Secreted products of Fasciola hepatica inhibit the induction of T cell responses that mediate allergy.

There is evidence from epidemiology studies of a negative association between infection with helminth parasites and the development of allergy and asthma. Here, we demonstrate that the excretory/secretory products of the helminth Fasciola hepatica (FHES) protected mice against ovalbumin (OVA)-induced allergic asthma when administered at time of allergen sensitization. FHES reduced the accumulation of mucus, eosinophils and lymphocytes into the airways of allergen-challenged mice. Furthermore, FHES treatment suppressed Th2 responses in the airways. Interestingly, systemic administration of FHES at allergen challenge had no effect on airway inflammation, demonstrating that alum-induced Th2 response is set following initial allergen sensitization. Our findings highlight the immunomodulatory potential of molecules secreted by F. hepatica.

A critical role for the TLR signaling adapter Mal in alveolar macrophage-mediated protection against Bordetella pertussis.

Bordetella pertussis causes whooping cough, an infectious disease of the respiratory tract that is re-emerging despite high vaccine coverage. Here we examined the role of Toll-like receptor (TLR) adapter protein Mal in the control of B. pertussis infection in the lungs. We found that B. pertussis bacterial load in the lungs of Mal-defective (Mal(-/-)) mice exceeded that of wild-type (WT) mice by up to 100-fold and bacteria disseminated to the liver in Mal(-/-) mice and 50% of these mice died from the infection. Macrophages from Mal(-/-) mice were defective in an early burst of pro-inflammatory cytokine production and in their ability to kill or constrain intracellular growth of B. pertussis. Importantly, the B. pertussis bacterial load in the lungs inversely correlated with the number of alveolar macrophages. Despite the maintenance and expansion of other cell populations, alveolar macrophages were completely depleted from the lungs of infected Mal(-/-) mice, but not from infected WT mice. Our findings define for the first time a role for a microbial pattern-recognition pathway in the survival of alveolar macrophages and uncover a mechanism of macrophage-mediated immunity to B. pertussis in which Mal controls intracellular survival and dissemination of bacteria from the lungs.

Inhibition of human monocyte function by prophylactic doses of chloroquine.

The effect of prophylactic doses of chloroquine on the phagocytic function of human monocytes was studied in young healthy male volunteers. They received placebo, 300, or 600 mg of chloroquine base/week for six weeks. In each subject, the phagocytic function was tested before and at the end of the chloroquine intake period. In the 600-mg chloroquine group, it was also tested six weeks after receiving the last dose. Chloroquine at both doses inhibited the phagocytosis of IgG-coated sheep red blood cells and of zymosan particles. The effect was more pronounced with the 600 mg dose of chloroquine. The phagocytic activity returned to normal values six weeks after the end of treatment.

Immunodiagnosis of human fascioliasis by enzyme-linked immunosorbent assay using excretory-secretory products.

In sera from patients with fascioliasis the enzyme-linked immunosorbent assay (ELISA) was used to detect antibody using excretory-secretory products (ES) from Fasciola hepatica adult worms. The specificity of ES-ELISA (with OD values greater than 0.38) allowed the differentiation among fascioliasis, schistosomiasis, clonorchiasis, and other human parasite infections.

Detection of specific anti-giardia serum antibody by an immunofluorescence test in children with clinical giardiasis.

In 50 of 100 children with clinical giardiasis studied, the presence of Giardia lamblia was proven by stool and/or duodenal aspirate examination; the presence of the parasite was not demonstrated in the rest. Serum antibodies to G. lamblia were investigated in all children by an indirect immunofluorescent test (IIF) with G. lamblia trophozoites as antigen. It was found that all the children with positive duodenal aspirate had positive serology (titers greater than or equal to 1/32). The best correlation between parasitological and serological procedures were in the 1-5-year-old age group, and there was an age dependent increase of the antibody titer. Most of the parasitologically negative children were serologically negative. Our results support the use of IIF as a useful diagnostic procedure in the diagnosis of giardiasis in children.

Longitudinal study of giardiasis in three day care centres of Havana City.

