Extracellular like-gregarine stages of Cryptosporidium parvum.

The present study confirms the existence of extracellular stages of Cryptosporidiumparvum during in vitro culture on MDCK, HCT 8 and Vero cells as well as alveolar macrophages, by optic, Nomarski and transmission electron microscopy images. Extracellular trophozoite/gamont, stages in syzygy, zygotes and spores with eight sporozoites were seen in the supernatant of the cultures. The first ultrastructural images of extracellular stages of C. parvum are shown in this study. The morphology of these stages, which have characteristics similar to those of some gregarines, support the contention that Cryptosporidium has closer affinity with gregarines. It also supports the necessity of reconsidering the life cycle of Cryptosporidium and the classification within the coccidia.

Transplante pulmonar: evolución y complicaciones: experiencia de Clínicas las Condes / Lung transplantation: evolution and complications: clinical experience

El transplante pulmonar es una alternativa terapéutica con enfermedad pulmonar severa sin respuesta a tratamiento. Objetivo: Evaluar los resultados y complicaciones de los pacientes transplantados en Clínica las Condes. Materia y método: 21 pacientes transplantados entre abril 1999 a mayo de 2003: 10 transplantes simples y 11 bipulmonares, edad x48 años (13-70); 13 hombres; patología predominante fibrosis idiopática: 10 (48 por ciento). Espirometría basal en patología restrictiva CVF: x 1.827 ml (43 por ciento) (1.170 - 2.430 ml) y obstructiva: VEF1 x 818 ml (24 por ciento) (352 - 1.756 ml), todos dependientes de oxígeno. Inmunosupresión triasociada: prednisona, azatioprina y ciclosporina o tacrolimus. Resultados: 3 pacientes fallecen 30 días por hemorragia cerebral, hemoptisis masiva y sepsis respectivamente. Seguimiento de 2 a 45 meses. Evolución espirométrica (VEF1): 1 año 2.107 ml (67 por ciento), 2 años 2.012 ml (65 por ciento) y 3 años 2.440 ml (74 por ciento). Todos suspenden oxígeno y realizar actividad física. Complicaciones; Disfución primaria de injerto 2, rechazo agudo 4, lesión vía aérea 2, neumonia por Citomegalovirus 2, síndrome de bronquiolitis obliterante (SBO) 3 y fallecen dos. Conclusiones: El transplante pulmonar mejora calidad de vida en pacientes con enfermedad pulmonar terminal y nuestros resultado corfirman que es una alternativa posible en nuestro país.

Seroprevalence of anti-cysticercus antibodies among the children living in the urban environs of Maputo, Mozambique.

Blood and faecal samples were collected from 269 children (aged 0-15 years) who lived in the urban environs of Maputo, the capital city of Mozambique. Antibodies against Cysticercus cellulosae were detected, at a titre of at least 1:100, in 56 (20.8%) of the blood samples. When the stool samples were checked for Taenia solium and other helminths, both as direct smears and after formalin-ether concentration, 180 (67.0%) were found to contain at least one helminth species. The parasites most commonly detected in the faecal samples were Trichurus trichiura (36.0%) and Ascaris lumbricoides (35.7%). Only in one sample (0.4%) were gravid proglottids of Ta. solium detected, but Hymenolepis nana (1.1%) and H. diminuta (0.4%) were also found. A positive correlation between seropositivity for anti-cysticercus antibodies and subject age, and positive associations between such seropositivity and infection with A. lumbricoides and infection with Tr. trichiura were observed. None of the other demographic and environmental factors investigated--the child's sex, religion and access to toilets and/or piped water, the type of house in which he or she lived, the number of individuals in the household to which he or she belonged, and whether that household had pets or raised livestock--showed any apparent association with either the seroprevalence of anti-cysticercus antibodies or infection with any intestinal helminth. The use of water from the common sewage-drainage system for agricultural irrigation in the study area probably causes most of the contamination with intestinal parasites.

In vitro culture of Glugea sp.

Recently the low host specificity of some microsporidians has been demonstrated and it has been indicated that many of these micro-organisms could be transmitted from invertebrates to mammals and adapt to changes in temperature. In this work, we demonstrate the first successful in vitro culture of a fish microsporidia of the genus Glugea on larval cells of the mosquito Aedes albopictus at 28 degrees C, and we show ultrastructural aspects of the different life cycle stages. It was impossible on salmon cells CHSE-214 at 21 degrees C. This study will be valuable for further work in biochemistry and immunology in addition to chemotherapy for microsporidiosis humans and animals.

Antigen incorporation on Cryptosporidium parvum oocyst walls.

