Toxoplasma gondii and cytomegalovirus: mixed infection by a parasite and a virus.

Human fibroblasts infected in vitro with cytomegalovirus are relatively resistant to infection by Toxoplasma gondii during the first 4 days of virus infection. After 5 days, however, the cytomegalovirus-infected cells become susceptible to the parasites. The toxoplasmas replicate in paracentral rosettes surrounded by host cell mitochondria. This growth configuration differs from that seen in human fibroblasts infected in vitro with toxoplasma only but resembles the pattern seen in doubly infected cells found in human necropsy tissue.

Construction of synthetic immunogen: use of new T-helper epitope on malaria circumsporozoite protein.

The circumsporozoite (CS) protein of Plasmodium falciparum is the focus of intense efforts to develop an antisporozoite malaria vaccine. Localization of sites for T-cell recognition on this molecule is critical for vaccine design. By using an algorithm designed to predict T-cell sites and a large panel of H-2 congenic mice, a major nonrepetitive T-cell site was located. When a synthetic peptide corresponding to this site was covalently linked to the major B-cell site on the molecule, an immunogen capable of eliciting a high-titer antibody response was formed. This peptide sequence could prime helper T cells for a secondary response to the intact CS protein. The new helper T-cell site is located outside the repetitive region of the CS protein and appears to be the immunodominant T site on the molecule. This approach should be useful in the rational design and construction of vaccines.

Chronic lymphadenopathic toxoplasmosis. A case with marked hyperglobulinemia and impaired delayed hypersensitivity responses during active infection.

A patient with lymphadenopathic toxoplasmosis characterized by prolonged symptoms and repeated relapses with isolation of toxoplasma from lymph nodes is described. As the disease persisted and progressed, striking immunologic changes occurred that ultimately resulted in a state of extreme hyperglobulinemia associated with impaired delayed hypersensitivity responses. The case in question illustrates that progressive infection may occur in the face of high antibody levels of all immunoglobulin types whereas the only demonstrable immunologic impairment was of delayed hypersensitivity.

A C1Q enzyme-linked immunosorbent assay (ELISA) using polyethylene glycol for immune complexes.

A C1q-ELISA for the detection of immune complexes is described in which the sensitivity was increased by the addition of polyethylene glycol (PEG). Although the ELISA without PEG adequately detected immune complexes in sera from patients with autoimmune disorders, when sera from patients with filariasis were tested, there was little correlation between values obtained with ELISA and the 125I-C1q binding assays. The addition of PEG to the filariasis sera before reacting the bound complexes to the enzyme conjugated anti-IgG increased the sensitivity to allow detection of immune complexes in those sera. This could be done without adversely affecting the reaction with normal sera or sera from patients with systemic lupus erythematosus. The solid phase C1q-ELISA with the PEG modification can be used for the detection of immune complexes in filariasis and should be adaptable for use with sera from other parasitic infections.

Enzyme-linked immunosorbent assay (ELISA) for detecting IgM and IgE antibodies in human schistosomiasis.

Sera from patients with acute or early and chronic schistosomiasis were examined for IgG, IgM, and IgE antibody by an enzyme-linked immunosorbent assay technique, using soluble egg antigen from Schistosoma mansoni. Cercarial/adult IgG antibody ratios were determined, using soluble cercarial and adult worm antigens. Sera with cercarial/adult ratios indicative of acute or early schistosomiasis also contained specific IgM antibodies. Schistosome IgE antibody was found in sera from patients with acute schistosomiasis, but in only 1 of 10 sera from patients with chronic schistosomiasis. The inability of ELISA to detect IgE antibodies in chronic sera indicates that it may be a relatively insensitive measure of IgE antibodies in those patients with chronic schistosomiasis.

Filarial antigen in circulating immune complexes from patients with Wuchereria bancrofti filariasis.

