Encephalocytozoon intestinalis-specific monoclonal antibodies for laboratory diagnosis of microsporidiosis.

Two monoclonal antibodies which can be used for the unambiguous identification by fluorescence microscopy of Encephalitozoon intestinalis spores in clinical specimens are described. Monoclonal antibody Si91 is specific for the extruded polar filament, and Si13 recognizes the surfaces of E. intestinalis spores. No cross-reaction with spores of Encephalitozoon hellem was observed. Immunogold electron microscopy confirmed the specific reactivities of both antibodies. Combined in an indirect immunofluorescence assay, these antibodies are used to identify spores in feces. Although there was some cross-reaction with fecal bacteria and fungi, the typical morphology of the extruded polar filaments enabled proper identification of the E. intestinalis spores. Parasites could also be demonstrated to be present in urine, nasal swabs, lung brush biopsy specimens, and bronchoalveolar lavage fluid from a patient with disseminated infection with E. intestinalis. The use of these monoclonal antibodies facilitates the detection and species determination of E. intestinalis in clinical specimens.

Association between anti-Pfs48/45 reactivity and P. falciparum transmission-blocking activity in sera from Cameroon.

Pfs48/45, a sexual stage parasite protein doublet of P. falciparum, is a target of antibodies which inhibit the development of the parasite in the mosquito. Twenty-eight (54%) out of 52 sera of gametocyte carriers from Cameroon reduced infectivity in the mosquito membrane feeding bioassay to less than 20% of the controls. These 52 sera were analysed by competition ELISAs for the presence of antibodies capable of competing the binding of six monoclonal antibodies (MoAbs) directed against five different epitopes on Pfs48/45. The percentage of these 52 Cameroon sera that competed with one of the MoAbs ranged from 13% (epitope I) to 33% (epitope IIc). Comparison of activity in the transmission-blocking assay (> or = 80%) and in the Pfs48/45 competition ELISA show a relative specificity of 100% (24 of 24) and a relative sensitivity of 75% (21 of 28). Non-blocking sera showed no competition with any of the MoAbs. These MoAbs were further used to study the diversity of epitopes among isolates of P. falciparum using a two-site ELISA. MoAbs against epitope I, III and V reacted with four different isolates whereas epitope II could be subdivided into three epitopes. None of the isolates reacted with MoAb 3G12 (epitope IV). Using these four different isolates, the competition ELISA titre varies from 1/20 to 1/80 and no significant differences are found between the isolates except for epitope II where only three out of 11 positives for epitope IIa were also positive for epitope IIc.

Plasmodium falciparum: a comparison of the activity of Pfs230-specific antibodies in an assay of transmission-blocking immunity and specific competition ELISAs.

The activity of monoclonal antibodies (mAbs) that specifically recognize the Plasmodium falciparum sexual stage-specific protein Pfs230 was analyzed. All mAbs reacted with the surface of extracellular sexual forms of the parasite in a suspension immunofluorescence antibody reaction and precipitated the Pfs230 protein from an NP-40 extract of surface radioiodinated macrogametes/zygotes. Only mAb that bound complement blocked transmission, whereas mAb that did not bind complement but competed with the complement-binding mAb for binding to the same epitope did not block transmission. These mAbs were used to develop Pfs230-specific competition ELISAs to analyze epitope diversity and to analyze the binding characteristics of anti-Pfs230 antibodies in human serum. Transmission-blocking (TB) antibodies in test/field sera competed in the competition ELISA for binding with epitope-specific, labeled mAbs against Pfs230. At least five different epitope regions could be defined with the competition ELISAs. All 46 sera from gametocyte carriers immunoprecipitated the Pfs230 molecule, while 19 of these sera blocked transmission in the bioassay. Five of the transmission-blocking and one of the nonblocking sera competed with monoclonal antibodies. A method comparison analysis was used to determine agreement between reactions in a competitive ELISA and the TB activity examined in the bioassay. The index of agreement kappa between outcomes of the bioassay and ELISA was fair to poor (kappa = 0.25) but since its range includes values below 0 the relation between the data obtained by the bioassay and the competition ELISA can be explained by chance alone. The serological data did not reveal a correlation between immunoprecipitation of Pfs230 and TB activity.

