Transfer factor: delayed hypersensitivity to Schistosoma mansoni and tuberculin in Macaca mulatta.

Delayed hypersensitivity in Macaca mulatta infected with either Schistosoma mansoni or mycobacteria was demonstrated by biopsies of skin test sites. Both dialyzable and nondialyzable leukocyte extracts from infected donors transferred delayed hypersensitivity to recipient monkeys. In two recipients, skin test conversion was associated with in vitro transformation of the recipients' lymphocytes.

Quantitative capacities of glutaraldehyde and sodium m-periodate coupled peroxidase-anti-human IgG conjugates in enzyme-linked immunoassays.

Covalently linked peroxidase-anti-human IgG conjugates were prepared by either glutaraldehyde or NaIO4 coupling techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows that the glutaraldehyde coupled conjugate is composed of generally lower molecular weight components than the NaIO4 coupled product. The NaIO4 conjugate, when used to quantitate human immunoglobulin (Ig) in enzyme-linked immunoassays, appears to be highly sensitive in that small amounts of Ig elicited relatively high reactivities. The quantitative range of this type of conjugates, where reactivities are linearly proportional to the amount of human Ig present, is, however, extremely narrow (0.01-0.10 micrograms/ml of human IgG). Conversely, the glutaraldehyde coupled type conjugate is capable of sustaining a much wider range of linearity (0.01-0.6 micrograms/ml), but with a more gradual rise of reactivity which corresponds well to the amount of human Ig present. Conjugates prepared with glutaraldehyde are thus more useful in quantitative assays where wide quantitative ranges are desirable. NaIO4 conjugates on the other hand, are more suited to qualitative assays where sensitivity is more important.

Hypersensitivity to parasite proteolytic enzyme in schistosomiasis.

A proteolytic enzyme which hydrolyses hemoglobin was obtained from the supernatant fraction of homogenized Schistosoma mansoni. This enzyme elicited histaminic skin reactions in various animals, including man, which were infected with S. mansoni. It failed to induce reactions in monkeys harboring S. haematobium, S. japonicum, or S. intercalatum. In a preliminary field trial in the Caribbean, the skin test proved to be somewhat less sensitive than the customarily used extract of adult worms in Coca's solution. However, the enzyme appeared to induce fewer false positive reactions and delayed responses than did the Coca's extract. A new diagnostic test for schistosomiasis probably could be developed by using specific parasite enzymes against which the host has become sensitized in the course of infection.

Prevalence of Entamoeba histolytica in patients with schistosomal colonic polyposis.

Two hundred and fifty-seven Egyptian patients were classified into three groups: patients with schistosomal colonic polyposis, those with simple schistosomiasis without polyposis, and a non-schistosomal group. A diagnosis of schistosomiasis was made by clinical history and examination plus three fresh stool examinations or a rectal biopsy. The presence of schistosomal colonic polyps was established by sigmoidoscopy and biopsy of polyps. Stool examinations were made on all individuals, using the merthiolate-iodine-formaldehyde technique to detect Entamoeba histolytica. We found the prevalence of amebiasis in the group with schistosomal colonic polyposis (37%) to be significantly higher than that in the non-schistosomal group (11%) and in the schistosomal group without polyposis (15%). The difference in prevalence of amebiasis between the simple schistosomal and non-schistosomal groups was not significant.

Serodiagnosis of Schistosoma mansoni with microsomal adult worm antigen in an enzyme-linked immunosorbent assay using a standard curve developed with a reference serum pool.

A standardized microtest plate enzyme-linked immunosorbent assay was developed using the microsomal fraction of adult worms of Schistosoma mansoni (MAMA) as antigen. The standard reference serum pool was prepared from acutely and chronically infected rhesus monkeys and was shown to be appropriate as a standard for measuring the levels of reactivity of the unknowns. The standard serum pool was arbitrarily designated as having 100 activity units per microliter. The levels of reactivity of the unknowns were expressed as activity units per microliter. Serum specimens were obtained from 190 patients infected with S. mansoni in the Caribbean, South America, and Africa. Serum was obtained from small numbers of patients infected with S. haematobium, S. japonicum, or S. mekongi. Controls were 136 patients with other helminthic infections, 142 patients with protozoal or other diseases with liver involvement, and 81 healthy serum donors. The J index (or predictability) of the assay was calculated to determine the significant level of reactivity. The assay has a predictability of 95% for both patients with S. mansoni infections and those with other infections. The sensitivity of the assay for S. mansoni infections was 96%, and the specificity (in terms of cross-reactions with infections with other parasite genera or with other liver diseases) was 99%. The heterologous Schistosoma species showed a markedly lower level of reactivity, with an overall sensitivity of 55%. This is in accord with the species-specificity previously recognized in MAMA, and emphasizes the need for standard reference pools of human sera prepared from patients infected with single species of each of the Schistosoma. Use of these pools in assays with antigens of the respective schistosome species would allow optimum serologic evaluation.