Detection of filarial antigen in circulating immune complexes from patient sera was performed by an enzyme immunoassay in which the immune complexes were precipitated in the cold with polyethylene glycol and then dissociated in an acid pH buffer before being added to an ELISA plate. The dissociated antigen bound to the plate where it could be detected by peroxidase-labeled polyclonal rabbit antifilarial antiserum. Control sera used for defining the specificity of the assay included sera with immune complexes not related to parasite infection with and without free parasite antigen added prior to polyethylene glycol precipitation as well as sera from normal individuals. Filarial antigen was detected in the circulating immune complexes from 10 of 28 patients with bancroftian filariasis residing in either the Cook Islands (subperiodic Wuchereria bancrofti) or India (periodic W. bancrofti). By immunoblotting, the most frequently identified filarial antigen in these complexes was an approximately equal to 200 kDa circulating antigen.

Cellular hypersensitivity to toxoplasmal and retinal antigens in patients with toxoplasmal retinochoroiditis.

To investigate the possible role of hypersensitivity to toxoplasmal and retinal antigens in patients with toxoplasmal retinochoroiditis, we examined their in vitro lymphoproliferative responses to antigens prepared from Toxoplasma gondii and human retina. The magnitude of patients' responses, determined by incorporation of [3H]-thymidine, was compared to those of Toxoplasma seropositive and seronegative controls. Patients were indistinguishable from seropositive controls in terms of antitoxoplasmal antibody titer (dye test, indirect hemagglutination and enzyme-linked immunosorbent assay) and in vitro lymphoproliferative responses to toxoplasmal antigens. Furthermore, there was no relationship between antibody titer and the magnitude of proliferative responses in seropositive individuals. Four of four patients with active eye disease and six of 13 with inactive disease, but none of the seropositive or seronegative controls, had significant lymphoproliferative responses to human retinal antigens. These observations raise the possibility of an autoimmune component in the pathogenesis of relapses in toxoplasmal retinochoroiditis.

Serological differences between acute and chronic schistosomiasis mansoni detected by enzyme-linked immunosorbent assay (ELISA).

Sera from patients with acute and chronic schistosomiasis mansoni, and from laboratory-infected monkeys, were examined by an enzyme-linked immunosorbent assay technique using antigens prepared from eggs, cercariae, and adult worms. Sera from patients with acute schistosomiasis and from monkeys 2 months post-infection reacted more positively to cercarial antigen than to adult worm antigen whereas sera from both patients with chronic schistosomiasis and monkeys infected for longer than 4 months reacted more positively to adult worm antigen. These differential responses to antigen serologically differentiated between acute and chronic schistosome infections.

Endemic filariasis on a Pacific Island. II. Immunologic aspects: immunoglobulin, complement, and specific antifilarial IgG, IgM, and IgE antibodies.

Sixty-eight individuals from a Pacific island hyperendemic for subperiodic bancroftian filariasis were selected from a larger study population to include the entire clinical spectrum of filarial infection in that region and also an "endemic control" group without clinical or parasitologic evidence of filarial infection. Analysis of their blood leukocyte and humoral immune responses yielded the following major findings: 1) levels of specific antifilarial antibodies of three different immunoglobulin class (IgG and IgM measured by ELISA and IgE determined by radioimmunoassay) were significantly greater in the "endemic control" population than in the patients with filariasis, an observation true for both children and adults; 2) the endemic controls also had significantly higher levels of serum IgM and C3 than did the filariasis patients 3) while individuals with "filarial fevers" and "chronic (lymphatic) pathology" did have significantly lower IgG antibody responses to filarial antigen than the controls, the lowest antibody levels were found in the patients with microfilaremia; 4) symptomatic patients (i.e., those with filarial fevers or lymphatic obstruction) regularly showed higher specific antibody responses to filarial antigens than asymptomatic, infected individuals, although the difference did not reach statistical significance. These findings are in concert with our previously reported, intriguing observation that lymphocyte proliferative responsiveness to filarial antigens was much greater in individuals of the "non-infected" endemic control population than in patients with filariasis; furthermore, they indicate the important issues that must be approached and resolved to define the immunologic determinants leading both to the various filarial clinical syndromes and to protective immunity.

Development of antibodies to Plasmodium vivax as measured by two different serologic techniques.

Sera from individuals infected with Plasmodium vivax were tested for the presence of malarial antibodies using the indirect fluorescent antibody (IFA) and the indirect hemagglutination (IGA) tests. The primary infection resulted in the conversion of all sera to a positive response in the IFA test, whereas only 50% gave a positive IGA response. There was a direct relationship between the duration of the primary parasitemia and percentage giving positive IGA response. Relapse resulted in high level positive IFA and IGA responses.

