Idiotypic replica of a Toxocara canis excretory/secretory antigen epitope.

This study describes the production, characterization and use of an anti-idiotype serum raised against the monoclonal antibody TC-1 which recognizes a T. canis excretory/secretory antigen (ES Ag) epitope. Anti-idiotypic (anti-Id or Ab2) antibodies were produced in rabbits using TC-1 F(ab')2 fragments; these anti-Id inhibited ES Ag binding to biotinylated TC-1, and also inhibited a larval microprecipitation assay using TC-1. Assays show that the Ab2 beta or "internal image" of a T. canis ES Ag epitope was obtained. The antibodies have been used as an idiotypic copy of ES Ag in a diagnostic ELISA for murine toxocariosis. Affinity-purified anti-Id antibodies were used to raise a homologous anti-anti-Id (Ab3) response in rabbits. Antibody formation was followed in the sera of BALB/c mice inoculated with embryonated eggs of T. canis during a 12-month infestation. A 3-week latency period was observed before specific anti-TC-1 epitope antibodies were detected. High levels were reached at 7 weeks post-inoculation with a maximum at the ninth month, and were then maintained until the end of the experiment. The results show the possible utility of anti-Id antibodies as an ES Ag molecular replica.

Evaluation by larval recovery of mebendazole activity in experimental murine toxocariasis.

We have studied the action of mebendazole on experimental murine toxocariasis using different formulations and vehicles. The treatment efficacy was evaluated by larval recovery after digesting several tissues. In the first part of the experiment, the drugs were administered on days 1-3 post infection (p.i.) inclusive (hepato-pulmonary phase of the migration), then the animals were sacrificed by the 1st week p.i. In the second part of the experimental protocol, the treatments were administered on days 4-6 p.i. inclusive (myotropic-neurotropic phase of the migration), with sacrifice of the animals by the 7th week p.i. The 2nd pattern of treatment reduced more effectively the total larval recoveries compared with control animals. However, the larval distribution by organs was scarcely affected. The first pattern of treatment reduced less effectively the total number of larvae, but it significantly inhibited migration.

Cross-reactions of sera from Toxocara canis-infected mice with Toxascaris leonina and Ascaris suum antigens.

The ELISA method using larval excretory-secretory (E/S) products and homogenized Toxocara canis, Toxascaris leonina and Ascaris suum adult worm extract were used to determine possible cross-reactions in BALB/c and C57BL/10 mice, inoculated with embryonated eggs or adult worm extract of T. canis in single and multiple doses. When we used sera of mice infected with embryonated eggs of T. canis against different heterologous antigens, we observed no cross-reactions in BALB/c mice against A. suum E/S and adult worm extract antigens with a single dose. In multiple doses this was absent too against T. leonina adult worm extract in BALB/c mice, and in both strains against A. suum E/S and adult worm extract. In BALB/c mice inoculated with adult worm extract of T. canis we did not observe cross-reactions with A. suum E/S antigen with both inoculation doses. In the remainder of the experiments, we observed cross-reactions of different intensities.

Cross-reactions of sera from Toxascaris leonina and Ascaris suum infected mice with Toxocara canis, Toxascaris leonina and Ascaris suum antigens.

The ELISA method using larval ES products and homogenized Toxocara canis, Toxascaris leonina and Ascaris suum adult worms extract, was used to determine the possible cross-reactions in BALB/c and C57BL/10 mice inoculated with embryonated eggs or adult worms extract of T. leonina of T. leonina or A. suum in single and multiple doses. When we used sera of mice infected with embryonated eggs of T. leonina against different heterologous Ag, no cross-reactions against T. canis ES and A. suum ES Ag were observed using a single dose. Similarly, in multiple doses no response against T. canis ES Ag was observed. In mice inoculated with adult worms extract of T. leonina cross-reactions with T. canis ES and A. suum ES Ag did not occur. Sera from BALB/c mice infected with embryonated eggs of A. suum, was tested using ES Ag from both A. suum and T. canis and no reactions were observed. This fact confirmed the resistance of this murine strain to A. suum embryonated eggs. When we used sera of susceptible C57BL/10 mice infected against different heterologous Ag, we observed no cross-reactions against T. canis ES Ag. In the case of both BALB/c and C57BL/10 and C57BL/10 mice immunized with a single dose of A. suum adult crude extract no cross-reactions were seen against ES T. canis Ag and with sera from C57BL/10 mice against ES T. leonina. These facts confirmed the specificity of the ES T. canis Ag.

Persistence of immune response in human toxocariasis as measured by ELISA.

The antibody titer was followed in a group of patients, clinically diagnosed with toxocariasis, during a 5 year period. We observed that larvae can survive for at least 5 years in humans. Antigenic stimulation was enough to keep high levels of immunoglobulins over this period. Antibody levels decreased slowly and this pattern is similar to that shown by animal models.

