Polyamine synthesis and interconversion by the Microsporidian Encephalitozoon cuniculi.

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.

The effects of elevated temperatures and various time-temperature combinations on the development of Brachiola (Nosema) algerae N. Comb. in mammalian cell culture.

Nosema algerae Vávra and Undeen 1970, a microsporidian known to cause infection in mosquitoes, develops in mammalian cell cultures at 24-35 degrees C and in the tails and footpads of athymic mice. More recently it has been reported to grow at 38 degrees C in human cell culture. The present study is a two-part temperature/development examination. The first part examines the development of N. algerae in rabbit kidney cell culture at 29 degrees C, which permits the formation of functional spores within 72 h, and compares the effect of elevated temperatures (36.0, 36.5, 37 degrees C) on parasite development. At these elevated temperatures, N. algerae infects but undergoes only one or two proliferative divisions, with no evidence of sporogony by 72 h post-inoculation. During this time, however, the host cells continue to divide resulting in fewer infected cells over time and giving the appearance of a diminished parasitemia. Additionally, at 37 degrees C some organisms degenerate/hibernate by 72 h while others remain viable/active. It is not until 96 h that the parasites appear in large clusters of proliferative stages in the few host cells that are infected. By 120 h post-inoculation, proliferative cells, sporoblasts, and early spores are observed. These results suggest that elevated temperatures impede proliferation rates and the onset of sporogony. The second part of this study evaluates developmental changes in N. algerae when incubation temperatures and times are varied during parasite growth, resulting in abnormal parasite morphology. These abnormalities were not present when parasites were grown at constant temperature (29-37 degrees C). This report demonstrates that N. algerae can successfully develop at high temperatures (37 degrees C), justifying its taxonomic relocation to the genus Brachiola.

Polar tube proteins of microsporidia of the family encephalitozoonidae.

Encephalitozoonidae are microsporidia associated with human infections including hepatitis, encephalitis, conjunctivitis, and disseminated disease. Microsporidia produce a small resistant spore containing a polar tube which serves as a unique vehicle of infection. Polar tube proteins (PTPs) from Encephalitozoon hellem. Encephalitozoon (Septata) intestinalis, and Encephalitozoon cuniculi were purified to homogeneity by HPLC. By SDS-PAGE, the Mr of E. hellem PTP was 55 kDa, while the Mr of E. intestinalis and E. cuniculi PTP was 45 kDa. Polyclonal rabbit antiserum to these purified PTPs localized to polar filaments by immunogold electron microscopy and immunofluorescence, and demonstrated cross-reactivity by both immunoblotting and immunogold electron microscopy. These PTPs have similar solubility properties, hydrophobicity, and proline content to a 43-kDa PTP we have previously purified from Glugea americanus, a fish microsporidium. As the polar tube is critical in the transmission of this organism, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.

The molecular characterization of the major polar tube protein gene from Encephalitozoon hellem, a microsporidian parasite of humans.

The microsporidia are obligate intracellular protozoan parasites of increasing importance as human pathogens, which are characterized by a small resistant spore with a single polar filament that coils around the sporoplasm. When stimulated, the polar filament rapidly everts out of the spore to form a hollow polar tube through which the sporoplasm passes, thus serving as a unique mechanism of transmission. A genomic library of the human microsporidium Encephalitozoon hellem was screened using a polyclonal rabbit antibody (anti-PTP Eh55) produced to the major HPLC purified polar tube protein (PTP) of E. hellem. This antibody localized to intrasporal polar filaments and extrasporal polar tubes of E. hellem by immunogold electron microscopy confirming the polar tube specificity of the antibody. A total of 14 anti-PTP Eh55 reactive genomic clones were identified and purified. A PTP gene was identified consisting of 1362 bp coding for 453 amino acids. The N-terminus of the translated protein consists of aputative N-terminal signal sequence of 22 amino acids, which when cleaved results in a mature protein of 431 amino acids with a predicted molecular mass of 43 kDa. The protein has a high proline content (14.6%) and contains a central domain of six alternating tandem repeats of 20 amino acids. After ligation of the gene into a glutathione S-transferase (GST) expression vector, a fusion protein was produced that reacted by immunoblotting with the polar tube specific anti-PTP Eh55. The gene was present as a single copy in the genome and there was no homology with other known genes. As the polar tube is a critical structure for the transmission of this organism to a new host cell, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.