Determination of IgM and IgG antibodies to Toxoplasma using the IFA test, ELISA, and Dot-ELISA procedures.

The dot enzyme-linked immunosorbent assay (Dot-ELISA) and the enzyme-linked immunosorbent assay (ELISA) were compared with the immunofluorescent antibody test (IFA) for detection of IgM- and IgG-specific antibodies to human toxoplasmosis. Reciprocal titers were determined in all three assays using sera from 56 patients with suspected toxoplasmosis or with symptoms and diseases requiring exclusion of toxoplasmosis and control sera from 56 healthy persons. Using the Dot-ELISA, six patient sera (10.7%) were positive at titers of greater than equal to 1024 for IgM antibodies (titer range 1024-16 384) and 47 sera (84%) were positive for IgG antibodies (titer range 16-262 144) at a titer of greater than or equal to 16. One control serum was reactive for IgM (titer 1024) and 10 control sera (18%) were positive for IgG in the Dot-ELISA. In the ELISA, at titers of greater than or equal to 128, five sera (9%) were reactive for IgM (titer range 128-512) and 52 sera (92.8%) were reactive for IgG (titer range 32-8192) at a titer of greater than or equal to 32; no control sera gave positive reactions for IgM while 10 sera (18%) were positive for IgG in the ELISA. Using the IFA test at reciprocal titers of greater than or equal to 16, four sera (7.1%) were positive for IgM (titer range 32-512), and 51 sera (91%) were positive for IgG (titer range 16-8192). None was reactive for IgM, and eight sera (14%) were positive for IgG (titer range 32-128) in the IFA test. The Dot-ELISA correlated well with the IFA test (correlation coefficient = 0.895) and the ELISA correlated slightly higher with the IFA test (correlation coefficient = 0.910) for detection of IgG antibodies to Toxoplasma gondii.

Antigenic differences between endozoites and cystozoites of Toxoplasma gondii.

Antigenic differences between the cystozoite and endozoite of Toxoplasma gondii were found using fluorescent antibody staining. Antisera against the cystozoite reacted against only the cystozoite, whereas antisera against the endozoite reacted against both endozoite and cystozoite. Absorption of sera with endozoites removed only positive reactions with endozoites. These findings are the first to suggest antigenic differences between these two forms of Toxoplasma.

Serologic test for antibody to Sarcocystis in cattle.

Soluble antigen was prepared from Sarcocystis zoites obtained from heart muscle of a bovine inoculated with sporocysts from canine feces and killed 120 days after infection. The antigen was used in an indirect hemagglutination (IHA) test and an agar gel diffusion test to detect antibody to Sarcocystis in experimentally infected cattle. IHA serum titers began to rise 30 to 45 days after infection and reached levels up to 1:39,000 90 days after infection. Sera collected under field conditions from 21 dairy cows had IHA titers ranging from 1:54 to 1:486. Since all cows appeared in good health, titers of 1:486 or less can probably be considered nonsignificant with regard to diagnosis of clinical disease. No positive Sarcocystis IHA titers were obtained with human sera previously found to be IHA positive for toxoplasma, indicating a lack of cross reactivity between antigens. Precipitins in the agar gel diffusion test appeared 30 days postinoculation and became very pronounced at 65 to 90 days.

Changes in serum and plasma proteins and in IgG and IgM antibodies in calves experimentally infected with sarcocystis from dogs.

Fifteen calves were used in three experiments to determine changes in serum and plasma proteins and IgG and IgM levels after oral inoculation with Sarcocystis sporocysts from dogs. Total serum or plasma protein levels in inoculated calves decreased during the acute phase of infection (4 to 5 weeks after inoculation) and then increased and became greater than the levels of control calves 7 to 8 weeks after inoculation. The initial decrease in total protein reflected reduced serum albumin and the subsequent increase reflected increased immunoglobulin levels. Immunoglobulin levels increased in both IgM and IgG fractions. Specific antibody activity against Sarcocystis antigen was 6 to 9 times greater in the IgM than in the IgC fraction 5 weeks after inoculation, but IgG activity became approximately 17 to 27 times greater than IgM activity 10 to 13 weeks after inoculation.