The prevalence, incidence and reinfection of giardiasis were studied in 365 children attending three day care centres (DCCs) in Havana City. Three stool samples were obtained from each child every 6 months during an 18-month period. We identified three distinct groups of children according to their patterns of infection. In the largest group (51%) children were never found infected. In the second group, they were found infected once or twice during the study period, and in the third and the smallest group (9%) they were found infected in most or all the study periods. This last group seems to be children 'predisposed' to Giardia lamblia infection. The prevalence of giardiasis (20%) remained almost constant throughout the study period. The incidence declined from 16 to 11%, and reinfection increased from 36 to 49%. All the children had normal nutritional status and the only clinical manifestation that correlated strongly with the frequency of cross-sectional surveys positive to Giardia was the number of diarrhoeal episodes recorded during the last 6 months of the study period. Further studies will be necessary to ascertain the causes that determine the 'predisposition' to giardiasis in children.

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica.

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fa(g)) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fa(g) and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica.

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fag) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fag and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.

Identification of Dirofilaria immitis-infected dogs with a coagglutination assay.

The presence of Dirofilaria immitis excretory-secretory (ES) products was detected in the urine of infected dogs using a coagglutination assay. Urine samples from 30 naturally infected dogs were positive. Seventeen of them were microfilaremic, whereas 13 had become amicrofilaremic after receiving 2 courses of diethylcarbamazine. Urine samples from 20 dogs infected with other parasites, Dipetalonema reconditum (7), Toxocara canis (5), and Ancylostoma caninum (8), and urine samples from 20 healthy dogs were negative. The assay detected 200 ng/ml or more of ES products. This assay is simple, easy to perform with minimum training, and requires no equipment. Therefore it should be useful to detect canine filariasis under field conditions.

Partial characterization of the epitope on excretory-secretory products of Fasciola hepatica recognized by monoclonal antibody ES78.

This report contains a partial characterization of the epitope recognized by monoclonal antibody (MAb) ES78 produced against excretory-secretory (ES) antigens of Fasciola hepatica. ES78 is currently used for the detection of ES antigens in serum and stool samples of cattle and humans with fasciolosis, using a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA). The epitope was characterized by periodate oxidation, alkaline borohydride reduction, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, and enzymatic proteolysis. These results, together with those of the 2-site ELISA, lectin immunoassays, and beta-galactosidase digestion, showed that MAb ES78 reacts with a partly protein/partly carbohydrate antigenic determinant that is found on several ES molecules of adult specimens of F. hepatica and contains at least 1 disulfide bond and beta-galactose probably as galactose-beta(1-3)-N-acetylgalactosamine disaccharide.

Detection of Fasciola hepatica antigen in cattle faeces by a monoclonal antibody-based sandwich immunoassay.

A monoclonal antibody-based sandwich immunoassay (mAb sandwich ELISA) was developed for the detection of Fasciola hepatica antigen in the faeces of cattle. The assay was applied to samples from 100 cattle infected with F hepatica, 56 animals with parasitologically proven infections of other parasites and 100 uninfected animals. F hepatica antigen was detected in all the faecal samples from animals with fasciolosis, but none of the samples from the uninfected animals or from those with other parasitic infections had significant levels of F hepatica antigens. The results indicate that the mAb sandwich ELISA is a rapid, simple and useful method for the diagnosis of active F hepatica infection in cattle.

A longitudinal study of enterobiasis in three day care centers of Havana City.

Pinworm infection was prospectively studied during one year in 469 children attending three day care centers. Each child was examined at six months intervals using up to three perianal swabs with adhesive tape. Those found infected were treated with mebendazole. At the beginning of the study we found a prevalence of 28% that dropped to 13% and 12% in the following study periods. The reinfection rate was twice the incidence rate in both study periods. We also found a small percentage (10%) of the children reinfected in most or all study periods. There was a high correlation between reinfection and perianal itching. Our results add further knowledge to the epidemiology of intestinal parasites in day care centers.

[Training for diagnosis of intestinal parasitic diseases in the national laboratory system of Cuba]. / Adiestramiento en el diagnóstico de las parasitosis intestinales en la red de laboratorios de Cuba.