Bilateral ocular toxocariasis demonstrated by aqueous humor enzyme-linked immunosorbent assay.

PURPOSE/METHODS: A 14-year-old girl with a history of contact with puppies and chronic bilateral panuveitis was examined. The fundus of the right eye showed an inferotemporal pars plana exudate. The fundus of the left eye showed an elevated posterior mass. RESULTS/CONCLUSION: Toxocara antibodies in the aqueous humor detected by enzyme-linked immunosorbent assay had a Goldmann-Witmer coefficient of 8.63 in the right eye and 8.94 in the left eye. Toxocariasis may be a cause of bilateral panuveitis.

Cross-reactivity between Anisakis simplex sensitization and visceral larva migrans by Toxocara canis.

The aim of this work was to study cross-reactivity in the diagnosis of two related ascaridosis. Nineteen patients diagnosed with recidivous acute urticaria (RAU) caused by Anisakis simplex and 26 patients diagnosed with visceral larva migrans (VLM) caused by Toxocara canis were studied employing commercial diagnostic kits and "in house" assay kits. Cross-reactivity observed was greater when using "in house" assay kits, suggesting that T. canis excretory-secretory antigens were not only recognized by antibodies from patients with RAU but with greater intensity compared to the A. simplex excretory-secretory antigens.

N3-arylspiroimidazolidine-2,4-diones N3-arylspiroimidazolidine-2-thio-4-ones and 4-hydroxy derivatives. Synthesis and anthelmintic activity.

The synthesis of new N3-arylciclohexanespiroimidazolidine-2,4-diones, N3-arylciclohexanespiroimidazolidine-2-tio-4-ones and the 4-hydroxy derivatives is described and their structures discussed on the basis of I.R. and 1H-N.R.M. data. The anthelmintic activity of these compounds was tested.

[Cryoglobulinemia following liver transplantation for cirrhosis secondary to hepatitis C virus infection]. / Crioglobulinemia tras trasplante hepático por cirrosis secundaria al virus de la hepatitis C.

Hepatitis C virus (HCV) infection has been linked with some extrahepatic immunologic abnormalities. Cryoglobulinemia is one of the most frequently reported. Nevertheless, there are only a few reports of cryoglobulinemia in the setting of liver transplantation. More studies are needed to clarify the frequency of post-OLT cryoglobulinemia in patients with HCV-related cirrhosis and its impact on OLT outcome. A case of a patient who underwent liver transplantation because of HCV end-stage liver disease and in whom cryoglobulinemia appeared 3 years after transplantation is reported. Treatment with cyclophosphamide and steroids was attempted but patient died of septicemia 3 years after liver transplantation.

Larval distribution of Toxocara canis in BALB/c mice at nine weeks and one year post-inoculation.

The migratory route of Toxocara canis in the mouse includes two phases: a hepato-pulmonary phase and a myotropic-neurotropic phase. A study was made of the migratory behaviour of larvae one year post-inoculation (p.i.) comparing the results to those obtained at the end of an initial experiment (day 63 p.i). The larval distribution per organ was already definitive at 9 weeks p.i. The total parasitic load showed a reduction when the experiment was prolonged for one year.

Comparative study of assays detecting circulating immune complexes and specific antibodies in patients infected with Toxocara canis.

A sandwich ELISA method using previously described E/S antigen-specific monoclonal antibodies has been developed to detect circulating immune complexes in patients infected with Toxocara canis. This technique could be used for the study of the dynamics of the parasite-host relationship, as we believe the detection of immune complexes and/or soluble antigen to be an improvement over detection of antibodies only. In this parasitosis, antibodies may be present in residual levels for prolonged periods after active infection.

Excretory/secretory antigen of Toxocara canis: recognition profiles of polyclonal and larvicidal monoclonal antibodies.

Five monoclonal antibodies against the excretory/secretory (E/S) antigen of Toxocara canis were obtained and characterized. Immunoprecipitating activity was demonstrated in an in-vitro microprecipitating assay using live T. canis larvae. Their capacity to kill larvae was also shown in an in-vitro assay. Two zones of reactivity were observed in 7.5 and 12.5% SDS-PAGE (177-77 kD, 43-15 kD) of immunoprecipitates of human and mouse positive polyclonal antisera. The murine monoclonal antibodies showed a common pattern of reactivity with the proteins in the 177-77 kD range.

Serological evidence of toxocariasis in patients from Spain with a clinical suspicion of visceral larva migrans.

Sera from patients with clinical characteristics of toxocariasis were assayed using the ELISA method and larval excretory-secretory antigen. Four hundred and seven samples of Toxocara serology were received at the laboratory of Ciudad Sanitaria Juan Canalejo Hospital of Corunna, Spain, from 1984 to 1989. Of these, 30 were from adults, 332 from children and 45 from patients of unknown age, resulting in Toxocara seroprevalences of 23.3%, 32.8% and 17.7% respectively. The reasons for these serological differences in the rural and urban areas of Galicia, Spain are discussed.

