Eduardo Dei-Cas

Polymorphism of the Thymidylate Synthase Gene of Pneumocystis carinii from Different Host Species

Pneumocystis carinii is an opportunistic agent found in the lung of various mammals which often causes severe pneumonia in immunocompromised humans, especially in AIDS patients. In the past several years significant additions have been made to the collection of knowledge we have concerning the genetic diversity of P. carinii. These additions provide new understanding of Pneumocystis transmission and the effect of possible reservoirs of Pneumocystis in the various species. In this study, a 400‐bp fragment of the thymidylate synthase (TS) gene of P. carinii has been amplified by PCR from 43 parasite isolates obtained from 4 mammalian host species: rat, mouse, rabbit and human. A probe selected from the TS gene sequence of rat‐derived P. carinii was hybridized with the amplified products from rat‐ and mouse‐derived P. carinii, but not with rabbit or human P. carinii DNA. Restriction profiles were performed on amplified fragments from all isolates, and the 4 nucleotide sequences of the TS gene fragment amplifed from rat, mouse, rabbit and human P. carinii were determined. Differences were detected in the gene fragment in P. carinii isolates from the 4 host species; however no difference was revealed in P. carinii isolates within a single host species, whatever the host strain or its geographic origin. Thus, the sequence differences of the P. carinii TS gene appeared as host‐species specific. A specific probe which recognized all human P. carinii isolates was defined.

In vitro attachment of Pneumocystis carinii from mouse and rat origin

Summary— The attachment of Pneumocystis carinii to lung cells could play a role in the pathophysiology of P carinii pneumonia. The trophozoite attaches to type I alveolar epithelial cells. Physical, chemical, and extracellular matrix factors, involved in the mouseor rat‐derived P carinii attachment to fibroblastic cells in culture, were examined using a new model of in vitro adherence. The development of parasite filopodia penetrating deeply the host cell cytoplasm was observed using transmission electronic microscopy. Killed P carinii organisms were unable to attach to cultured cells. Also, parasites were unable to attach to killed target cells. The P carinii in vitro attachment was partially inhibited by cytochalasin B. In contrast, the parasite attachment was not affected when the target cell cytoskeleton was altered. In our work conditions, sialic acids were not involved in the attachment process. Present results showed that fibronectin (Fn) plays a role in the parasite attachment, and suggest that a specific Fn‐binding receptor is present at the surface of mouse‐derived P carinii organisms.

In vitro systems in pneumocystis research

Most groups involved in Pneumocystis research need large quantities of well preserved, viable Pneumocystis organisms free of host cell contamination. Biological, biochemical, immunological, genetic or other studies on Pneumocystis usually involve the separation of Pneumocystis from lung tissue as well as elimination of host cell debris from parasite extracts. In other investigations, such as transmission, infectivity, life cycle, biochemical, in vitro culture or drug-screening studies, viable and infectious Pneumocystis organisms are urgently required. However, there is no generally accepted methodology for obtaining Pneumocystis from experimental hosts or from human clinical samples; methods are still far from reaching standardization, as discussed here by the members of the European Concerted Action (ECA) on Pneumocystis carinii, which is co-ordinated by Eduardo Dei-Cas and Jean-Charles Cailliez.

Pneumocystis carinii organisms from in vitro culture are highly infectious to the nude rat

Many in vitro systems have been used to cultivatePneumocystis, but only limited parasite growth has been obtained by different authors. A reliable in vitro system enabling a sustained propagation ofPneumocystis appears to be an important condition for a better definition of the transmission ofP. carinii pneumonia. In this work,Pneumocystis in vitro culture was performed on monolayers of L2 rat lung epithelial-like cells. Ultrastructural assessment revealed that culture parasites were structurally intact.Pneumocystis culture samples were intratracheally inoculated into corticosteroid-treated nude rats (nonlatently infected byP. carinii), which developedP. carinii pneumonia at 40 days postinoculation. The infectious power of parasites obtained in vitro was 7–10 times higher than that of parasites freshly extracted from parasitized rat lung. In summary, the present results show that it is possible to obtain in vitro highly infectiousPneumocystis forms, and this study provides a promising infectivity test for use by investigators working onPneumocystis in vitro systems.