Cryptosporidium parvum oocysts are the infective stages responsible for transmission and survival of the organism in the environment. In the present work we show that the oocyst wall, far from being a static structure, is able to incorporate antigens by a mechanism involving vesicle fusion with the wall, and the incorporation of the antigen to the outer oocyst wall. Using immunoelectron microscopy we show that the antigen recognized by a monoclonal antibody used for diagnosis of cryptosporidiosis (Merifluor(R), Meridian Diagnostic Inc.) could be found associated with vesicles in the space between the sporozoites and the oocysts wall, and incorporated to the outer oocyst wall by an unknown mechanism.

Contribution of steroidogenic factor 1 to the regulation of cholesterol synthesis.

Steroidogenic factor 1 (SF-1) is an orphan member of the nuclear receptor family expressed in steroidogenic tissues, where it has an essential role in the regulation of the steroid hormone biosynthesis, adrenal and gonadal development and endocrine responses fundamental for reproduction. Here we show that SF-1 regulates the transcription of cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene, which is essential for the endogenous synthesis of cholesterol. We have identified an element located 365 bp upstream of the gene for cytosolic HMG-CoA synthase; SF-1 binds as a monomer to this element and confers SF-1 responsiveness to homologous and heterologous promoters. It has been shown that in tissues with a high demand for cholesterol to be used in steroid synthesis, there is a lack of correlation between the cholesterol levels and the activity of the limiting enzymes of the mevalonate pathway. In accord with those results, we observed that cholesterol synthesis from acetate and either cytosolic HMG-CoA mRNA expression or transcriptional activity were not changed in response to 25-hydroxycholesterol in the SF-1-expressing steroidogenic Leydig tumour MA-10 cells. Moreover, the overexpression of SF-1 in non-steroidogenic CV-1 cells renders them less sensitive to the regulatory effects of cholesterol. This observation led to the hypothesis that in steroidogenic tissues the expression of SF-1 permits high levels of endogenous synthesis of cholesterol irrespective of the intracellular levels of this metabolite.

Inhibition of cryptosporidium parvum in vitro by 9-(alkylthio)acridine derivatives.

The synthesis of several acridinic thioethers is described. Compounds prepared were tested in vitro as potential drugs against the opportunistic infection known as cryptosporidiosis. With a view to predict activity, the quantitative structure-activity relationships were investigated. Correlations between experimental data and either log P or pKa are discussed.

Sterol regulatory element binding protein-mediated effect of fluvastatin on cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A synthase transcription.

The effects of acute treatment with fluvastatin, a hypocholesteremic drug, on the mRNA levels of several regulatory enzymes of cholesterogenesis and of the LDL receptor were determined in rat liver. Fluvastatin increased the hepatic mRNA levels for HMG-CoA reductase up to 12-fold in 5 weeks of treatment at a daily dose of 6. 3 mg/kg. The effect was less marked in cytosolic HMG-CoA synthase, farnesyl-PP synthase, squalene synthetase, and LDL receptor. SREBP-2 mRNA levels were also increased, but SREBP-1 were not. De novo synthesis of cholesterol in several cultured cells was reduced by increasing concentrations of fluvastatin, and the IC(50) values of fluvastatin in HepG2, CV-1, and CHO cells were respectively 0.01, 0. 05, and 0.1 microM. When CHO cells stably transfected with a chimeric gene composed of the promoter of cytosolic HMG-CoA synthase and the CAT gene as a reporter were incubated with fluvastatin, the CAT gene was overexpressed, an effect which was similar to the cotransfection with the processed form of SREBP-1a. Both ALLN and fluvastatin increased the transcriptional activity of cytosolic HMG-CoA synthase. Mutation in either SRE or NF-Y boxes abolished the increase in transcriptional rate caused by fluvastatin in the promoter of cytosolic HMG-CoA synthase. These results indicate that the increase in transcriptional activity in the HMG-CoA synthase gene attributable to fluvastatin is a consequence of the activation of the proteolytic cleavage of SREBPs by reduced levels of intracellular cholesterol.

Acute intestinal anisakiasis in Spain: a fourth-stage Anisakis simplex larva.

A case of acute intestinal anisakiasis has been reported; a nematode larva being found in the submucosa of the ileum of a woman in Jaén (Spain). The source of infection was the ingestion of raw Engraulis encrasicholus. On the basis of its morphology, the worm has been identified as a fourth-stage larva of Anisakis simplex. In Spain, this is the ninth report of human anisakiasis and also probably the first case of anisakiasis caused by a fourth-stage larva of A. simplex.

Control of human muscle-type carnitine palmitoyltransferase I gene transcription by peroxisome proliferator-activated receptor.

