B. Chumpitazi

Purification and immunochemical study of Plasmodium falciparum exoantigens.

Plasmodium falciparum, in in vitro culture, elaborated many antigens including soluble exoantigens that are released into the culture medium. Anionic and cationic methods of isolating these antigens offer a great potential for large scale purification from medium that is rich in proteins but contains relatively low concentrations of P. falciparum specific antigens. These exoantigens have cationic and anionic dependent elution profiles (pI between 3.7 and 4.8). Five apparent molecular weight entities (58, 80, 145, 200, and 290 kdaltons) have been determined by GEDELISA. Susceptibility to lipase and to a proteolytic enzyme confirmed the proteinaceous nature of the antigens. They were isolated from 4 strains of different geographic origin, indicating their ubiquitous nature. The analogy of these exoantigens to circulating antigens in patients with acute malaria and their potential usefulness in immunodiagnosis and immunoprophylaxis are discussed.

Characteristic and clinical relevance of Candida mannan test in the diagnosis of probable invasive candidiasis.

The gold standard laboratory tests used to diagnose invasive Candida infection (ICI) are based on the in vitro culture of blood or samples from other sterile sites. However, these tests have limited sensitivity (Se) and are generally not diagnostic until late in the infectious process. The Serion Candida mannan kit was evaluated for the diagnosis of ICI at Grenoble University Hospital (France) between 2007 and 2011. The results were then compared with worldwide data published between 1997 and 2011. This retrospective study was based on follow-up from the investigation of 162 patients of whom 91 had proven ICI; 13 had Candida colonization index (CCI) scores ≥0.42, positive mannan tests, with nonconcomitant infections; and 58 had no evidence of Candida infection. Candida albicans, C. glabrata, C. tropicalis, and C. parapsilosis were the etiologic agents in 104 patients. For patients with or without ICI, the 12-week mortality rates were 35/104 (33.7%) and 6/58 (10.3%), respectively. The mannan diagnostic specificity was 51% and Se was 77%. However, in the meta-analysis (n = 1,536), values were 86% and 62%, respectively. Positive mannan test results may appear early (median 6 days) in the development of candidemia and have moderate diagnostic value for ICI, with a negative predictive value of 83%. In patients at risk of ICI with negative candidemia, the combination of Candida mannan test data with a CCI score ≥0.42 may improve the diagnosis of probable ICI.

Immunological and biological characterization of Plasmodium falciparum exoantigens.

Abstract Exoantigens (E-antigens) of P. falciparum prepared by cationic exchange chromatography from asynchronous culture supernatant were evaluated by High Performance Liquid Chromatography (HPLC), Chromatofocusing, Enzyme Linked ImmunoSorbent Assay (ELISA), Gel Electrophoresis of Derivated ELISA (GEDELISA) and Immunoblotting. Their mol. wt after denaturation and immunoblotting were: 170,000,135,000 and 100,000. Their isoelectric points (pI) in their native forms were: ⩾ 6.3, 4.1 and ⩽ 3.7. Thermostability tests showed that 65% of their antigenic activity remained after 5 min at 100°C. Metabolic labelling with 3 H glucosamine, periodate oxidation and borohydride reduction showed that they were in part glycoproteins. These antigens are excreted or secreted during merozoite release and invasion of erythrocytes. Metabolically labelled ( 3 Ft leucine) culture fluid from synchronous P. falciparum cultures was analysed by Inhibition ELISA (ELISA-I) and CounterImmunoElectrophoresis (CIE) to determine the presence of E-antigens. These antigens inhibited the in vitro growth of Plasmodium falciparum .

Isolation and characterization of toxoplasma exo-antigens from In vitro culture in MRC5 and vero cells

Abstract Exo-antigens from the supernatant medium of in vitro cultures of Toxoplasmsa gondii on Vero and MRC5 cells have been characterized by different immunological techniques: enzyme-linked immunosorbent assay (ELISA), counter immunoelectrophoresis (CIE), twodimensional immunoelectrophoresis (2-DIEP) and gel electrophoresis of derivated ELISA (GEDELISA). These exo-antigens are similar in molecular sizes, thermostability and of proteic nature with glucidic components. Their enzymatic properties and precipitation arcs differ. The exo-antigens, produced on Vero cells (monkey kidney cells), seem to be more antigenic than those produced on MRC5 cells (human foetal lung cells). The formaldehyde treatment stabilizes the exo-antigens, in spite of eliminating certain epitopes, and increases their migration rate; it has practically no effect on their enzymatic or antigenic activities. The pI of the formolated exo-antigens produced on MRC5 cells are mainly under 5.06.