Different ultrastructural morphology of Pneumocystis carinii derived from mice, rats, and rabbits

Pneumocystis carinii (PC) is a fungus present in the lungs of many mammal species. Even though studies of the genome, the isoenzymes, and the antigens have proved some host‐species‐linked heterogeneity, the existence of distinct Pneumocystis species or subspecies has still not been accepted. Comparative studies of the ultrastructural morphology of pneumocysts derived from several host species may support evidence of host‐species‐linked heterogeneity. We have compared the ultrastructural morphology of pneumocysts derived from mice, rats, and rabbits. The density of membrane‐limited electron‐dense cytoplasmic granules was found to be higher in mouse‐derived pneumocysts than in rabbit‐derived pneumocysts, and furthermore the average diameter of the granules from mouse pneumocysts was larger than that of granules from rabbit‐derived pneumocysts. The average diameter of the filopodia of mouse‐derived pneumocysts was smaller than that of filopodia from rat‐derived pneumocysts, which was smaller than that of filopodia from rabbit‐derived pneumocysts. Globular electron‐dense bulbous dilatations at the tip of the filopodia were described for the first time and they were only found on filopodia of mouse‐derived pneumocysts. These distinct host‐species‐linked morphological differences of pneumocysts from mouse, rat, and rabbit may support previous biochemical data indicating the existence of different Pneumocystis species or subspecies.

Inhibitory effect of a Pichia anomala killer toxin on Pneumocystis carinii infectivity to the SCID mouse

Abstract A Pichia anomala killer toxin has been demonstrated to have a specific inhibitory effect on the in vitro attachment of Pneumocystis carinii. The results presented herein show that this yeast toxin is also effective against P. carinii infectivity in reducing parasite colonization in the lungs of SCID mice. The specificity of this inhibitory effect was controlled using a monoclonal antibody neutralizing the killer properties of the yeast toxin.

Erythrocytes carrying mutations in spectrin and protein 4.1 show differing sensitivities to invasion byPlasmodium falciparum

The role of the erythrocyte skeleton in the invasion process ofPlasmodium falciparum was evaluated using genetically variant erythrocytes containing well-defined molecular defects in α spectrin (αSp) or protein 4.1 from eight unrelated families. Invasion into red cells from subjects of three black families with hereditary pyropoikilocytosis (HPP) due to inheritance of αI/74 mutant spectrin was significantly reduced in cells both from the patients and from the relatives of these who carried asymptomatic hereditary elliptocytosis (HE). Like-wise, reduced invasion was also seen in red cells from two families with HE in which the αI/65 variant spectrin was present. Resistance to invasion was not absolute in any sample and varied between 38% and 71% of that seen in normal cells. The decreased invasion correlated with the percentage of spectrin dimers present within the membrane of variant cells. In contrast, invasion into elliptocytes from three families that had a partial deficiency in protein 4.1 (HE/4.1+) but a normal percentage of spectrin dimers was either unchanged or increased. The precise mechanism and molecular basis behind the reduced invasion into HPP and HE red cells bearing SpαI domain variants remains to be elucidated but might relate to alterations in merozoite/red cell-receptor interactions and/or merozoite endocytosis. The occurrence of elliptocytosis with spectrin defects (in particular, SpαI/65 and SpαI/46 variants in West Africa) suggests that these mutations of the αSp gene could be related to some protection against malaria.

Golgi complex and lysosomes in rabbit derived Pneumocystis carinii.

The ultrastructural morphology of Pneumocystis carinii obtained from nonimmunosuppressed rabbit is described in details. Golgi complex and primary lysosomes of P carinii are described here for the first time. They are easily revealed by the zinc iodide‐osmium tetroxide cytochemical reagent. Thiamine pyrophosphatase and β‐glycerophosphatase activities are found in the parasite but cytidine 5′ monophosphatase activity is not observed. A weak thiamine pyrophosphatase activity is detected in Golgi vesicles. An endomembranous saccular structure, present from the intracystic body stage to the precystic stage, apparently plays the role of secondary lysosome. A second type of endomembranous saccular structure, only present in the well developed trophozoitic and precystic forms is also described. The presence of carbohydrates in the cell wall of the parasite was demonstrated by periodic acid‐thiosemicarbazide‐silver proteinate staining and lectin concanavalin A labeling. The development of Golgi vesicles preceded the transition from double‐layered to three‐layered parasite stages.