A national training project in the diagnosis of intestinal parasites was conducted in 1997. An initial national course was followed respectively by courses in the Central, Eastern, and Western Provinces. Our results showed that Cryptosporidium parvum, Cyclospora cayetanensis, and leukocytes showed a significantly lower percentage of errors after the training than before (p < 0.01). The same occurred with Entamoeba histolytica/E. dispar and Chilomastix mesnilii (p < 0.05). Among the helminths, Taenia spp., Fasciola hepatica, and hookworm showed significantly fewer errors after the training (p < 0.01). In the other specimens, few mistakes were found both at the beginning and after the training, and the percentage of errors did not change (p > 0.05). Furthermore, when comparing scores before and after training, a significant increase in median scores appeared in the Central Provinces (p < 0.05), Western Provinces (p < 0.01), and in the entire series (p < 0.01). The results showed the effectiveness of this intervention; these periodic mandatory training courses should be developed together with national programs for quality assessment in Parasitology.

Sandwich enzyme-linked immunosorbent assay for detection of excretory secretory antigens in humans with fascioliasis.

A sandwich enzyme-linked immunosorbent assay has been developed for the detection of Fasciola hepatica excretory secretory (ES) antigens in stool specimens of infected humans. The assay uses antibodies against F. hepatica ES antigens. A monoclonal antibody (ES78, mouse immunoglobulin G2a) was used to capture ES antigens, and a rabbit polyclonal antibody, peroxidase conjugate, was used to identify ES antigens. Thirteen of 14 patients with parasitological evidence of fascioliasis had a detectable concentration of ES antigens (more than 15 ng/ml). None of the stool specimens from controls and from patients with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay. When the 14 patients were retested 2 months after treatment, all of the specimens from the 11 parasitologically cured patients were negative by the antigen detection assay while the specimens from the 3 patients with persisting F. hepatica eggs in their stools remained positive.

The action of Clofazimine on the level of lysosomal enzymes of cultured macrophages.

Mouse peritoneal and calf alveolar macrophage cultures were exposed to various concentrations of Clofazimine, 3 (p-chloroanilino)-10-p-Chlorophenyl 2, 10-dihydro-2-isopropylimino, for 120 hr and an increase of four lysosomal enzymes were found with 0 . 3 micrograms/ml of the drug. In mouse peritoneal macrophage cultures, higher concentrations were toxic. Cycloheximide inhibited the lysosomal enzyme activity increase found. No change in enzymatic activity was observed when a lysosomal enriched granular fraction was incubated with various drug concentrations. Our results strongly suggest that Clofazimine at concentrations close to therapeutic serum levels induces de novo synthesis of lysosomal enzymes in macrophage cultures.

Phagocytosis and intracellular degradation of 125I-labelled immune complexes by Clofazimine treated macrophage cultures.

Mouse peritoneal and calf alveolar macrophage cultures were exposed to various Clofazimine concentrations for 5 days. Cultures exposed to drug concentrations near the blood therapeutic level phagocytize and digest more immune complexes than control cultures. Our results allow us to suggest that the beneficial action of Clofazimine in lepromatous leprosy could be at least partially mediated by the removal and degradation of circulating immune complexes by macrophages.

In vivo effect of clofazimine in the lysosomal enzyme level and immune complex phagocytosis of mouse peritoneal macrophages.

The effects of clofazimine on macrophages obtained from mice fed by gavage with various drug concentrations were studied. The results obtained demonstrated an increase in the activity of various lysosomal enzymes and in the amount of labeled immune complexes phagocytosed at drug concentrations of 1 mg/kg and 10 mg/kg body weight. This confirms and extends the effects reported by us of clofazimine's action on the lysosomal apparatus.

Detection of circulating excretory secretory antigens in human fascioliasis by sandwich enzyme-linked immunosorbent assay.

We developed a sandwich enzyme-linked immunosorbent assay to detect circulating parasite antigen in humans with fascioliasis. The assay uses antibodies against Fasciola hepatica excretory secretory (ES) antigens. A monoclonal antibody was used to capture circulating ES antigens, and a human polyclonal antibody peroxidase conjugate was used to identify circulating ES antigens. Optimal dilutions of all reagents were determined by block titration. The antigen concentration in sera from patients was estimated by comparing the optical density at 492 nm of test sera with a standard curve. All of the serum samples from 25 patients with parasitological evidence of fascioliasis had a detectable antigen concentration (more than 10 ng/ml). None of the serum samples from 80 patients infected with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay.