Identification of distinct accumulation sites of 4-aminoquinoline in chloroquine sensitive and resistant Plasmodium berghei strains.

We report the synthesis of an analogue of chloroquine (CQA) which can be used as a probe to visualize accumulation of 4-aminoquinoline by electron microscopy. A mouse monoclonal antibody against CQA was raised and used for immunodetection by the protein-A gold method on ultrathin cryosections, of CQA treated parasites. We demonstrate that in a P. berghei chloroquine(CQ)-sensitive strain (N strain) the chloroquine analogue used accumulates in the endocytic vacuoles where hemoglobin (Hb) degradation is occurring. In contrast, in a P. berghei CQ-resistant strain (RC strain) the probe was found scattered all over the cytoplasm of the parasite. This result suggests that endocytic vacuoles of the parasite could constitute the site of antimalarial action of CQ.

Characterization of a family of rhoptry proteins of Toxoplasma gondii.

A monoclonal antibody (McAb 4A7) raised against a rhoptry enriched subcellular fraction of Toxoplasma gondii tachyzoites reacted in immunofluorescence studies with rod-like organelles located in the anterior part of the organisms and gave specific labeling of rhoptries in immunoelectron microscopy. On immunoblots, two major proteins of 55 and 60 kDa were identified by McAb 4A7. Similar results were obtained both by immunodetection and immunoblotting with tachyzoites, bradyzoites and sporozoites. Pulse chase analysis of [35S]methionine labeled tachyzoites demonstrated that the 55 and 60 kDa rhoptry proteins derived from a 66-68 kDa doublet which was processed approximately 30 min after biosynthesis. Two other monoclonal antibodies (McAb 2F8, McAb 2H3) respectively specific for rhoptry proteins of 55 kDa having a 66 kDa precursor and 60 kDa having a 68 kDa precursor were also obtained; we suggest that they recognize separately the two components of the 55-60 kDa rhoptry protein family of Toxoplasma.

A 50 kilodalton exoantigen specific to the merozoite release-reinvasion stage of Plasmodium falciparum.

The immunoglobulins G of a human plasma inhibiting in vitro Plasmodium falciparum merozoite reinvasion have been purified and used to immunoprecipitate the antigens released into the culture medium by an [35S]methionine-labeled synchronous culture. Several of the major exoantigens identified were found throughout the entire life cycle; they were also immunoprecipitated from the labeled parasitized cells. Some antigens were found only after the reinvasion stage, and especially a major one of molecular mass 50 kDa and pI 5.5. The latter was not found in the parasitized cells but derived most likely from the processing of a major 126 kDa antigen which disappeared from the parasites during the reinvasion period and which was immunoprecipitated by an anti-50 kDa monoclonal antibody.

Localization, biosynthesis, processing and isolation of a major 126 kDa antigen of the parasitophorous vacuole of Plasmodium falciparum.

Monoclonal antibodies prepared against a 50 kDa antigen found in Plasmodium falciparum culture supernatants identify a 126 kDa polypeptide which can be localized by immunofluorescence and immunoelectronmicroscopy at the periphery of the schizonts. This polypeptide is released from the infected erythrocytes by mild saponin lysis and is probably a component of the parasitophorous vacuole. Pulse chase kinetic analysis demonstrated its disappearance from the parasitized red blood cell from 6 to 10 h after being synthesized and the concomitant appearance of the 50 kDa molecule in the culture supernatant. Purification of metabolically labeled, schizont infected cells demonstrated that spontaneous release of merozoites is needed for the processing of the 126 to the 50 kDa whereas reinvasion is not. Polyclonal antibodies were raised in rabbit against affinity purified 126 kDa protein. These antibodies, together with another 126 kDa specific monoclonal antibody have enabled us to characterize two other cleavage products of the 126 kDa antigen in culture supernatants, namely 47 and 18 kDa polypeptides. We believe that the processing of the 126 kDa protein into low molecular weight fragments reflects a proteolytic event which may participate in merozoite release.