Ring-infected erythrocyte surface antigen (Pf/155RESA) induces tumour necrosis factor-alpha production.

Cerebral malaria is probably related to an overstimulation of the immune system and the cytokinc network. We have previously demonstrated that tumour necrosis factor (TNF) secretion by human macrophages can be induced by soluble and heat‐stable malarial antigens. Indirect evidence from epidemiological and in vitro studies suggests that Pf155/RESA can be considered as a candidate for triggering TNF secretion. Thus we conducted experiments to investigate the relationship between Pf155/RESA and TNF production. The SGE1 strain of Plasmodium falciparum was compared with the P. falciparum FCR3 strain, which does not express Pf155/RESA protein, for ability to induce TNF secretion by normal human macrophages in vitro. Synthetic peptides from the Pf155/RESA antigen ((EENV)4, (EENVEHDA)4, (DDEHVEEPTVA)3), were used in some experiments. TNF levels were measured by an immunoradiometric assay. We observed that the RESA‐defective strain induces lower levels of TNF after schizont rupture than the SGE1 strain. Moreover, substantial TNF secretion was detected when macrophages were incubated with all three peptides, maximum levels being obtained with the(EENV)4 peptide. Although previous reports have described TNF‐inducing activity of phospholipid from P. falciparum, these findings strengthen the evidence for Pf155/RESA antigens also being involved in TNF production during malaria.

Relationships between clinical protection and antibodies to Plasmodium falciparum RESA (ring-infected erythrocyte surface antigen) peptides.

A longitudinal study involving 76 individuals living in Dafinso and Vallée du Kou (near Bobo-Dioulasso, Burkina Faso, West Africa) was performed in June 1987 (beginning of the transmission period), August-September 1987 (during) and January 1988 (after). The serological antibody (Ab) responses against synthetic peptides representing repeat amino acid sequences of the P. falciparum Ring-Infected Erythrocyte Surface Antigen (RESA): (EENV)5, (EENVEHDA)4, (DDEHVEEPTVA)2 were evaluated by ELISA. The clinical longitudinal study during the transmission period allowed us to define three different groups in terms of age and occurrence of clinical malarial attack (greater than 5000 parasites mm-3 of blood and axillary fever greater than 37.7 degrees C). Levels (A620) of Ab to (EENVEHDA)4 and (DDEHVEEPTVA)2 were correlated with age. The adult group (III) had the highest prevalences of Ab to RESA peptides. No significant difference was found between groups of children with or without malaria attack. Nevertheless, at the beginning of the transmission period, children who had at least one malaria attack during the study presented the lowest level of antibodies to RESA peptides.

The immunodiagnostic value of six serological techniques in hydatidosis

Abstract Six different serological methods were tested for their ability to diagnose human hydatidosis. These were indirect immunofluorescence, indirect hemagglutination, counter immunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA). In all, 298 sera were examined: 151 sera from negative control subjects, 78 sera from patients with proven hydatidosis, and 69 sera from patients with disease not due to hydatidosis. The last group of samples were used to define the techniques specificity. The specificity and sensitivity of the two recent techniques: enzyme-linked immunoelectrodiffusion assay (ELIEDA) and immunoblotting (electrophoretic migration under denatured and reducing conditions of hydatid fluid antigens, blot and immunoenzymatic assay) were tested mainly on doubtful or weakly positive sera from patients with proven hydatidosis and with filariasis. The combination of enzymelinked immunosorbent assay, indirect immunofluorescence and counter immunoelectrophoresis or ELISA and ELIEDA gave the most dependable and reliable serodiagnostic results with our antigens. ELIEDA is the most useful technique if only one can be used. Our preliminary results with ELIEDA confirm the good specificity and sensitivity of this technique and allow, in some cases, an interpretation of false positive values obtained with ELISA and indirect immunofluorescence. This method also confirms, in most cases, the disease by revealing the arc 5 in patients with low specific IgG content. Immunoblotting in our experimental conditions has a lower sensitivity than ELIEDA but has a specificity for certain proteins (10–12, 15–18, 220, 230 kDa).