A comparison of transmission-blocking activity with reactivity in a Plasmodium falciparum 48/45-kD molecule-specific competition enzyme-linked immunosorbent assay.

Monoclonal antibodies (MAbs) 32F1 and 32F3 react with two independent epitopes of a protein doublet with molecular weights of 48 and 45 kilodaltons (kD) expressed on the surface of Plasmodium falciparum (Pfs48/45) macrogametes and zygotes; only 32F3 blocks transmission. These MAbs were used to develop a Pfs48/45-specific competition enzyme-linked immunosorbent assay (ELISA) using 32F1 to capture antigen and labeled 32F3 for quantification and analysis of the contribution of antibodies in human serum to transmission-blocking activity. A comparison analysis was used to determine agreement of competition ELISA titers and transmission-blocking activity as observed in the bioassay in three groups of serum samples: 37 from European travelers with previous exposure to malaria, 56 from gametocyte carriers, and 66 from schoolchildren from a malaria-endemic area in Cameroon. The index of agreement between outcomes of the ELISA and transmission-blocking assay in gametocyte carriers and in travelers was specifically defined as fair-to-moderate; in schoolchildren the agreement was not significant. The combined analysis of all sera showed a significant and fair-to-moderate agreement between the results of the competition ELISA and the transmission-blocking assay, with a relative specificity of 94% (of 105 cases negative in the transmission-blocking assay, 99 were also negative in the competition ELISA) and a relative sensitivity of 44% (of 54 cases positive in the transmission-blocking assay, 24 were also positive in the competition ELISA). This study shows that a positive C48/45-ELISA is indicative for transmission-blocking activity in the mosquito assay, while a negative result does not exclude transmission-blocking activity.

Pneumocystis carinii pneumonia in renal transplant recipients.

In 1991 and 1992 Pneumocystis carinii pneumonia (PCP) was diagnosed in 28 renal transplant recipients. The incidence of PCP in our renal transplant centre was remarkably increased from 1.1% before 1991 to 11.5% in 1991-1992. We compared 28 PCP patients with a control group of 27 renal transplant recipients, matched for transplantation day and without an episode of PCP. The mean age was significantly higher in the PCP group (50 +/- 13 versus 38 +/- 13 years). We observed no differences in basic immunosuppressive and rejection treatment nor in antibiotic consumption, number of hospitalization days, and incidence of CMV infection. In March 1993 we introduced PCP prophylaxis. More than 140 renal transplant recipients received co-trimoxazole, starting 1 day after transplantation and continued for a period of 4 months. To the time of writing no one in this group had developed PCP.

Cloning and expression of the gene coding for the transmission blocking target antigen Pfs48/45 of Plasmodium falciparum.

The gene encoding the gametocyte/gamete-specific membrane protein Pfs48/45 of Plasmodium falciparum has been cloned. The Pfs48/45 gene is a non-interrupted, single copy gene that codes for a hydrophobic, non-repetitive protein of 448 amino acid residues containing a putative signal peptide at the N-terminus, a hydrophobic C-terminus and 7 potential N-glycosylation sites. Antibodies directed against a Pfs48/45-glutathione-S-transferase fusion protein reacted with both the 45-kDa and 48-kDa proteins of gametocytes. When Pfs48/45 is expressed in the baculovirus-insect cell system the recombinant Pfs48/45 protein is targeted and exposed to the insect cell surface in such a configuration that it is recognized by transmission-blocking anti-45/48-kDa monoclonal antibodies.

Comparison of serological tests and bio-assay for malaria transmission blocking capacity in field sera.

Competition ELISAs have been developed for natural transmission blocking antibodies. Approximately 50% of the sera blocking in the conventional mosquito feeding experiments, gave positive results in these competition ELISAs. Attempts to adapt competition ELISAs to a field application have been partly successful.