Characterization of a 225 kilodalton rhoptry protein of Plasmodium falciparum.

A monoclonal antibody (24C6 4F12) raised against Plasmodium falciparum culture supernatant antigens gave a multiple dot picture on schizonts when assayed by immunofluorescence on P. falciparum erythrocytic stages. The corresponding antigen was localized in the peduncle of rhoptries by immunoelectronmicroscopy. On Western blots of P. falciparum schizonts, a major antigen of 225 kDa and a minor one of 240 kDa were recognized by this McAb. Pulse chase analysis of [35S]methionine biosynthetic labeling of P. falciparum culture demonstrated that the 240 kDa molecule was the precursor of the 225 kDa and that its processing occurred between 0 and 4 h after synthesis. Biosynthesis of the 240-225 kDa antigen occurred only during schizogony.

Characterization of microneme proteins of Toxoplasma gondii.

Three microneme proteins of Toxoplasma gondii have been characterized using 3 monoclonal antibodies and a recombinant protein specific antiserum. In all cases, apical labeling of tachyzoites and bradyzoites was observed by indirect immunofluorescence assay. Immunogold localization on ultrathin sections of bradyzoites or tachyzoites showed a specific labeling of micronemes. The following proteins were characterized using 2-dimensional gel electrophoresis and Western immunoblotting: Mic 1 (60 kDa, Pi 6.5), Mic 2 (120 kDa, Pi 5) and Mic 3 (90 kDa, Pi 6.75). The 90-kDa protein (Mic 3) is a heterodimer of two 38-kDa polypeptides (Pi 6.7 and 6.75 respectively) linked by disulfide bridges. Metabolic labeling and immunoprecipitation assays showed that at least one of the 38-kDa polypeptides was processed from a 40-kDa precursor. No processing was observed during the biosynthesis of the 120- and 60-kDa polypeptides.

Successful reinfection of chronically infected mice by a different Toxoplasma gondii genotype.

It is generally assumed that primary infection by Toxoplasma gondii protects from reinfection. A recent study using a murine model has questioned this dogma using indirect procedures to detect the reinfecting strain. We have reinvestigated this issue using a transfected strain of T. gondii (Prugniaud beta galactosidase: Pru beta gal) which expresses Escherichia coli beta-galactosidase. Detection of enzyme activity on fixed parasites allows a direct distinction between transfected and untransfected strains. We have found that in OF1 mice primary infection with the 76 K strain of T. gondii fully protects mice against tissue cyst production upon reinfection with the Pru beta gal T. gondii strain whereas primary infection with the Pru beta gal T. gondii strain does not impair tissue cyst formation upon reinfection with the Ned strain of T. gondii, which belongs to another T. gondii genotype. These results suggest that the immune protection conferred by one strain of T. gondii can be breached by reinfection with a strain belonging to another genotype; which can have significant consequences in human or veterinary medicine.

Immunochemical analysis of a major antigen of Plasmodium falciparum (P126) among ten geographic isolates.

Protein P126, a parasitophorous vacuole major antigen of Plasmodium falciparum and precursor of 3 major exoantigens (50, 47, and 18 Kd in strain FCR-3) has been studied in 10 culture-adapted isolates originating from various endemic areas. Two monoclonal antibodies (specific for 50 and 47 Kd exoantigens, respectively) were used to immunoprecipitate culture supernatants and parasitized erythrocytes in each case. It was observed that all the parasite isolates reacted with both monoclonal antibodies, indicating the ubiquity of the epitopes analyzed. Further, two of the exoantigens (the 50 and 18 Kd of FCR-3) were found to have a stable molecular mass in all the isolates tested, whereas, the other one (47 Kd in FCR-3) was found to have a variable molecular mass, from 47 to 50 Kd. The molecular mass of the precursor varied from 126 Kd to 128 Kd. No correlation was found between geographic origin and antigenic size.

Electric and chemical fusions for the production of monoclonal antibodies reacting with the in-vivo growth phase of Candida albicans.