Isotypic analysis of humoral immune responses in rhesus monkeys to an adult microsomal antigen of Schistosoma mansoni: an indicator of successful treatment.

A study was undertaken to examine the potential role of immunodiagnostic methods in determining successful chemotherapy in schistosomiasis. Fifteen rhesus monkeys were infected with 1,500 Schistosoma mansoni (Puerto Rico strain) cercariae, and 10 of the monkeys were then treated with a curative dose of praziquantel 13 weeks after infection. Five monkeys remained untreated. One monkey was not successfully cured, as confirmed by the presence of both male and female worms at the time of perfusion. Serum samples were longitudinally collected and specific Ig isotypes were quantified with an adult microsomal antigen of S. mansoni using the FAST-ELISA. Specific isotypes were detected with monoclonal antibodies specific for each human Ig isotype, followed by a peroxidase-conjugated anti-mouse Ig. Longitudinally, all monkeys showed similar isotype patterns. Isotypes increased for the first nine weeks following infection, and then began to decrease. Ten to 14 days following treatment, all isotypes increased. The Ig isotype responses of all monkeys followed classic patterns of isotype expression. A ratio of pretreatment (week 13) IgG1 absorbance values to post-treatment IgG1 absorbance values was generated for each monkey. All successfully treated monkeys, determined to be worm-free by perfusion, had IgG1 ratios at week 53 greater than 2.4 (range 2.4-181). The untreated monkeys and the single monkey that was a treatment failure had IgG1 ratios less than 2.1 (range 0.09-2.05) for the same time period.

Schistosoma mansoni infection in intact and B cell deficient mice: the effect of pretreatment with BCG in these experimental models.

The course of infection with Schistosoma mansoni was determined in B cell deficient mice by means of a schistosomule lung recovery assay 6 days after infection or by determination of the adult worm burden 7 weeks after infection. The intensity of infection was not significantly different from that in age- and sex-matched intact controls. B cell deficiency was demonstrated by absence of surface immunoglobulin-bearing cells in the spleen and by absence of B cell areas in the lymphoid follicles of the spleen and mesenteric lymph nodes. In addition, B cell deficient mice infected for 7 weeks with S. Mansoni were unable to form anti-schistosome antibodies detectable by the Cercarienhüllenreaktion. A normal granulomatous response, however, was observed around schistosome eggs. Pretreatment with BCG suppressed infection with S. mansoni comparably in intact and B cell deficient mice. A marked depletion of eosinophils occurred in the schistosome egg granuloma of all BCG treated mice.

Diagnosis of paragonimiasis by immunoblot.

A sensitive and specific immunoblot assay was used to rapidly and accurately diagnose paragonimiasis. The immunoreactivity of a complex Paragonimus westermani Chaffee antigen was evaluated by SDS-PAGE and Western blot analysis. Initial probing with pooled human serum from proven Paragonimus infections revealed many bands, including a significant antibody response to an approximately 8,000 molecular weight (8 kDa) protein. Forty-three of 45 proven paragonimiasis serum specimens had antibodies to this diagnostic band. Of 29 normal serum specimens and 210 serum specimens from patients with other parasitic and nonparasitic infections, only 1 serum, from a schistosomiasis haematobium patient, reacted positively. These results indicate that our immunoblot for paragonimiasis, which uses a comparatively crude antigen, is highly sensitive (96%) and specific (99%).

Acquired immunity in B cell-deficient mice to challenge exposure following primary infection with Schistosoma mansoni.

