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L H Kasper

Publications and source records attributed to L H Kasper.

84 records · Page 5Linked to original sources
PubMed1985

Recognition and characterization of stage-specific oocyst/sporozoite antigens of Toxoplasma gondii by human antisera.

Human infection with Toxoplasma gondii is presumed due to the ingestion of either tissue cysts containing bradyzoites or oocyst/sporozoites that are excreted in the feces of infected cats. The incidence of human infection in the general population by either of these routes is unknown. We have previously described unique stage-specific oocyst/sporozoite antigens identified by murine hybridoma monoclonal antibodies. We obtained acute and convalescent antitoxoplasma antisera from patients in an epidemiologically well-documented outbreak of oocyst-transmitted infection associated with the ingestion of contaminated water. An enzyme-linked immunosorbent assay comparing equal numbers of tachyzoites (invasive stage) and oocyst/sporozoite (excreted stage) indicated that these antisera recognized antigens from both life forms. Absorption of pooled antisera with purified oocyst/sporozoites reduced both the antioocyst immunoglobulin G (IgG) and immunoglobulin M (IgM) titer but had only minimal effect on the antitachyzoite titer. Absorption of the antisera with tachyzoites reduced the IgG and IgM antioocyst and antitachyzoite titer. A sodium dodecyl sulfate-polyacrylamide gel analysis of radioiodinated oocyst/sporozoites shows that the principal stage-specific surface proteins of the oocyst/sporozoite have approximate Mr of 67,000 and 25,000. Periodic acid and silver stain of purified oocyst/sporozoite identified bands of similar molecular weight not present in the tachyzoite preparation. Western blot analysis of purified parasites assayed with human antioocyst antisera identified specific oocyst/sporozoite antigens not present on the tachyzoites. At least two major stage-specific oocyst/sporozoite antigens of approximate Mr of 67,000 and 190,000 were identified by the infected patients' antisera and not by the normal controls. Reaction to these oocyst/sporozoite antigens was seen primarily in the IgM fraction of the acute phase and the IgG fraction of convalescent phase antisera. Neither absorption of the antisera with tachyzoites nor periodate treatment of the oocyst/sporozoites reduced the antibody recognition of these stage-specific antigens. These data suggest that individuals infected by a presumed oocyst-transmitted route develop antibodies against unique stage-specific oocyst/sporozoite antigens.

Acute Disease↗
PubMed1985

An unexpected response to vaccination with a purified major membrane tachyzoite antigen (P30) of Toxoplasma gondii.

A purified Toxoplasma gondii tachyzoite membrane protein (P30) and a monoclonal antibody directed against this antigen were used to immunize mice. The P30 protein has an apparent m.w. of 30,000 and is the major radioiodinated tachyzoite membrane antigen identified by human and mouse antitoxoplasma antisera. Polyclonal and monoclonal antibody to the P30 antigen are parasiticidal in the presence of human serum. A series of mice were immunized with affinity column-purified P30 protein. This produced a dose-dependent, antigen-specific IgG and IgM response. The mice were challenged with the less virulent C strain tachyzoite. Immunized mice showed a statistically significant increase in mortality over nonimmunized control mice. In addition, vaccinated mice had an increased number of intracerebral tissue cysts when compared with the control group. Similar results were obtained with passive transfer immunization by using monoclonal antibody directed against the P30 antigen. Immunofluorescence assay of brain tissue cyst bradyzoites revealed a total absence of P30 antigen. Bradyzoites were also deficient in another major tachyzoite antigen of approximate m.w. 22,000 (P22). Mouse antibradyzoite serum absorbed with tachyzoites recognized bradyzoites but failed to identify tachyzoites. This suggests that there are stage-specific bradyzoite antigens of Toxoplasma gondii.

Animals↗
PubMed1984

Identification of stage-specific sporozoite antigens of Toxoplasma gondii by monoclonal antibodies.