Peptides reactive with a transmission-blocking monoclonal antibody against Plasmodium falciparum Pfs25: 2000-fold affinity increase by PEPSCAN-based amino acid substitutions.

Based on PEPSCAN analyses, a peptide derived from the 25-kDa surface protein of P. falciparum sexual stages (LDTSNPVKT, amino acids 122-130) was recently shown to react with transmission-blocking monoclonal antibodies. Subsequently, a pinbound construct (FDDTDPIKK; six amino acids substituted) was designed that reacted 1000 times better with the transmission-blocking monoclonal antibody 32F81 than with the parent LDTSNPVKT peptide. While this construct was obtained through single amino acid replacement studies, it now is shown that the combined contribution of the various substitutions is necessary to give the total effect. Free peptides comprising LDTSNPVKT or FDDTDPIKK were shown to inhibit the interaction of the transmission-blocking monoclonal antibody with plate-bound peptides, when prepared by conventional synthesis procedures. Compared with peptides that contained LDTSNPVKT, similar levels of inhibition could be achieved with an average 500-fold lower molar amount of peptides containing the FDDTDPIKK sequence. Affinity constants of the peptides for the transmission-blocking monoclonal antibody ranged from 1.3 x 10(5), for peptides that contained LDTSNPVKT, to 2.6 x 10(8), for peptides that contained FDDTDPIKK. The structural relationship between the epitope of the 25-kDa surface protein and the peptides was demonstrated by competition experiments with the transmission-blocking monoclonal antibody 32F81. Again, peptides comprising the newly designed sequence FDDTDPIKK competed about 200 times better than peptides with the parent LDTSNPVKT sequence.

The in vitro cultivation of P. falciparum ookinetes, and their enrichment on Nycodenz density gradients.

Employing a simple method of growing ookinetes of Plasmodium falciparum in culture, 40% of mature gametocytes convert to macrogametes, 4% reach the retort-form ookinete stage and 0.45% become mature ookinetes. A single-step gradient centrifugation method on 12.5% Nycodenz enriches both gametocytes and ookinetes.

Recent developments in the immunobiology of Pneumocystis.

Available data on the immune responses to Pneumocystis are discussed leading to the general conclusion that T lymphocytes and monocytic cells are involved in host defence. Recent progress in characterization of parasite antigens is reviewed with special reference to the development of monoclonal antibodies specific for Pneumocystis.

Sequential expression of antigens on sexual stages of Plasmodium falciparum accessible to transmission-blocking antibodies in the mosquito.

Plasmodium falciparum gametocytes contain specific antigens, some of which (Mr 230,000, 48,000, 45,000) are expressed on the surface of the newly emerged macrogamete. A different antigen (Mr 25,000) surrounds the surface of the ookinete and, although present to some extent in the developing gametocyte, is synthesized in high quantities by the macrogamete/zygote and expressed progressively on the transforming zygote surface. These antigens are targets of transmission blocking antibodies that are effective at two distinct points after gametogenesis: fertilization of the macrogamete and ookinete to oocyst development. The antigens involved in the fertilization blockade are the Mr 48 and 45 proteins, which are expressed on the macrogamete surface. The Mr 230 K coprecipitating protein probably plays no part in transmission block. mAb directed against the Mr 25 K ookinete surface protein blocked transmission without inhibiting ookinete formation, indicating that this protein has an important role in the transformation of ookinete into oocyst. A combination of mAb recognizing different epitopes on the same protein molecule acted synergistically in inhibiting oocyst formation. Using a mixture of two blocking mAb reacting against the Mr 48/45 and 25 K proteins, respectively, an additive blocking effect could be demonstrated.

Plasmodium falciparum transmission blocking monoclonal antibodies recognize monovalently expressed epitopes.

Target proteins of transmission blocking monoclonal antibodies (MoAbs) are present on the surface of Plasmodium falciparum macrogametes with Mr of 48,000 and 45,000 and on the surface of developing ookinetes with Mr of 25,000. Other MoAbs directed against the same proteins were not able to reduce the number of oocysts in mosquitoes. A combination of a blocking MoAb with a non-blocking one potentiated the transmission blocking effect. This implies that at least two different epitopes are present on the target antigens. This was confirmed using a sandwich immunoradiometric assay. The results demonstrated that on the 48/45 kD proteins as well as on the 25 kD protein a "blocking" and a "non-blocking" epitope exist which are not repeated anywhere else in the molecule.

Developmental biology of Pneumocystis carinii, and alternative view on the life cycle of the parasite.

In this paper we present, based on elaborate ultrastructural studies, data on the existence of both intracellular and extracellular stages of Pneumocystis carinii, which result in a proposal of a new life cycle of the parasite. Up to now the formation of daughter cells in thick-walled pneumocysts is supposed to be the only way of multiplication. The present study shows that in rats treated with cortisone acetate the formation of daughter cells also takes place within thin-walled pneumocysts. In our opinion this way of multiplication is important for the understanding of the rapid increase in number of the parasites in an infected lung. The presence of pneumocysts inside the alveolar epithelial cells suggests that intracellular development of the parasites can occur, but the method of cell penetration, intracellular multiplication and parasite liberation is still unknown. Moreover our observations for the first time indicate a direct pathogenicity of the parasites in host cells.

Parasitologic and serologic observations of infection with Pneumocystis in humans.

More than 500 specimens of lung tissue were examined for Pneumocystis. Of the 38 infections detected, most were in immunodeficient patients. Samples of serum from approximately 600 healthy normal subjects and 117 children with acute lymphatic leukemia were examined by an indirect fluorescent antibody test. The age-related data from the normal children suggested that nearly 100% of children are infected with Pneumocystis during the first two years of life. Groups of patients with leukemia who had symptoms of pneumocystis pneumonia had significantly higher titers of IgG antibody than groups of patients with leukemia who did not have clinical symptoms and normal subjects. Nevertheless, the diagnostic value of the indirect fluorescent antibody test is limited, but serologic follow-up study can be useful. Groups of children with leukemia had lower mean titers of IgM antibody regardless of their clinical condition.

New aspects of the life cycle of Pneumocystis carinii.

Pneumocystis is recognized as a cause of pneumonitis in vertebrates, including man. So far an extra-cellular life cycle has been proposed; thick-walled cysts are the site of the formation of so-called intracystic bodies. These intracystic bodies are set free and are supposed to develop into free living thin-walled Pneumocysts ("trophic" stage) (Seifert and Pliess, 1960; Vavra and Kucera, 1970; Campbell, 1972). In this paper we present, based on elaborative ultrastructural studies, data on the existence of both intra-cellular and extra-cellular stages of the parasite which result in a new proposal for the life cycle of the parasite. This short communication will be followed by a more detailed description of our observations.

The effect of changes in the respiratory metabolism upon genome activity. A correlation between induced gene activity and an increase in activity of a respiratory enzyme.

The in vitro regression of experimentally induced chromosome puffs was investigated in explanted salivary gland chromosomes of Drosophila hydei. It was observed that the regression of the puffs 2-32A, 2-36A, 2-48C, and 4-81B is accelerated if substrates for the respiratory metabolism are supplied to the cells. A similar effect can be produced by addition of KCN or oligomycin to medium in which intact salivary glands are incubated. The acceleration of puff regression by these substances occurs not only if the puff-inducing stimulus is removed but as well under conditions in which the stimulus is maintained. Regression of the puffs 2-32A, 2-36A, and 4-81B is inhibited if cycloheximide is present in the incubation medium. Chloramphenicol has no effect on puff regression. Measurements on nicotinamide adenine dinucleotide-dehydrogenase activity in homogenates of salivary glands revealed an increase in enzyme activity of 41 %. Maximum increase is attained at 30 min after the induced puffs have reached their maximum size. The increase in enzyme activity does not occur if the glands are kept in a medium containing either actinomycin D or cycloheximide. Chloramphenicol does not inhibit the increase in enzyme activity. The possible relationship between puff activity and its control as a result of changes in the respiratory metabolism is discussed.