Seroprevalence of toxocariasis in children and adults in Madrid and Tenerife, Spain.

A study on the seroprevalence of toxocariasis, using ELISA with Toxocara larval excretory-secretory antigens, was carried out on human populations in two regions of Spain. Sera from a population of 195 children from Madrid and 143 children from Santa Cruz de Tenerife (Canary Isles), showed a prevalence of 0% and 4.2% respectively. Sera from a population of 272 adults from Madrid and 803 adults from Santa Cruz de Tenerife showed a prevalence of 3.6% and 17.4%. Reasons for these differences in the seroprevalence of Toxocara in the different age groups from the two regions are discussed.

Evaluation of chemotherapy in experimental toxocarosis by determination of specific immune complexes.

Parasitism by the larval phase of Toxocara canis is a chronic process in which the larvae survive in the tissues, resulting in the constant stimulation of the immune system. As a result, the detection of specific antibodies may not reflect the active state of the parasite. We have studied the dynamics of the production of specific immune complexes by ELISA with the monoclonal antibody TC-1 in rabbits inoculated with single and multiple doses of T. canis eggs. We also compared this with the production of specific antibodies and their possible modification after treatment with mebendazole. The specific antibodies against excretory-secretory antigen were detected with peaks at 10 and 12 weeks depending on the dose and remained positive during the entire experiment (62 weeks). Treatment caused an increase in the level of detectable antibodies dropping to similar levels to the controls. Specific immune complexes were detected only in multiple doses, and were then positive during the entire experiment. From the beginning of treatment the values of immune complexes fell quickly, remaining at undetectable levels during the rest of the experiment. For this reason the detection of specific immune complexes is a valid technique for monitoring the efficiency of treatment.

Cross-reactivity induced by Anisakis simplex and Toxocara canis in mice.

The aim of this study was to verify whether cross-reactivity appeared between Toxocara canis and Anisakis simplex in an experimental rodent model. No cross-reactions were detected using sera from mice infected with T. canis eggs. When responses obtained against T. canis ES antigen using sera from BALB/c and C57BL/10 mice infected with T. canis eggs were compared with those obtained by testing sera from mice infected with one A. simplex L3, an increase in cross-reactions was observed using the C57BL/10 strain.

Evaluation of mebendazole activity on experimental murine toxocariasis by immune complexes determination.

The mebendazole action on experimental murine toxocariasis, using different formulations and vehicles, was studied by means of the detection of antibodies and immune complexes by ELISA. After inoculation with 1000 embryonated Toxocara canis eggs, BALB/c mice were submitted to the anthelmintic treatment as follows: group 1 (control without treatment); group 2 (mebendazole (MBZ), Lomper, Steve Laboratories, Barcelona, Spain, suspended in 1% sodium carboxymethylcellulose (CMC) at a dose of 100 mg/kg/day); group 3 (MBZ, pure compound suspended in 1% CMC at a dose of 100 mg/kg/day); group 4 (MBZ, pure compound suspended in water at a dose of 100 mg/kg/day); group 5 (MBZ, pure compound, formulated to a solid dispersion at 10% in polyethylene glycol 6000 (PEG) then dissolved in water at a dose of 100 mg/kg/day); group 6 (MBZ, pure compound, formulated to a solid dispersion at 10% in PEG then dissolved in water at a dose of 50 mg/kg/day); group 7 (MBZ, pure compound, formulated to a solid dispersion at 10% in PEG then dissolved in water at a dose of 25 mg/kg/day). The treatments were administered on days 5-7 post-inoculation (p.i.) inclusive. The dynamics of the production of the specific antibodies for both excretory-secretory (ES) antigen or crude extract showed a similar profile as compared to the control group. In groups 2 and 6, from the beginning of the treatment, values of immune complexes fell rapidly and were undetectable for the remainder of the experiment. Reductions of immune complex levels by the 4th-6th, 2nd-3rd and 2nd-5th weeks p.i. were observed from groups 3, 5 and 7, respectively. In the other groups, similar profiles as compared to the control group were observed in the dynamics of the specific immune complexes. The evaluation of chemotherapy by immunological methods is a valid technique for the efficiency of the treatment without the disadvantages of larval recovery from several digested tissues. Mebendazole, pure compound, formulated to a solid dispersion in PEG, then dissolved in water reduced immune complexes from the beginning of the treatment. The larval immobilization produced by MBZ should entail a reduction in their metabolic activity with a reduction in the production of excretory-secretory substances which are responsible for the formation of immune complexes. The rapid clearance of specific immune complexes together with a total larvae reduction would explain the decrease in specific immune complexes, which detection is a valid technique for monitoring the efficiency of treatment.