding a VH region specific to 4-hydroxy-3-nitrophenylacetyl hapten, and express VHT-encoded antigen receptors on 80-85% of their B cells. Consistent with the frequency of VHT-positive B cells, more than 80% of IgE Abs induced in QBF1 B cells that were cultured with LPS plus IL-4 were found to bear VHT-encoded H chains. In contrast, when QBF1 mice were infected with Nb, less than 10% of Nb-induced IgE Abs were found to use VHT. The QBF1-derived IgE did not react with Nb antigens either. Taken together, data suggest that Nb-induced IgE response in mice is not merely the result of polyclonal activation of B cells, but may involve a mechanism that revises Ig genes secondarily.     

Low-level infection with the nematode Nippostrongylus brasiliensis induces significant and sustained specific and non-specific IgE antibody responses in rats.

Specific and non-specific IgE antibody responses were studied in SD rats infected with between 5 and 2500 Nippostrongylus brasiliensis (NB) larvae. In rats with 2500 NB larvae, specific IgE antibody, measured by enzyme-linked immunosorbent assay (ELISA) using NB excretory/secretory substance as antigen, reached a peak at 4 weeks of infection and gradually declined. On the other hand, in rats infected with 10 or 100 NB larvae, specific IgE was induced at 4 weeks of infection and the level continued to rise until at least 8 weeks after infection. The level at 8 weeks was significantly higher in rats infected with 10 or 100 larvae than in rats infected with 2500 larvae. The results indicate that the low-level infection induced a much more sustained specific IgE response than that induced after heavy infection. However, the level of specific IgG was correlated with the dose of infection, and reached a plateau 6 weeks after infection. Total serum IgE increased significantly even in rats infected with five larvae, a dose which did not induce detectable specific IgE. The kinetics of the production of total IgE was different in rats with light and heavy infections. In rats infected with five or 10 larvae, total IgE increased slowly and reached a plateau 4 weeks after infection. On the other hand, rats infected with more than 500 larvae showed a remarkable rise in total IgE at 2 weeks of infection; this rise gradually declined thereafter. Six weeks after infection, total IgE levels were almost the same (2-3 micrograms/ml) in rats infected with 10-2500 NB larvae. These results show that low-level NB infection induces a significant and sustained specific and non-specific IgE response in rats.     

Cysteine protease of the nematode Nippostrongylus brasiliensis preferentially evokes an IgE/IgG1 antibody response in rats.

Some cysteine proteases such as papain and those of mites and schistosomes have potent allergenic properties. To clarify the allergenicity of nematode cysteine proteases, the enzyme was purified from the intestinal nematode Nippostrongylus brasiliensis using cation exchange chromatography and gel filtration chromatography. The purified protease, of 16 kD and pI 8.5, showed maximum enzyme activity at pH 5.5 and substrate preference for Z-Phe-Arg-MCA. The specific inhibitors of cysteine protease leupeptin, iodoacetic acid, and E-64, completely suppressed the activity, indicating that the purified enzyme belongs to the cysteine protease family. Cysteine protease activity was found not only in somatic extract, but also in the excretory-secretory (ES) product of the nematode. When anti-cysteine protease immunoglobulin isotypes were examined in sera from rats infected with N. brasiliensis, a high level of IgG1 and a lower level of IgE antibody were detected. Depletion of IgG antibodies from the sera using protein G affinity columns resulted in a marked increase in reactivity of anti-cysteine protease IgE with the antigen, possibly due to the removal of competing IgG antibodies. In contrast to IgE and IgG1, production of anti-cysteine protease IgG2a was negligible. These results indicate that the nematode cysteine protease preferentially evokes an IgE/IgG1 antibody response.     

Reaginic antibodies and immunity to Nippostrongylus brasiliensis in the rat: II. Some properties of the antibodies and antigens

1. Passive transfer of immunity to Nippostrongylus brasiliensis with pooled antiserum from immune rats was neutralized in vivo by intravenous injection of small amounts of a saline extract of adult worms. This inhibition of protection was associated with systemic anaphylaxis and appeared to result from the neutralization of protective antibodies.

2. Serum from infected rats was fractionated by G-200 Sephadex gel-filtration. Reaginic antibodies were shown to be intermediate in molecular size between 7S and 19S globulins in sera from both singly and multiply infected animals. In immunoelectrophoresis they migrated with fast immunoglobulins but could not be related to either IgG or IgA rat immunoglobulins. The same serum fractions gave both homologous passive cutaneous anaphylaxis (PCA) and systemic anaphylaxis.

3. Blocking antibodies were found both in the 7S and 19S fractions after separation on G-200 Sephadex. These antibodies were found in sera from rats immunized with worm extracts as well as in sera from singly and multiply infected animals.

4. The saline extract of adult worms was fractionated on G-200 Sephadex. The isolated antigenic material (allergen) for both homologous PCA and systemic anaphylaxis seemed to be a protein with a molecular weight of approximately 12,000–17,000.