The expression of several genes involved in intra- and extracellular lipid metabolism, notably those involved in peroxisomal and mitochondrial beta-oxidation, is mediated by ligand-activated receptors, collectively referred to as peroxisome proliferator-activated receptors (PPARs). To gain more insight into the control of expression of carnitine palmitoyltransferase (CPT) genes, which are regulated by fatty acids, we have examined the transcriptional regulation of the human MCPT I gene. We have cloned by polymerase chain reaction the 5'-flanking region of this gene and demonstrated its transcriptional activity by transfection experiments with the CAT gene as a reporter. We have also shown that this is a target gene for the action of PPARs, and we have localized a PPAR responsive element upstream of the first exon. These results show that PPAR regulates the entry of fatty acids into the mitochondria, which is a crucial step in their metabolism, especially in tissues like heart, skeletal muscle and brown adipose tissue in which fatty acids are a major source of energy.

Secretory immune response to membrane antigens during Giardia lamblia infection in humans.

The secretory immune response in humans infected with Giardia lamblia was studied by using saliva samples and a membrane-rich protein fraction. The membrane fraction, studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed 24 antigen bands, ranging from 170 to 14 kDa. Saliva samples from giardiasis patients showed a heterogeneous response against the membrane fraction when they were assayed by immunoblotting. Among the antigens recognized by patient saliva samples, those of 170, 105, 92, 66, 32, 29, and 14 kDa stood out. These antigens were not recognized by saliva samples from healthy individuals. They may be of importance in future studies of protection from or diagnosis of G. lamblia infections.

Ultrastructural details of Cryptosporidium parvum development in calf intestine.

Cryptosporidium parvum and C. muris appear to be different species found in calves, with different oocysts size and distribution on the gastrointestinal tract. This work presents new images of C. parvum ultrastructure in calf intestine, mainly its development in nonmicrovillous cells and the presence of microtubular structures in the membrane enveloping the macrogamonts and immature oocysts.

Glycolytic enzyme activities in Cryptosporidium parvum oocysts.

Oocysts of Cryptosporidium parvum were obtained from an experimentally infected newborn goat. After purification, the oocysts were homogenised and the activities of the glycolytic enzymes measured in the different subcellular fractions. All of the activities of the Embden-Meyerhoff pathway were located in the non-sedimentable, cytoplasmic fraction. Under the conditions used, hexokinase activity was below the limits of detection. The pathway is also characterised by the presence of a pyrophosphate-dependent phosphofructokinase and a carbon dioxide-fixing cycle comprising phosphoenolpyruvate carboxylase, malate dehydrogenase and malate dehydrogenase (decarboxylating) activities. The data presented in this paper suggest that the infective stage of this parasite probably relies on substrate-level phosphorylation for energy generation.

A nonsense mutation in the 3-hydroxy-3-methylglutaryl-CoA lyase gene produces exon skipping in two patients of different origin with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

A novel nonsense mutation associated with the skipping of constitutive exon 2 of the 3-hydroxy-3-methylglutaryl-CoA lyase gene was found in two patients, from Portugal and Morocco, with 3-hydroxy-3-methylglutaric acidemia. By reverse transcriptase PCR and single-strand conformational polymorphism a G-T transversion was located, at nucleotide 109, of the 3-hydroxy-3-methylglutaryl-CoA lyase cDNA, within exon 2. Two mRNAs were produced as a result of this nonsense mutation: one of the expected size that contains the premature stop codon UAA, and the other with a deletion of 84 bp corresponding to the whole of exon 2. This deletion produced the loss of the last seven amino acids of the leader peptide and the first 21 amino acids of the mature protein. The nonsense mutation was found in a purine-rich GGAAG sequence, which is equal to, or similar to, others reported to be exonic splicing enhancers (ESE). We suggest that the nonsense mutation may affect a possible ESE on exon 2, which would hinder the splice site selection and facilitate an aberrant splice with the skipping of this exon. Determination by quantitative PCR shows that the ratio of mRNA with the nonsense mutation to the mRNA with the deletion is approx. 3:1.

The effects of IFN-gamma activated mouse peritoneal and alveolar macrophages on Cryptosporidium parvum development.

Mouse peritoneal and alveolar macrophages were interacted in vitro with C. parvum oocysts and cultured in normal medium and in medium with IFN-gamma. The results showed that in vitro activation of macrophages by IFN-gamma limits C. parvum development although the inhibitory effect is not as potent as in other intracellular parasitic protozoa.

Anti-oxidant enzymes in Cryptosporidium parvum oocysts.

Oocysts of Cryptosporidium parvum showed relatively low levels of SOD activity. The SOD which had a pI of 4.8 and an approximate molecular weight of 35 kDa appeared to be iron dependent. Catalase, glutathione transferase, glutathione reductase and glutathione peroxidase activity could not be detected, nor could trypanothione reductase. No NADH or NADPH oxidase activity could be detected, nor could peroxidase activity be demonstrated using o-dianisidine, guaiacol, NADPH or NADH as co-substrates. However, an NADPH-dependent H2O2 scavenging system was detected in the insoluble fraction.