To obtain monoclonal antibodies (MAbs) directed preferentially against the pathogenic phase of Candida albicans, mice were immunised with germ tubes of C. albicans serotype A, strain VW.32, killed by exposure to ultraviolet (UV) irradiation. Fusions were performed either by the standard chemical procedure with polyethylene glycol, or by electric discharge following linkage of the myeloma and lymphocyte cells with a Concanavalin A-mannoprotein bridge. The preliminary characteristics of one MAb obtained from each of these fusions are described. An IgM antibody (3B7) obtained from the chemical fusion reacted with a polysaccharide antigen that was heterogeneously distributed on both in-vitro and in-vivo forms of C. albicans. This MAb agglutinated different strains of C. albicans irrespective of their serotype. An IgG1 antibody (3G6) that had been obtained from the electric fusion was found to react in vitro with a proteinaceous antigen located only on the germ tubes of strain VW.32. However, MAb 3G6 displayed strong reactivity against all growth forms of C. albicans in vivo and reactivity extended to other strains.

Comparison of an ELISA technique with quantal micro-neutralization test for serotyping of HSV-1 or HSV-2-infected patients.

An enzyme-linked immunosorbent assay (ELISA) using semi-purified herpes simplex antigens extracted from cell nuclei was employed for typing of 32 sera collected from patients infected with HSV-1 or HSV-2. The results were in agreement with those obtained by the quantal micro-neutralization test. Moreover, sera with antibodies which could not be detected by micro-neutralization test could be typed by the ELISA technique according to the HSV isolates.

Antibody determination by ELISA in rats with retinal S antigen-induced uveoretinitis.

Enzyme-linked immunosorbent assay was applied to the determination of the serum IgG antibody contents in rats immunized with the organ-specific autoantigen (S antigen) of the retina. Optimal conditions (i.e. S antigen concentration, serum and conjugate dilutions, enzymatic reaction time) were determined. The assay required only a single serum dilution and was well reproducible. It was very sensitive, allowing the detection of low antibody contents in animals immunized with 1 microgram of S antigen. The time course of the antibody response and its variations according to the dose and the species of the immunizing S antigen were considered.

[Acute myocarditis caused by toxoplasmosis simulating infarction. Apropos of a case]. / Myocardite aiguë à toxoplasme simulant un infarctus. A propos d'un cas.

Acute myocarditis due to toxoplasmosis infection has been previously reported, usually in patients suffering from immuno-depression. Cardiac involvement by toxoplasmosis is rare in subjects with a normal immunological status. The authors report the case of a 16-year-old patient without immuno-depression with acute myocarditis caused by toxoplasmosis simulating myocardial infarction.

[Guillain-Barré syndrome with favorable outcome in a case of recent human immunodeficiency virus infection]. / Syndrome de Guillain et Barré d'evolution favorable dans un cas d'infection récente par le virus de l'immunodéficience humaine.

An acute polyradiculoneuropathy occurred in a 30 years homosexual male. E.L.I.S.A. test and Western Blot showed recent infection by H.I.V. Besides, endogenous reinfestation by cytomegalovirus was found: high concentrations of specific IgG antibodies and presence of the virus in the blood. T4 helper cells were severely reduced, without any other sign of cellular immunity failure. None of the two viruses was found in the nervous biopsy. This Guillain-Barre syndrome with a subsequent cellular reaction in the CSF, is probably to be related to an immunoallergic mechanism. Brief increase of antibodies specific for HBsAg and Borrelia Burgdorferri and the beneficial effect of plasmapheresis, supported this view. Two months later, the patient showed superficial lymph nodes hyperplasia, without any other symptom of pre-Acquired Immuno-Depression Syndrome.

[Evaluation of the Immulite 2000 Toxoplasma quantitative IgG et Toxoplasma IgM for the diagnosis of human toxoplasmosis]. / Utilisation des trousses Immulite 2000 Toxoplasmose IgG et Toxoplasmose IgM pour le diagnostic de la toxoplasmose humaine.