Stage-specific antigens of Toxoplasma gondii have not been reported. We developed a panel of monoclonal antibodies that identified unique surface membrane antigens on the sporozoite and oocyst wall of T. gondii. These monoclonal antibodies failed to react with T. gondii tachyzoite surface membrane antigens in several immunologic assays. A comparison of surface membrane radioiodinated tachyzoites and sporozoites by one- and two-dimensional electrophoresis showed that T. gondii sporozoites have two major membrane proteins of approximate m.w. 67,000 and 25,000, not present in the tachyzoite stage. Two of the stage-specific monoclonal antibodies (2E4 and 5A4) were directed against the sporozoite 67,000 m.w. protein. In addition, T. gondii sporozoites are deficient in the major radioiodinated tachyzoite protein P30, which is identified by both human and mouse convalescent antitoxoplasma antisera. A convalescent antitoxoplasma serum failed to recognize sporozoite antigens by both immunoprecipitation and reaction with nitrocellulose transfer blots.

Animals↗
PubMed1983

Toxoplasma gondii: use of mutants to study the host-parasite relationship.

Toxoplasma gondii, an obligate intracellular parasite, is readily grown in nearly all cultured cells. The host-parasite relationship in these cultures can often be explored by using mutant host cells or mutant parasites. Host cells incapable of incorporating uracil or hypoxanthine, which were excellent precursors for T. gondii, allowed the demonstration that the host cell had no access to the purine or pyrimidine nucleotide pools of the parasite. Conversely, a T. gondii mutant that was defective in the principal pyrimidine salvage pathway allowed the demonstration that the parasite had no access to pyrimidine nucleotide or deoxynucleotide pools of the host cell. One metabolite that must pass from the host cytoplasm to T. gondii is a purine. An absolute defect in purine biosynthesis by the parasite was disclosed by growing T. gondii in a mutant host cell that was, itself, incapable of purine synthesis. T. gondii grew normally at 40 degrees C in a mutant host cell that was incapable of protein synthesis at that temperature. Thus, the parasite did not depend on concomitant protein synthesis in the host cell. An antigenic mutant of T. gondii was isolated with the aid of parasiticidal monoclonal antibody. This mutant lacked a major parasite surface protein, of relative molecular mass (Mr) 22 000. The antibody used to select this mutant immunoprecipitated a protein of this Mr from the wild-type parasite.

Animals↗
PubMed1983

Toxoplasma gondii: genetic crosses reveal phenotypic suppression of hydroxyurea resistance by fluorodeoxyuridine resistance.

Mutants resistant to sinefungin (SF) and hydroxyurea (HU) were isolated from an oocyst-producing strain of Toxoplasma gondii with the aid of mutagenesis with ethylnitrosourea. These mutants were used with previously described mutants resistant to adenine arabinoside (araA) and fluorodeoxyuridine (FUDR) in genetic crosses in cats. In order to interpret the data from crosses in which all four mutants were used to infect the same cat, it was necessary to devise a mathematical expression to predict the recombination frequency for unlinked markers. This frequency was shown in theory to be half of the product of the two parental phenotype frequencies. A series of crosses in which the parental frequencies were systematically varied yielded frequencies of recombination that were in accord with this calculation. The four-way crosses in the same cat showed unlinked recombination between all markers except HU and FUDR. This pair of markers yielded no doubly resistant recombinants, suggesting complete linkage. However, linkage was excluded when a binary cross between the HU- and FUDR-resistant mutants resulted in the normal number of doubly sensitive recombinants. The lack of doubly resistant recombinants was shown to be a consequence of phenotypic suppression of HU resistance by FUDR resistance. This suppression was first demonstrated by showing that an FUDR-resistant mutant selected from an HU-resistant parasite lost the HU resistance. The phenotypically suppressed HU-resistant gene was revealed by genetic crosses with wild type T. gondii. Although both parental stains were sensitive to HU, some of the progeny parasites were resistant.

Animals↗
PubMed1983

Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody.