Contribution of anti-Hsp70.1 IgG Antibody Levels to the Diagnostic Certainty of Clinically Suspected Ocular Toxoplasmosis

PURPOSE Laboratory diagnosis of ocular toxoplasmosis, the major cause of posterior uveitis worldwide, can be improved. Heat shock protein (Hsp) 70 is involved in cellular infection by Toxoplasma gondii but also in the immune response to this parasite. The authors postulate that infected patients may exhibit serum IgG anti-Hsp70.1 antibodies and that determining the presence of these antibodies could improve the diagnosis of suspected ocular toxoplasmosis. METHODS This retrospective case-control study included 26 laboratory-confirmed cases of ocular toxoplasmosis (group A), 41 clinically suspected cases (group B), and 67 currently healthy blood donors who were chronically infected with T. gondii (group C). Laboratory and clinical data were analyzed according to the ocular presentation and Goldmann-Witmers coefficient. Serum and aqueous humor were sampled at the time of uveitis. Serum anti-Hsp70.1 antibody levels were obtained by ELISA. The probability of ocular toxoplasmosis was estimated by a logistic regression analysis that combined data from serum IgG anti-Hsp70.1 and aqueous-humor IgG anti-T. gondii antibody levels. RESULTS Serum IgG anti-Hsp70.1 antibody levels were significantly increased in groups A and B when compared to the levels in control group C (P ≤ 0.0034). These levels correlated with the retinal lesion size (r = 0.301; P < 0.0349). Logistic probability and anti-Hsp70.1 antibodies in sera confirmed that 10 of 23 cases in group B were true ocular toxoplasmosis. CONCLUSIONS Anti-Hsp70 may play a role in the immunopathogenesis of ocular Toxoplasma infection. This study showed that the anti-Hsp70.1 antibody and the logistic probability test can confirm clinically suspected ocular toxoplasmosis.

Correlation between β2-microglobulin and soluble interleukin-2 receptor levels in plasma of individuals living in a malarial endemic region.

beta 2-Microglobulin (beta 2m) levels were related to the expected immunoprotection in 81 individuals living in a malarial mesoendemic area near Bobo-Dioulasso (Burkina Faso), who were longitudinally followed. Soluble interleukin-2 receptor (sIL-2R) levels were positively correlated to those of beta 2m (r = 0.44; n = 237; P less than 0.001). This suggests that most of the beta 2m could have originated from activated T and B cell membrane turnover. In our study, both beta 2m and sIL-2R were inversely related to IgG antibodies (Ab) against somatic antigen of Plasmodium falciparum (Som-Ag). Therefore, these molecules at high levels could have a down regulating activity, directly or indirectly, on B cells producing this kind of Ab.

Circulating stable antigens at higher levels down-regulate antibody responses toPlasmodium falciparum

A study involving 169 schoolchildren (5–14 years old) living in Manarintsoa near Antananarivo (Madagascar, East Africa) was performed during the seasonal malaria transmission period. For the whole population examined, the prevalence ofPlasmodium falciparum and the rates of spleen enlargement and of circulating stable antigen (S-Ag) were found to be 60.9%, 71.7%, and 46,8%, respectively. The prevalence of IgG antibody to RESA (ring-infected erythrocyte surface antigen) was 42.7% and that of IgG and IgM antibodies to E-Ag (exoantigens) was 44.9% and 2.9%, respectively. The positive rates for IgG and IgM antibodies to Som-Ag (somatic antigen) were 48.5% and 5.9%, respectively. Concerning S−Ag, no significant relationship was observed for parasitemia, spleen size, age, or IgM antibody responses to exoantigens (E-Ag) or to somatic antigen (Som-Ag). Levels of S−Ag were found to be related to IgG antibodies to E-Ag. Our results suggest that S−Ag at low levels may participate in the mechanisms involved in the development of the IgG antibody responses to E-Ag and to Som-Ag, whereas at a comparative population level, higher quantities of S−Ag down-regulate antibody responses toP. falciparum. The data we obtained were compared with those gathered in another malaria mesoendemic area (Bobo-Dioulasso, Burkina Faso, West Africa), where lower levels of S−Ag were found.