Four hundred and ninety five human sera with clinical and biological data were tested for the evaluation of Immulite 2000 Toxoplasma Quantitative IgG and Immulite 2000 Toxoplasma IgM produced by Diagnostic Products Corporation (Los Angeles, USA) for the diagnosis of human toxoplasmosis. The results of these kits were compared to those of the University Hospital of Nancy where the reference assays were Enzygnost Toxoplasmosis IgG and Enzygnost Toxoplasmosis IgM (Berhing-Dade, Germany), Toxoscreen (bioMérieux, France), ISAgA Plus (IgM et IgA) (bioMérieux, France). The sensitivity and the specificity of IgG detection by Immulite 2000 Toxoplasma Quantitative IgG were 98% and 100%, respectively. The high sensitivity of IgM detection by Immulite 2000 Toxoplasma IgM was adapted to the early diagnosis of toxoplasmic primo-infection and to the pediatric diagnosis or follow-up of congenital toxoplasmosis but could reveal IgM a long time after primary infection.

[Detection of early humoral response in experimental toxoplasmosis in mice]. / Détection du début de la réponse humorale de la toxoplasmose expérimentale murine.

Experimental infection of mice by Toxoplasma gondii is the best way for antigen production or direct diagnosis. Early serological response from sera and peritoneal exudate of mice infected by T. gondii has been studied. In this report the authors show that specific antibodies (IgM) are found in the serum at 24 hours after intraperitoneal inoculation. Serological methods were unable to detect free antibodies in the peritoneal exudate. Specific antibodies coating the parasite surface were detected by direct immunofluorescence as soon as 48 hours post infection. Biochemical treatment usually used for antigen production (trypsin, pepsin) were unable to destroy this antibodies which where no longer detected after 2 mercapto-ethanol treatment. Control experiments showed that, in these conditions, 2 mercapto-ethanol treatment had no effect on the reactivity of surface antigens with antibodies. Therefore, this results show that checking for specific antibodies absorbed at the surface of T. gondii is a prerequisite of the use of antigen produced in vivo.

[Value of the immunoenzyme method (ELISA) in determining serum and aqueous humor antiherpes immunoglobulin levels (apropos of 155 cases)]. / Intérêt de la méthode immuno-enzymatique (ELISA) pour le dosage des immunoglobulines antiherpès dans l'humeur aqueuse et le sérum (à propos de 155 cas).

Immuno-enzymatic assay of aqueous humor and serum antiherpetic antibodies was performed in 155 patients with all types of uveitis and with keratitis. Globulin levels were also determined in the two fluids, employing an original nephelometry-laser technique. This immuno-enzymatic technique, which possesses greater reliability, reproducibility, and sensitivity than passive hemagglutination, appears suitable for microassay in aqueous humor, and demonstrates the production of anti-herpes antibodies in this fluid to the exclusion of other specificity. Applying limits of at least 1/40e for H.A. antibody levels, and at least 10 for the immunity load coefficient, these antibodies could be demonstrated in half of the cases of clinically confirmed herpes, in one-third of clinically suspected cases, and in two intermediary cases of uveitis where no predictive signs of herpes were present. In contrast, specific antibodies were never detected in 70 cases of anterior or total uveitis. Measuring anti-herpes antibodies in aqueous humor and serum by the ELISA method, in association with immunity load coefficients determination in the two fluides, appears to be a very useful method for the future etiological diagnosis of anterior and intermediary uveitis when the etiology is uncertain but clinical signs suggest a possible herpetic origin.

[Study of developing clinical outbreak and serological rebounds in children with congenital toxoplasmosis and follow-up during the first 2 years of life]. / Etude des poussées cliniques évolutives et des rebonds sérologiques d'enfants atteints de toxoplasmose congénitale et suivis durant les 2 premières années de vie.