Mice were made B cell-deficient by injections of globulin prepared from goat antimouse mu-chain serum. Anti-mu globulin was administered throughout the experiments (18 or 23 weeks). B cell deficiency was determined at the time of assay of worm burden levels (7 weeks after challenge with Schistosoma mansoni) by quantitation of serum IgM and IgG, by assaying the specific antibody response to cercarial and adult worm antigens in enzyme-linked immunosorbent assay and by histologic examination of the spleen and mesenteric (regional) lymph nodes. Four-week-old mice were exposed to S. mansoni and 8 weeks later were challenged with a second exposure. The B cell-deficient mice developed a degree of resistance (79%) similar to that of the intact controls (81%). The IgM and IgG levels of the B cell-deficient mice were markedly suppressed. Follicular development was not detected in their lymph nodes; but in the spleen of some animals clusters of cells morphologically similar to B cells were observed peripheral to a central T cell-like area. B cell-deficient mice developed schistosome egg granulomas comparable to those of the intact controls. Control animals developed an antibody response with titers of 1:64 to 1:1,024 against cercarial and adult worm antigens; B cell-deficient animals were nonreactive in these assays. The data suggest that specific antibody does not play a major role in resistance acquired within 8 weeks as a result of a primary infection in murine schistosomiasis.

A specific diagnostic antigen of Echinococcus granulosus with an apparent molecular weight of 8 kDA.

An echinococcus antigen with an apparent molecular weight of 8 kDa was identified as diagnostically important. An immunoblot assay using this antigen was 91% sensitive for surgically confirmed Echinococcus granulosus hydatid disease of the liver. Specificity was 100% for echinococcosis. Marked cross-reactivity was observed with serum specimens from patients with E. multilocularis and E. vogeli infections. The 8 kDa component was not related to the widely recognized echinococcus antigen 5.

Gross and histological studies of immediate, Arthus, and delayed skin test responses to schistosome antigen, and of delayed response to ubiquitous antigens in Egyptians.

Gross studies of skin reactions to adult antigen of Schistosoma mansoni were made on 156 hospitalized patients with schistosomiasis and 114 subjects from the nonendemic area of Hurghada in Egypt. Wheal areas equal to or greater than 1.0 cm2 indicated a positive immediate (15-min) reaction to adult worm antigen; the criterion of positivity for both 24-hour and 48-hour delayed reactions was an area of induration equal to or greater than 0.6 cm2. Immediate reactions with adult worm antigen were observed in 99% of the patients with schistosomiasis and 11% of the subjects from Hurghada: the percentages with delayed reaction were 58% and 2%, respectively. Biopsies of skin test sites at various intervals after antigen injection were done on 87 individuals. Eosinophilic and mononuclear infiltrates were characteristic of immediate and delayed skin responses, respectively. Biopsies from 22 patients with marked skin reactions 5 hours after antigen injection showed that a neutrophilic response indicative of Arthus reactivity was present in only 18. Thus, Arthus reactivity could not be determined on gross appearance alone. The studies did not show any evidence of delayed basophilic hypersensitivity to schistosome antigen. Immunofluorescent studies on a small number of biopsies suggested that a late phase (5-hour) reaction due to IgE may occur in some patients. Delayed reactivity to mumps and/or monilia skin test antigens was observed in 91% of Egyptians in a nonendemic area of schistosomiasis. Delayed hypersensitivity to PPD was detected in 44% of the same group.

The present status of serodiagnosis and seroepidemiology of schistosomiasis.

The literature of the past 4-5 yr on serodiagnosis and seroepidemiology of schistosomiasis is reviewed. A variety of assays with different antigens are being used for serodiagnosis. Several purified antigens appear to be sensitive and specific, but have little if any capability of indicating duration of infection, parasite burden, or effect of chemotherapy. The results of long-term posttherapy field studies indicate that serology has a role in monitoring control programs. Standardized serologic assays and the need for International Standard Reference Sera are emphasized. A standardized enzyme-linked immunosorbent assay based on the Falcon Assay Screening Test system (FAST-ELISA), and involving a standard reference serum pool, is suitable for both serodiagnosis and field studies. Measurement of circulating antigens as a parameter of active infection is considered to have increased potential, compared with antibody measurement, in management of clinical disease and in control programs. Recombinant DNA technology may be useful for producing standard antigens for use in assays measuring antibody or circulating antigen. Time-resolved immunofluorescence involving europium-labeled conjugates may provide the increased assay sensitivity needed for measurement of circulating antigen.

Evaluation of serologic tests for Pneumocystis carinii antibody and antigenemia in patients with acquired immunodeficiency syndrome.

Sera from 68 patients with acquired immunodeficiency syndrome and 135 controls were used to evaluate the indirect immunofluorescence and enzyme-linked immunosorbent assays for detection of antibodies to Pneumocystis carinii and a counterimmunoelectrophoresis assay for detection of circulating Pneumocystis antigen. None of these assays was helpful in the diagnosis of P. carinii pneumonia. An improved assay for antigenemia is needed to differentiate between clinical and subclinical infection.

