Anisakis simplex and Kudoa sp.: evaluation of specific antibodies in appendectomized patients

High prevalence and intensity of infection with anisakid larvae has been reported in commercially important fish in Spain. Likewise, Kudoa-infected fish have lately been detected in both fresh and frozen fish. In the present study the possible relation between appendectomy and specific antibodies to these fish parasites was investigated. One hundred and sixty patients were enrolled in this study. They were divided into two groups of eighty patients each and matched for sex and age: Group 1 (appendectomized) and Group 2 (control group). Total immunoglobulins (Ig’s), IgG, IgM, IgA and IgE against Anisakissimplex or Kudoa sp. antigens were analysed by ELISA. The mean values of the specific antibodies were lower in the appendectomy group, although significant differences were not observed in the case of IgG, IgA and IgE anti-A. simplex and IgE anti-Kudoa sp. In summary, appendectomy significantly decreased serum specific immunoglobulin levels against these food borne parasite antigens. This decrease was detectable from three months to three years post-appendectomy. It is necessary to study the influence of the surgical removal of other important parts of the GALT on these anti-parasite humoral immune responses.     

Trypanosoma cruzi: homocytotrophic antibody response in mice immunized with unrelated antigens.

The homocytotropic antibody response to unrelated antigens of mice with acute or chronic infection with Trypanosoma cruzi was studied. The production of IgG1 and IgE antibodies was observed in animals immunized with ovalbumin. The levels of IgG1 and IgE antibody were determined by passive cutaneous anaphylaxis. There was a depression in both IgG1 and IgE when infection and immunization were simultaneous. This depression was more intense when the animals were immunized 3 days after infection. A depression of IgG1 but not of IgE was observed in the animals with chronic infection and in the animals infected during the course of the humoral antibody response (12 days after immunization). It is suggested that a loss of T-cell regulatory mechanism may explain these results.     

Relationships between cell-mediated immunity and the IgE antibody response: II. Delayed hypersensitivity and antibody production to DNP-Ascaris conjugates

Rats of the W/F strain were immunized with DNP-Ascaris conjugates using complete Freund's adjuvant (CFA), Al(OH)₃ gel (alum), or B. pertussis vaccine as adjuvants. Cell-mediated immunity was assessed by lymphotoxin in vitro and by delayed hypersensitivity in vivo. IgE and IgG antibody determinations were made on serum pools obtained at various times during the primary and secondary responses. Although delayed hypersensitivity appeared earlier than lymphotoxin, these two parameters correlated during the primary but not during the secondary response. The discrepancies suggested that different cells may be responsible for these two phenomena. Antibody production was influenced by the adjuvant used. CFA led to IgG antibody responses to both hapten and carrier but not to IgE antibody production. The use of B. pertussis resulted in both IgE and IgG antibody production. In the case of alum, anti-hapten antibodies appeared during the primary response while anti-carrier antibodies of both IgE and IgG classes were detected after booster. The results indicated that cell-mediated immunity, IgE, and IgG antibodies appeared independently in an ordered, temporal sequence, and that these responses were not mutually exclusive but were under strong modulatory influences of the various adjuvants used.     

The prevalence and identity of Chlamydia-specific IgE in children with asthma and other chronic respiratory symptoms

Background

Recent studies have confirmed the presence of viable Chlamydia in the bronchoalveolar lavage (BAL) fluid of pediatric patients with airway hyperresponsiveness. While specific IgG and IgM responses to C. pneumoniae are well described, the response and potential contribution of Ag-specific IgE are not known. The current study sought to determine if infection with Chlamydia triggers the production of pathogen-specific IgE in children with chronic respiratory diseases which might contribute to inflammation and pathology.

Methods

We obtained BAL fluid and serum from pediatric respiratory disease patients who were generally unresponsive to corticosteroid treatment as well as sera from age-matched control patients who saw their doctor for wellness checkups. Chlamydia-specific IgE was isolated from BAL and serum samples and their specificity determined by Western blot techniques. The presence of Chlamydia was confirmed by species-specific PCR and BAL culture assays.