Bradyzoite development in Toxoplasma gondii and the hsp70 stress response.

Toxoplasma gondii is a well-described ubiquitous Apicomplexan protozoan parasite that is an important opportunistic pathogen. The factors affecting the transition of tachyzoites to the latent bradyzoite stage remain to be defined. The induction of bradyzoite development in vitro has been linked to temperature, pH, mitochondrial inhibitors, sodium arsenite, and many of the other stressors associated with heat shock protein (hsp) induction. There is evidence for other organisms that hsps are developmentally regulated. Therefore, we examined whether hsp induction is an early event in bradyzoite differentiation. Extracellular and intracellular T. gondii cells, after exposure to pH 8.1 or 7.1, were analyzed for the expression of inducible hsp70 by using monoclonal antibody C92F3A-5 (specific to hsp70). Western blotting demonstrated that a 72-kDa protein reactive with C92F3A-5 (hsp70), which we believe is part of the hsp70 family, is induced during bradyzoite development. By immunofluorescence and immunoelectron microscopy, we were able to demonstrate that hsp70 staining colocalized to T. gondii expressing bradyzoite-specific antigens and the presence of hsp70 in bradyzoites isolated from mouse brain. Quercetin, a bioflavonoid which inhibits the synthesis of hsp90, hsp70, and hsp27, suppresses the induction of bradyzoite development in vitro. Reverse transcription-PCR with conserved hsp70 primers demonstrated an increase in hsp70 in T. gondii on exposure to conditions which induce bradyzoite formation. A T. gondii hsp70 was subsequently cloned and sequenced by using this amplified fragment. We believe our evidence suggests that hsps are important in the process of bradyzoite differentiation.

Brachiola vesicularum, n. g., n. sp., a new microsporidium associated with AIDS and myositis.

Brachiola vesicularum, n. g., n. sp., is a new microsporidum associated with AIDS and myositis. Biopsied muscle tissue, examined by light and electron microscopy, revealed the presence of organisms developing in direct contact with muscle cell cytoplasm and fibers. No other tissue types were infected. All parasite stages contain diplokaryotic nuclei and all cell division is by binary fission. Sporogony is disporoblastic, producing 2.9 x 2 microns diplokaryotic spores containing 8-10 coils of the polar filament arranged in one to three rows, usually two. Additionally, this microsporidium produces electron-dense extracellular secretions and vesiculotubular appendages similar to Nosema algerae. However, the production of protoplasmic extensions which may branch and terminate in extensive vesiculotubular structures is unique to this parasite. Additionally, unlike Nosema algerae, its development occurred at warm blooded host temperature (37-38 degrees C) and unlike Nosema connori, which disseminates to all tissue types, B. vesicularum infected only muscle cells. Thus, a new genus and species is proposed. Because of the similarities with the genus Nosema, this new genus is placed in the family Nosematidae. Successful clearing of this infection (both clinically and histologically) resulted from treatment with albendazole and itraconozole.

[Malformations of Wolffian duct derived male genital organs (epididymis, vas deferens, seminal vesicules, ejaculatory ducts)]. / Malformations des organes génitaux masculins issus du canal de Wolff (épididyme, déférent, vésicule séminale, canal éjaculateur).

OBJECTIVES: To analyse embryological phenomena, in order to define a practical approach to management. METHODS: The authors reviewed the files of eight boys with an abnormality of Wolffian duct derived genital organs, consisting of three cases of vas deferens agenesis, two of which were accompanied by homolateral renal agenesis, one case of ectopic vas deferens with contralateral renal agenesis, three cases of duplicated vas deferens and one congenital seminal vesicle cyst with renal agenesis. DISCUSSION: Congenital malformations of the epididymis consist of cysts and agenesis or partial atresia. Cysts essentially raise a problem of differential diagnosis. Treatment is only required in the case of severe discomfort. Vas deferens agenesis is the commonest lesion, an incidental finding in children. An experimental treatment is proposed in adults. Duplications, interdeferential communications and deferentomegaly are much rarer lesions. Seminal vesicle cysts are well visualized by transrectal ultrasonography and should be treated surgically. Lastly, ejaculatory ducts may present urethroseminal reflux or may be ectopic. CONCLUSION: The possibility of absent or ectopic vas deferens should be considered in the case of unilateral renal agenesis. Vas deferens agenesis justifies examination of the contralateral side and investigation to exclude renal agenesis. Congenital cysts of the epididymis only require surgery when they are symptomatic. The presence of ipsilateral renal agenesis should be investigated in the case of cystic dilatation of the seminal vesicle.