Serum immunoglobulin levels and malaria antibodies in Burkitt's lymphoma.

Data are presented to support a relationship between malaria infection and Burkitt's lymphoma in African children. IgG, IgM and IgA levels were measured in sera from Burkitt's lymphoma patients and from sex- and age-matched, nearest-neighbour controls. All three classes of immunoglobulins were present in significantly lower amounts in the sera from Burkitt's lymphoma patients than in the sera from controls. The mechanism of this apparent B-cell suppression is not yet clear. Malaria-specific IgG and IgM antibody titres were determined in the indirect immunofluorescence test. No significant difference in the IgG malaria-specific antibodies was detected between the two groups of sera. Malaria antibody levels measured using IgM specific conjugates were significantly lower in the sera from Burkitt's lymphoma patients in reactions with Plasmodium falciparum antigen. No significant difference was observed when P. malariae was used. Confirmation of this finding would serve as a positive link between Burkitt's lymphoma and P. falciparum infection.

Adoptive transfer of protective immunity in experimental schistosomiasis in the mouse.

Passive transfer of immune serum alone did not confer protection to recipient mice irrespective of the routes of serum transfer or cercarial challenge of Schistosoma mansoni. Mice that received both sensitized cells and immune serum were protected against challenge by subcutaneous injection of cercariae but not by percutaneous exposure. The immune serum could be transferred as late as 8 days after subcutaneous challenge, suggesting that the protection was afforded in part by a late parasite killing mechanism which functions after the schistosomula have migrated through the lungs.

Systematic fractionation of Schistosoma mansoni urea-soluble egg antigens and their evaluation by the single-tube, kinetic-dependent, enzyme-linked, immunosorbent assay (K-ELISA).

The application of a single-tube, kinetic-dependent, enzyme-linked, immunosorbent assay (k-ELISA) is described. The k-ELISA is simple, highly sensitive, and quantitative. With this test, the antigenic activities and cross-reactivities of several fractions from Schistosoma mansoni eggs were quantitated and compared. Particulate egg components were solubilized readily by 8 M urea, yielding antigenic fractions of high specific activities and low corss-reactivities. SDS-PAGE and activity profiles of these antigens clearly showed that they were separate and distinct form the soluble egg antigens (SEA) group. The urea-solubilized antigens appeared to be composed of two major protein bands of high molecular weights. The yield for these antigens was significantly greater than the SEA group, thus making them worthwhile candidates for serological antigens. The systematic and quantitative nature of the present study allows for the critical comparison of every antigenic fraction from S. mansoni eggs. From data of this type, a diagnostic antigen of high efficacy can be selected.

Standardization of FIAXtm for schistosomiasis using crude cercarial and adult worm antigens.

A fluoroimmunoassay (FIAXTM) has been developed for quantitating the antibody response to schistosomiasis by using cercarial and adult worm antigens of Schistosoma mansoni. The FIAXTM assay was calibrated by using an enzyme-linked immunosorbent assay (ELISA) performed with the same antigens. Studies of reproducibility and preliminary data on a battery of sera from proven cases of schistosomiasis an uninfected control sera are presented.

Immunologic studies on hamsters infected with Entamoeba histolytica.

The serologic and cell-mediated immune responses of hamsters exposed to 2 strains of Entamoeba histolytica (HM-1 and HM-19) were evaluated by a series of in vitro tests. The pathogenicity of the 2 strains was evaluated in terms of their ability to produce liver abscesses and spleen enlargement. Antibody response was evaluated by the indirect hemagglutination test. The cellular immune response was assayed by increased DNA synthesis by lymphocytes and migration inhibition of macrophages.

Studies on putative adult worm-derived vaccines and adjuvants for protection against Schistosoma mansoni infection in mice.

Intraperitoneal transfer of viable adult worms of Schistosoma mansoni did not confer protection against a challenge infection to recipient mice. Antigens of schistosome origin were evaluated for their ability, with and without concomitantly administered nonspecific adjuvants, to stimulate protective immunity against S. mansoni. Freshly perfused ground worms or a putative membrane antigen extracted with 0.5 M KC1 from adult worms, when injected together with Corynebacterium parvum (or in a single experiment with poly [A : U]), resulted in a significant reduction in worm burden of a challenge infection with S. mansoni as compared with that of untreated controls. The membrane antigen was maintained carefully at low temperatures in buffers capable of retarding enzymatic degradation while it was being prepared.