Results

Chlamydial DNA was detected in the BAL fluid of 134/197 (68%) patients. Total IgE increased with age until 15 years old and then decreased. Chlamydia-specific IgE was detected in the serum and/or BAL of 107/197 (54%) patients suffering from chronic respiratory disease, but in none of the 35 healthy control sera (p < 0.0001). Of the 74 BAL culture-positive patients, 68 (91.9%, p = 0.0001) tested positive for Chlamydia-specific IgE. Asthmatic patients had significantly higher IgE levels compared to non-asthmatics (p = 0.0001). Patients who were positive for Chlamydia DNA or culture had significantly higher levels of serum IgE compared to negative patients (p = 0.0071 and p = 0.0001 respectively). Only 6 chlamydial antigens induced Chlamydia-specific IgE and patients with C. pneumoniae-specific IgE had significantly greater levels of total IgE compared to C. pneumoniae-specific IgE negative ones (p = 0.0001).

Conclusions

IgE antibodies play a central role in allergic inflammation; therefore production of Chlamydia-specific IgE may prove significant in the exacerbation of chronic, allergic airway diseases, thus highlighting a direct role for Chlamydia in asthma pathogenesis.     

Variable Region Identical IgA and IgE to Cryptococcus neoformans Capsular Polysaccharide Manifest Specificity Differences

In recent years several groups have shown that isotype switching from IgM to IgG to IgA can affect the affinity and specificity of antibodies sharing identical variable (V) regions. However, whether the same applies to IgE is unknown. In this study we compared the fine specificity of V region-identical IgE and IgA to Cryptococcus neoformans capsular polysaccharide and found that these differed in specificity from each other. The IgE and IgA paratopes were probed by nuclear magnetic resonance spectroscopy with ¹⁵N-labeled peptide mimetics of cryptococcal polysaccharide antigen (Ag). IgE was found to cleave the peptide at a much faster rate than V region-identical IgG subclasses and IgA, consistent with an altered paratope. Both IgE and IgA were opsonic for C. neoformans and protected against infection in mice. In summary, V-region expression in the context of the ϵ constant (C) region results in specificity changes that are greater than observed for comparable IgG subclasses. These results raise the possibility that expression of certain V regions in the context of α and ϵ C regions affects their function and contributes to the special properties of those isotypes.     

A seroepidemiological survey for toxocariasis in apparently healthy residents in Gangwon-do,Korea

We investigated the sero-prevalence of toxocariasis among healthy Korean adults in 1999. A total of 314 sera from normal inhabitants in Whachon-gun, Gangwondo, Korea was examined for specific antibody levels against excretory-secretory products of second stage larvae of Toxocara (TES). The presence of cross-reactions with other helminthiases such as cysticercosis, paragonimiasis, sparganosis or clonorchiasis was also checked by specific IgG ELISA. Sera showing positive reaction against TES were also tested by IgG immunoblot and by IgE ELISA. Out of 314 subjects, 16 was found to be positive by TES IgG ELISA and immunoblot, among whom 12 were also positive by TES IgE ELISA. Among the 16 seropositive samples, two sera showed positive reaction against Paragonimus and sparganum antigen, respectively. These results inferred that cross-reactions were negligible between toxocariasis and other helminthiases. Toxocariasis seroprevalence among Korean rural adults was detected to be approximately 5%.     

Correlation between protective antibody response and patent infection with Hymenolepis nana in mice

Correlation between protective antibody response and patent infection with Hymenolepis nana in mice. International Journal for Parasitology16: 197–203. Mice inoculated with mouse-derived cysticercoids of Hymenolepis nana, as well as with eggs, produced IgG and IgE antibodies that were detected by double diffusion (DD) and passive cutaneous anaphylaxis (PCA), respectively. When mice inoculated with eggs (day 0) were challenged with eggs (day 66), all were resistant to the challenge (assessed by the failure of cysticercoid recovery in the intestinal tissue) and produced protective antibodies evidenced by passive transfer, as well as IgG and IgE isotypes. When mice inoculated with eggs (day 0) were treated with a highly efficacious cestocide, praziquantel on day 6 at the beginning of the lumen phase, all were also resistant to the egg challenge on day 66, however, IgE, IgG, and protective antibodies were not detected. When mice treated with praziquantel before patent infection were repeatedly challenged with high doses of eggs, some of them produced IgG and IgE antibodies. From these results, it is suggested that (1) the production of protective antibody is a secondary response after patency (which may be ascribed to eggs released from mature worms), and (2) mice initially given eggs are highly resistant to egg challenge showing that an effector mechanism of acquired resistance to egg challenge may be expressed without high titres of protective antibody, at least in the serum.     

Antigenemia and Specific IgM and IgG Antibody Responses in Rabbits Infected with Toxoplasma gondii

In this experiment, the correlation between antigenemia and specific antibody responses in Toxoplasma gondii-infected rabbits was assessed. We injected 1,000 T. gondii tachyzoites (RH) subcutaneously into 5 rabbits. Parasitemia, circulating antigens, and IgM and IgG antibody titers in blood were tested by ELISA and immunoblot. For detection of parasitemia, mice were injected with blood from rabbits infected with T. gondii and mice died between days 2 and 10 post-infection (PI). Circulating antigens were detected early on day 2 PI, and the titers increased from day 4 PI and peaked on day 12 PI. Anti-Toxoplasma IgM antibody titers increased on day 6 PI and peaked on days 14-16 PI. IgG was detected from day 10 PI, and the titers increased continuously during the experiment. The antigenic protein patterns differed during the infection period, and the number of bands increased with ongoing infection by the immunoblot analysis. These result indicated that Toxoplasma circulating antigens during acute toxoplasmosis are closely related to the presence of parasites in blood. Also, the circulating antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis.     

Detection of IgG and IgE antibodies to Aspergillus fumigatus in human sera by immunogold assay

An immunogold assay (IGA) was developed to detect IgG and IgE antibodies to Aspergillus fumigatus. Sixteen sera from patients with allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, and normal controls were studied. All sera were also evaluated for antibodies against A. fumigatus by biotin-avidin linked enzyme immunosorbent assay (BALISA) and by agar gel double diffusion method. A. fumigatus specific IgG and IgE antibodies could be detected by IGA in all the patients' sera but not in the sera of normal controls. Both IgG and IgE antibodies to A. fumigatus could be demonstrated in all the sera by BALISA and normal controls showed only low levels of these antibodies. There was a positive correlation between the degree of reactivity detected by IGA, the BALISA titer and the precipitins by agar gel diffusion. It can be concluded that IGA is a reliable, sensitive and simple method capable of detecting both IgG and IgE antibodies against A. fumigatus in patient serum.     

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to FcεRII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.     

Brugia pahangi: immunologic evaluation of the differential susceptibility of filarial infection in inbred Lewis rats

After inoculating inbred Lewis rats with infective larvae of Brugia pahangi, we found consistently that patent infection was established in only about two-thirds of experimental animals. To determine which aspects of the host response were responsible for this differential susceptibility to infection, we evaluated all animals with respect to blood leukocyte levels, antifilarial IgG and IgE antibody production, and specific lymphocyte responses to mitogens and filarial antigens during the first 50 weeks of infection. In the critical “prepatent” period of infection, each of these responses developed in an essentially identical fashion in the infected and resistant groups except for the production of specific IgE antibodies. While those animals destined to resist infection developed specific IgE antibodies between Weeks 5 and 8, none of the animals that became microfilaremic showed a similar IgE response. These findings suggest that the development of specific IgE antibodies played a role in protecting the animals from acquiring filarial infection.     

Trypanosoma cruzi: immunoglobulin isotype profiles during the acute phase of canine experimental infection with metacyclic or blood trypomastigotes

A detailed investigation has been carried out about the serological profiles of groups of dogs experimentally infected with metacyclic (MT) or blood (BT) trypomastigotes of Berenice-78 Trypanosoma cruzi strain. Peripheral blood was collected from infected dogs and uninfected controls, weekly during 35 days following the acute phase of infection, and immunoglobulin profiles were determined by ELISA. Dogs infected with BT exhibited unaltered levels of IgG2, increases in IgM, IgE, IgA, IgG and IgG1. In contrast, dogs infected with MT presented unaltered levels of IgE and IgG1 and an increase in IgM, IgA, IgG and IgG2 levels. Compared with the MT group, animals infected with BT showed significant increases in IgM on days 7, 14 and 28, in IgA on days 7, 14 and 21, in IgE on days 7 and 14, in IgG on days 14 and 28, and in IgG1 on days 7, 14 and 21. Parasitemia levels of the infected animals were measured over the same time period. No correlations were found between the immunoglobulin profiles and the parasitemia levels. The results demonstrated that the inoculum source (BT or MT) influence the immunoglobulin isotype profile that may drive distinct outcome of acute canine Chagas disease.     

Fasciola hepatica: IgG and IgA levels in the serum and bile of infected cattle

Bile and serum samples were collected from calves with an implanted cannula throughout a 20-week period of infection with Fasciola hepatica. Using indirect fluorescent antibody labelling and plastic-embedded sections of juvenile and adult flukes as antigens, estimates were made of the relative concentrations of IgG and IgA specific for fluke tegumental and gut antigens in the samples of serum and bile. In serum, antibodies against juvenile (t1) tegument and gut antigens reached peak concentrations 4–6 weeks postinfection and declined slowly thereafter as flukes became established in the bile ducts. IgG against adult tegument (t2) antigens appeared in the serum 6 weeks after infection, but no IgA against t2 was detected. In the bile, both IgG and IgA titres against t1 and gut antigens rose to peak values at 4–6 weeks after infection, but there was no activity against t2 antigen. The Ig levels in bile were considerably lower than in serum. Much more IgA relative to IgG occurred in bile as compared to serum (IgG/IgA ratio in serum was 16–32, in bile 1–2) suggesting a role for IgA in defence at mucosal surfaces. Comparison of the antibody profiles in bile and serum suggested that IgG in the bile was derived from circulating IgG whereas IgA may have been preferentially concentrated in the bile.     

Immunological follow-up of hydatid cyst cases

Hydatid disease is caused by Echinococcus granulosus. In this study, we aimed to investigate the benefit of monitoring cases with hydatid cyst by means of immune components in patients in a long-term follow-up after surgery. Eighty-four preoperative and postoperative serum samples from 14 cases undergoing surgery for hydatid disease were evaluated in terms of immune parameters, such as total and specific IgE, IgG, IgM, IgA and complement. Total and specific IgE were determined by ELISA. Specific IgG levels were measured by indirect hemagglutination.Total IgG, IgM, IgA and complement (C3 and C4) were detected by nephelometry. Imaging studies were also carried out during the follow-up. In none of the patients hydatid cysts were detected during the follow-up. Total IgE levels in the sera of the patients decreased to normal six months after surgery. Although specific IgE against echinococcal antigens decreased one year after operation, levels were still significantly high. There were no changes in the levels of anti-Echinococcus IgG and total IgG in follow-up period. Additionally, other parameters, such as IgA, IgM, C3 and C4, were not affected.     

Immune responses in Indonesian thin tail and Merino sheep during a primary infection with Fasciola gigantica: lack of a specific IgG2 antibody response is associated with increased resistance to infection in Indonesian sheep.

After a primary infection with Fasciola gigantica, the immune responses in a resistant (Indonesian thin tail) and a susceptible (Merino) breed of sheep were analysed. The number of adult flukes recovered from the livers of the Indonesian thin tail sheep were significantly lower than those found in the Merino animals. On days 8, 14 and 25 p.i., Indonesian thin tail sheep exhibited a significantly higher eosinophilia than Merino sheep, whereas neutrophilia was significantly elevated in the Indonesian thin tail sheep on days 36 and 48 p.i. Serum from both sheep breeds demonstrated IgM, IgG1 and IgE responses to F. gigantica. In contrast, the Indonesian thin tail sheep produced significantly lower levels of IgG2 antibodies relative to the high level detected in Merino sheep. The IgE response was biphasic in both sheep breeds with the first response detected by day 14 and the second response developing from days 30 to 60 p.i. Western blotting showed that a similar profile of adult fluke antigens was recognised by IgG1 and IgE antibodies in both the Indonesian thin tail and Merino sheep. The IgE response was directed to a major antigen at about 92 kDa. We postulate that IgG2 could act as a blocking antibody for protective effector responses against F. gigantica in sheep and that the Indonesian thin tail sheep, by downregulating IgG2 responses, have an enhanced capacity for killing F. gigantica in vivo.     

Prevalence of Toxoplasma gondii in Dogs in Zhanjiang,Southern China

Toxoplasmosis, caused by Toxoplasma gondii, is a parasitic zoonosis with worldwide distribution. The present study investigated the prevalence of T. gondii in dogs in Zhanjiang city, southern China, using both serological and molecular detection. A total of 364 serum samples and 432 liver tissue samples were collected from the slaughter house between December 2012 and January 2013 and were examined for T. gondii IgG antibody by ELISA and T. gondii DNA by semi-nested PCR based on B1 gene, respectively. The overall seroprevalence of T. gondii IgG antibody was 51.9%, and T. gondii DNA was detected in 37 of 432 (8.6%) liver tissue samples. These positive DNA samples were analyzed by PCR-RFLP at 3''- and 5''-SAG2. Only 8 samples gave the PCR-RFLP data, and they were all classified as type I, which may suggest that the T. gondii isolates from dogs in Zhanjiang city may represent type I or type I variant. This study revealed the high prevalence of T. gondii infection in dogs in Zhanjiang city, southern China. Integrated measures should be taken to prevent and control toxoplasmosis in dogs in this area for public health concern.     

Control of IgE and IgG1 antibody production in mice.

The production of IgE and IgG1 was studied in untreated, thymectomized. splenectomized, anti-thymocyte serum-treated, or sublethally X-irradiated mice. Dinitrophenyl Ascaris and ovalbumin were used as antigens, and aluminum hydroxide was used as adjuvant. A suppression of IgE production was observed in adult thymectomized mice, although the kinetic pattern of the antibody response was the same as in control animals. IgG1 antibody production was not affected by thymectomy. Splenectomy did not change either IgE or IgG1 production. A single dose of rabbit anti-thymocyte serum (ATS) given 8 days after immunization inhibited IgE antibody production. The effect of ATS was dose dependent and also varied with the amount of antigen used, the immune response to high doses being more susceptible to the effect of ATS. No alteration in IgG1 production was caused by ATS even when IgE antibody formation was completely inhibited. When preceding immunization, sublethal irradiation enhanced IgE antibody formation and partially suppressed IgG1 production; applied after immunization, irradiation caused an enhancement of IgE production which was inversely proportional to the interval elapsed between the two procedures. On the other hand, the IgG1 antibody production was fairly resistant to the same treatment. The results suggest a clearcut separation between the mechanisms regulating IgE and IgG1 production in mice.     

Production and properties of reaginic antibodies in rabbits infected with Clonorchis sinensis or Schistosoma japonicum

The production of reaginic antibodies detected by homologous passive cutaneous anaphylaxis (PCA) was demonstrated in all rabbits experimentally infected with either Clonorchis sinensis or Schistosoma japonicum. The antibodies appeared in the sera as early as 3 weeks after exposure and persisted with relatively high titers for at least 7 weeks in some animals. The antisera of rabbits infected with C. sinensis were found to be cross reactive against heterologous trematode antigens, although PCA titers were less than 3% of the titer by the homologous antigen; no cross reaction was observed between S. japonicum antiserum and the heterologous antigens. PCA activity of the antisera was completely destroyed in some samples by heat treatment at 56 C for 2 hr, but partially in the others even after heating for 6 hr. However, the physicochemical properties of these antibodies were analogous to human IgE; the PCA activity was eluted with 0.035 M phosphate buffer from a DEAE-cellulose column and recovered in the ascending portion of the IgG peak by Sephadex G-200 gel filtration. PCA activity was found in a β region in preparative agar electrophoresis.     

A human IgE bispecific antibody shows potent cytotoxic capacity mediated by monocytes

The generation of bispecific antibodies (bsAbs) targeting two different antigens opens a new level of specificity and, compared to mAbs, improved clinical efficacy in cancer therapy. Currently, the different strategies for development of bsAbs primarily focus on IgG isotypes. Nevertheless, in comparison to IgG isotypes, IgE has been shown to offer superior tumor control in preclinical models. Therefore, in order to combine the promising potential of IgE molecules with increased target selectivity of bsAbs, we developed dual tumor-associated antigen-targeting bispecific human IgE antibodies. As proof of principle, we used two different pairing approaches - knobs-into-holes and leucine zipper–mediated pairing. Our data show that both strategies were highly efficient in driving bispecific IgE formation, with no undesired pairings observed. Bispecific IgE antibodies also showed a dose-dependent binding to their target antigens, and cell bridging experiments demonstrated simultaneous binding of two different antigens. As antibodies mediate a major part of their effector functions through interaction with Fc receptors (FcRs) expressed on immune cells, we confirmed FcεR binding by inducing in vitro mast cell degranulation and demonstrating in vitro and in vivo monocyte-mediated cytotoxicity against target antigen-expressing Chinese hamster ovary cells. Moreover, we demonstrated that the IgE bsAb construct was significantly more efficient in mediating antibody-dependent cell toxicity than its IgG1 counterpart. In conclusion, we describe the successful development of first bispecific IgE antibodies with superior antibody-dependent cell toxicity–mediated cell killing in comparison to IgG bispecific antibodies. These findings highlight the relevance of IgE-based bispecific antibodies for clinical application.