The principal iodinatable surface protein (P30) of our cloned RH strain of Toxoplasma gondii has an apparent molecular weight of 30,000, as measured by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Monoclonal antibody B specifically immunoprecipitated protein P30 from a detergent extract of surface radioiodinated T. gondii. Monoclonal antibody B in the presence of complement was also parasiticidal for T. gondii, and this parasiticidal effect could be blocked by protein P30. Monoclonal antibody B was purified from mouse ascitic fluid and linked to cyanogen bromide-activated Sepharose. The resulting immunoabsorbent was used to purify 1.7 mg of protein P30 from a large number of parasites. The efficiency of recovery of protein P30 was measured by assays of radioactivity and of parasiticidal blocking activity. Protein P30 represented 3 to 5% of the total protein. It is also present in a recently isolated strain of T. gondii. A convalescent human antitoxoplasma serum immunoprecipitated radiolabeled protein P30. Three convalescent antisera when quantitated by an ELISA test had a high anti-protein P30 titer. Charge shift electrophoresis showed that protein P30 has an extensive hydrophobic region and thus is probably an integral membrane protein. Electrophoresis under nonreducing conditions showed no evidence that protein P30 exists as a disulfide linked homo- or heterodimer, although it probably has intramolecular disulfide bonds.

Absorption↗
PubMed1982

Hydroxyurea inhibition of growth and DNA synthesis in Toxoplasma gondii: characterization of a resistant mutant.

Hydroxyurea inhibited the growth and DNA synthesis of Toxoplasma gondii growing in human fibroblast cells. A concentration of 18 micrograms/ml totally suppressed plaque formation. The synthesis of T. gondii RNA was not acutely inhibited. The parasite was equally sensitive to hydroxyurea when grown in wild type or hydroxyurea resistant host cells. With the aid of chemical mutagenesis, we isolated a stable hydroxyurea resistant mutant of T. gondii. This mutant showed no increased ability to incorporate [3H]uracil into its pyrimidine deoxynucleotide pool. Hydroxyurea depressed the [3H]uracil labeling of the pyrimidine deoxynucleotide pool in the wild type parasite but not in the mutant, suggesting that the mutant ribonucleotide reductase was resistant to the inhibitory effect of the drug.

Animals↗
PubMed1982

Isolation and characterization of a monoclonal antibody-resistant antigenic mutant of Toxoplasma gondii.

Several monoclonal antibodies against Toxoplasma gondii were parasiticidal in the presence of normal human serum as measured by reduction in plaque titer or in assays that detected lysis. One monoclonal antibody, G, was used to select a resistant mutant from a large population of chemically mutagenized wild-type parasites. This mutant retained the wild-type sensitivity to other monoclonal antibodies and to polyclonal antisera. A comparison of surface radioiodinated wild-type and mutant parasites by using one and two-dimensional electrophoresis showed that the mutant lacked a protein (or proteins) of approximate m.w. 22,000. An immunoprecipitation procedure using monoclonal antibody G yielded a protein(s) of this m.w. from surface radioiodinated wild-type T. gondii but not from surface radioiodinated mutant parasites.

Animals↗
PubMed1979

Bilateral rhinocerebral phycomycosis.

A 51-year-old diabetic woman developed bilateral rhinoorbitocerebral phycomycosis. Successful treatment was accomplished by correction of the ketoacidosis, surgical debridement, and amphotericin B therapy. Rhinoorbitocerebral phycomycosis is a fulminant and frequently fatal disease most often seen in debilitated hosts. This survival suggests that aggressive therapy is indicated in patients with extensive fungal involvement.

Amphotericin B↗
PubMedDate not supplied

Detection of Toxoplasma gondii soluble antigen, SAG-1(p30), antibody and immune complex in the cerebrospinal fluid of HIV positive or negative individuals.

Active infection by T. gondii was evaluated by immunoassay for soluble SAG-1 (p30), the major surface antigen from T. gondii, specific antibodies and immune complexes in human cerebrospinal fluid (CSF) samples. A total of 263 samples of CSF were collected from hospitalized patients presenting neurological disorders and analyzed for antibodies to HIV. Patients were divided into two groups: HIV positive (n = 96) or HIV negative (n =167). The results of the assays showed that 45% of all samples were positive for soluble SAG-1. Toxoplasma Ag/Ab immune complexes were detected in 19% of the CSF samples and 62% were positive for T. gondii- specific IgG. A combination of these assays in the presence of clinical findings consistent with active Toxoplasma infection may predict the presence of toxoplasmic encephalitis. Moreover, detection of soluble SAG-1 in the CSF of these individuals appears consistent with active infection.

Animals↗