BACKGROUND: The survival of T gondii bradyzoites in cysts explains clinical recurrences and serological rebounds after birth in children with congenital toxoplasmosis. At the present time, management of such manifestations is not well defined. PATIENTS AND METHODS: Sixty-three infants with congenital toxoplasmosis were followed-up at the University Hospital of Lille (France) during the first two years of life. For each child, the treatment before and after birth was well defined. Clinical, ophthalmological, radiological and serological data were collected every third month. Serological assays specially adapted to this age bracket were used for the quantification of specific IgG, or for the detection of T gondii specific IgM and IgA. RESULTS: Seventy-six serological rebounds were reported in 55 of the 63 children (87%). They concerned essentially IgG (96%) and less frequently IgM (47%) or IgA (60%). At the same time, only five clinical recurrences were observed, four of them being preceded by a serological rebound. DISCUSSION: Treatment of fetuses or children with pyrimethamine and sulfonamides versus spiramycin alone was associated with a decrease in the frequency of serological rebounds during the first year of life (P < 0.001). Such a therapeutic regimen during the second year of life decreases the appearance of serological rebounds in children without rebound antecedent (P < 0.001). CONCLUSION: The increase in number of rebounds after the end of a course of pyrimethamine and sulfonamides necessitates the evaluation of such a long term treatment without interruption.

[Malaria and pregnancy. Therapeutic problems, a case report]. / Paludisme et grossesse. Problèmes thérapeutiques, à propos d'un cas.

Malaria is an old but still current parasitosis. Transmitted by the bite of the female anopheles, it can be revealed by more or less serious symptoms according to the species of Plasmodium. Plasmodium falciparum is responsible for the serious forms. It is the most frequent species, resistant to 4 amino-quinolein and sulfamides in some areas. P. vivax and P. ovale, usually harmless, are not so widely geographically spread as P. falciparum; and apparently are not drug resistant but they expose patients to the risk of relapses. During pregnancy, the choice of a curative and prophylactic therapy must take into account the supposed or confirmed species, the place of the stay and the duration of the exposure. On this case the authors call to mind the epidemiological criteria necessary for early diagnosis, the antimalarial drugs that can be used in pregnant women and draw attention to immuno-allergic and the classical secondary effects of quinine, which can also occur with chloroquine.

[Medullary aplasia during treatment for congenital toxoplasmosis in a twin pregnancy]. / Aplasie médullaire au cours du traitement d'une toxoplasmose congénitale lors d'une grossesse gémellaire.

The authors report a case of a patient who in the 24th week of a twin pregnancy became sero-positive for toxoplasmosis. This was diagnosed by cordocentesis as being infected, and the treatment was therefore started with pyrimethamine and sulfadiazine and folic acid at the 28th week of pregnancy. At 35 weeks, the patient had an acute medullary aplasia due to the absence of the folates. The mother's state was improved rapidly by giving her folinic acid and the twins were normal haematologically. In this case, the authors point out how important the folates are in a pregnancy, especially in twin pregnancies, and point out the precautions that have to be taken when treatment with pyrimethamine and sulfadiazine is started for congenital toxoplasmosis.

[Prevention and treatment of materno-fetal toxoplasmosis]. / Prévention et traitement de la toxoplasmose materno-foetale.

Preventing congenital toxoplasmosis is fundamental, and in France screening tests for primary infection are obligatory for pregnant women. All nonimmunized women should be encouraged to follow good dietary and general health rules until delivery. Nevertheless, the risk of seroconversion does exist but can be detected early through monthly serum tests. Spiramycin as initial treatment of maternal primary infection is an essential step in preventing parasite transmission to the fetus. Fetal antitoxoplasmosis antibodies and indirect signs of congenital abnormalities should be detected early by amniotic fluid and fetal blood tests before echographic evidence confirms the diagnosis. Pyrimethamine-sulfonamide therapy should be undertaken and modulated according to laboratory results in order to prevent or treat any possible fetopathy. Congenital toxoplasmosis can be prevented in utero through biological diagnosis and specific therapy.