High osmotic pressure enables fine ultrastructural and cytochemical studies onPneumocystic carinii

High osmotic pressure was used to preserve the ultrastructure of rabbit-, SCID mouse-, and rat-derivedPneumocystis carinii organisms from osmotic stress during fixation. Organelles and cytosol were well preserved within the tonicity range of 850–1,300 mosmol. Under these experimental conditions, we determined that the endoplasmic reticulum was well developed in all parasite stages and could observe the Golgi complex, autophagic vacuoles, dense bodies, type II endoplasmic saccules, and the recently described outer surface membrane, which was found in all parasite stages. The biological implications of these findings are discussed.

Pneumocystis carinii Detection in 158 HIV-seronegative Patients

Pneumocystis carinii pneumonia (PCP) is the most common severe pulmonary infection among AIDS patients living in Europe and in United States. Clinicians are also confronted with an yearly increasing number of PCP cases in patients without AIDS, immunodepressed by a well defined malignancy and/or a disorder treatedwith immunosuppressive agents [1,6]. In this population, PCP usually occurs in a more fulminant manner than during AIDS PI. Because of this short clinical course, the diagnosis must be estaWed quicldy and unequivocaUy. But it is quite difXcult to establish on clinical and radiological data a definitive diagnosis of PCP. Actually, P. carinii detection in pulmonary specimens, like bronchoalveolar lavage fluid (BAJI) or induced sputum, is needed The usual detection is performed using classical stainings like GiemsaWre, Toluidine Blue 0 or methenamine silver stains, as well as immunofluorescence assays. Since few years, PCR assays have been developed [7] and evaluated especially in HIVpositive populations, much less on immunodepressed patients Without AIDS. A highly sensitive detection assay like PCR appears particularly interesting because of the presence of considerably fewer parasites in the lungs of HIVuninfected patients than in those with HIV infection during PCP [4]. Moreover, it allows to investigate a potential pulmonary carriage, related to P. carinii air borne transmission. The main goal of our study was to investigate and to compare the PCP frequency with that of P. carinii camage, in an important HIV-negative populatim. The P. carinii detection was performed on 17 1 BALF specimens harvested on 162 patients, using microscopical staining methods compared with a PCR assay followed by hybridization with a digoxigenin-labeled probe. MATERIALS AND METHODS. The study was performed at the Universty Hospital Center of Lille, within one year. One hundred sixty two patients were included in this study. They were admitted to many clinical services. Clinical, radiological and biological data were obtained by retrospective medical chart survey. The P. carinii deteclion tests were performed on 17 1 BALFs. Five patients have been submitted to 2 BALs, 2 patients to 3 ones. Before any laboratory handling, BALF samples were divided in 2 aliquots. Smears were prepared with the first one. They were stained With a Giemsa-like staining, -5 5 5 , to visualize trophic and cystic forms, with Toluidine Blue 0 or GumoriGrocott to detect cyst~c forms. DNA extraction and amplification were perfomed on the second aliquot as previously described [S]. Briefly, the samples were incubated with SDS audprotekase K at 5OoC overnight. Then, conveational phenolchloroform extraction was pedormed, followed by ethanol precipitatioa. The amplitication of a thymidylate synthase gene 398bp-fragment was made as previously described [2,5]. After electrophoresis and capillary transfer, hyixidbtimswre perf‘ormed “ing a specific digoxigenin labeled probe, at 42OC for at least 4 hours. Then 3 successive washings were made: the first at room temperature in SSCZX, SDS 0.1%, the second at 68OC in SSC IX, SDS 0.1% and the third at 68OC in SSC 0.1X. The chemiluminescent revelation was performed following the manufacturer‘s recommendations. The luminescent signal was scored in a semi-quantitative fashion (+,++,+). To avoid contamination, the DNA preparation of the samples, the addition of the PCR reagents and the detection procedures were pefomed in physically separated areas with diEerent sets of micropipettes. Controls without template and positive controls were included in each experiment. Differences in independant variables were determined by using X’