Identification of a microsporidian polar tube protein reactive monoclonal antibody.

The microsporidia are characterized by spores containing a single polar tube that coils around the sporoplasm. When triggered by appropriate stimuli, the polar tube rapidly discharges out of the spore forming a hollow tube. The sporoplasm passes out of the spore through this tube serving as a unique vehicle of infection. Due to the unusual functional and solubility properties of the polar tube, the proteins comprising it are likely to be members of a protein family with a highly conserved amino acid composition among the various microsporidia. Polar tube proteins were separated from the majority of other proteins in glass bead disrupted spores of Glugea americanus using sequential 1% sodium dodecyl sulfate (SDS) and 9M urea extractions. The resultant spore pellet demonstrated broken, empty spore coats and numerous polar tubes in straight and twisted formations by negative stain transmission electron microscopy. After subsequent incubation of the pellet with 2% dithiothreitol (DTT), empty spore coats were still observed but the polar tubes were no longer present in the pellet. The DTT supernatant demonstrated four major protein bands by SDS-PAGE: 23, 27, 34 and 43 kDa. Monoclonal antibodies were produced to these proteins using Hunter's Titermax adjuvant. Mab 3C8.23.1 which cross-reacted with a 43-kDa antigen by immunoblot analysis, demonstrated strong reactivity with the polar tube of G. americanus spores by immunogold electron microscopy. This antibody will be useful in further characterization of polar tube proteins and may lead to novel diagnostic and therapeutic reagents.

A cell culture system for study of the development of Toxoplasma gondii bradyzoites.

Toxoplasma gondii is a ubiquitous apicomplexan parasite and a major opportunistic pathogen under AIDS-induced conditions, where it causes encephalitis when the bradyzoite (cyst) stage is reactivated. A bradyzoite-specific Mab, 74.1.8, reacting with a 28 kDa antigen, was used to study bradyzoite development in vitro by immuno-electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii. Bradyzoites were detected in tissue culture within 3 days of infection. Free floating cyst-like structures were also identified. Western blotting demonstrated the expression of bradyzoite antigens in these free-floating cysts as well as in the monolayer. Bradyzoite development was increased by using media adjusted to pH 6.8 or 8.2. The addition of gamma-interferon at day 3 of culture while decreasing the total number of cysts formed prevented tachyzoite overgrowth and enabled study of in vitro bradyzoites for up to 25 days. The addition of IL-6 increased the number of cysts released into the medium and increased the number of cysts formed at pH 7.2. Confirmation of bradyzoite development in vitro was provided by electron microscopy. It is possible that the induction of an acute phase response in the host cell may be important for bradyzoite differentiation. This system should allow further studies on the effect of various agents on the development of bradyzoites.

[Analysis of the failure of endoscopic treatment of vesico-renal reflux in children using injections of teflon and collagen and the preliminary results of injections of Macroplastic]. / Analyse des échecs du traitement endoscopique du reflux vésico-rénal chez l'enfant par injections de téflon ou de collagène et résultats préliminaires des injections de Macroplastique.

This study of 785 cases of vesicorenal reflux in 494 children treated endoscopically over a 7-year period was designed to evaluate the results obtained with three products used successively: Teflon, collagen and Macroplastic. Following Teflon injection, despite a 90% short-term success rate, recurrent reflux was subsequently observed in 16.71% of the ureters reviewed. The failure rate was 52.63% after collagen injection and 11.77% after Macroplastic. After one or two injections, complete resolution of reflux was obtained in 48% of children treated with collagen, versus 85.72% with Teflon and 93.33% with Macroplastic. In one half of cases, failure was related to the quality of the product and its modifications after injection. The marked resorption of collagen accounts for the poor results despite the large doses injected. Apart from one case of partial resorption of Teflon paste, the failures with this product were due to lateralisation or secondary elimination of the product from the injection site due to its fluidity. Macroplastic, due to its higher viscosity and absence of retraction, currently provides the best results with doses of less than 0.20 ml in children.