[PCR-based diagnosis of mucormycosis in tissue samples]. / PCR-Diagnostik von Mukormykosen aus Gewebeschnitten.

Mucormycosis is characterized by a rapid, often fatal progression. Early diagnosis of invasive mucormycosis is the key for timely therapeutic intervention and improved survival. Contrary to the more prevalent aspergillosis, effective antifungal therapy of mucormycosis is mainly limited to amphotericin B. Given the importance to guide the timely initiation of amphotericin B and possible surgical intervention, rapid and specific identification of fungal hyphae is essential. Conventional histopathology depends on abundance and morphology of the fungi as well as on the skills of the personnel, and usually shows an accuracy of 80 %. PCR assays targeting fungal ribosomal genes to identify Mucorales at least at genus level increase sensitivity, allow a rapid identification as well as detection of double mold infections. Thus, PCR assays are beneficial to complement existing approaches. They are recommended to rapidly specify tissue diagnosis and accurate identification of fungi. This will help to guide effective therapy and thereby, survival will increase. Retrospective analyses of mucormycosis by PCR help to evaluate therapeutic interventions and will optimize treatment options.

Superoxide dismutase expression and H2O2 production by hemocytes of the trematode intermediate host Lymnaea stagnalis (Gastropoda).

Snail hemocytes mobilise ROS-generating enzymes during oxidative burst similar to those of mammalian leukocytes. We report herein the identification of an inducible Cu/Zn superoxide dismutase, which converts O2- to H2O2, in hemocytes of the pond snail Lymnaea stagnalis. The deduced amino acid sequence with all characteristic residues (His44,46,61,69,78 and 118, Asp81, Cys55/144, Arg141 and the Greek Key loop region Glu119-Leu/Val142) includes an open reading frame of 155 AA. Changes in Cu/ZnSOD gene expression induced by stimulation with Zymosan or trematode larvae were examined in a time course. Activated hemocytes significantly up-regulate Cu/ZnSOD expression during 2-48 h upon stimulation with the maximal induction at 45 min during phagocytosis and at 12 h during encapsulations. This increase in Cu/ZnSOD expression paralleled the increasing production of hydrogen peroxide by hemocytes. Thus, intracellular or extracellular targets elicit an induced expression of Cu/ZnSOD and the generation of elevated amounts of hydrogen peroxide by L. stagnalis hemocytes, reflecting a significant activation of their host defense function.

Antioxidant enzymes in intramolluscan Schistosoma mansoni and ROS-induced changes in expression.

Killing of intramolluscan schistosomes by host haemocytes is mediated by reactive oxygen metabolites. Hence, defence against oxidative damage is essential for the parasite to survive. In this study, expression of three key antioxidant enzymes, superoxide dismutase (EC 1.15.1.1), glutathione peroxidase (EC 1.11.1.9) and glutathione-S-transferase (EC 2.5.1.18) was determined in Schistosoma mansoni miracidia, sporocysts and cercariae. Stage-dependent expression of these enzymes was shown to be regulated at the transcriptional level. Second, the influence on enzyme expression of reactive oxygen species (ROS) and of haemocytes from schistosome-resistant and -susceptible host snails was determined. Generation of ROS by xanthine/xanthine oxidase resulted in increased transcript levels for all three enzymes. Addition of hydrogen peroxide induced a significantly increased expression of GPx and SOD but not GST. Snail haemocytes induced an up-regulation of SOD and GPx at 12 and 18 h post-exposure, respectively. Susceptible haemocytes elicited a stronger induction of transcript expression than resistant haemocytes. After 36-48 h, SOD remained up-regulated in sporocysts encapsulated by haemocytes from susceptible hosts, whereas a down-regulation of SOD and GPx occurred in schistosomes encapsulated by haemocytes from resistant snails. These observations indicate that schistosomes express elevated levels of antioxidant enzymes in interaction with haemocytes from susceptible snail hosts in which they survive. On the other hand, haemocytes of resistant snails may interfere with reactive oxygen detoxification via down-regulation of schistosome antioxidant enzymes, thus shifting the balance towards parasite killing.

Differential display analysis of hemocytes from schistosome-resistant and schistosome-susceptible intermediate hosts.

Hemocytes from schistosome-resistant and schistosome-susceptible Biomphalaria glabrata differ fundamentally in their behavior towards an invading parasite. When the schistosome infects a resistant snail host it is quickly surrounded by hemocytes, encapsulated and destroyed. Hemocytes from susceptible hosts fail to kill the parasite. To detect the differences between these two host phenotypes, we used differential-display reverse-transcription PCR (DDRT-PCR), based on RNA extracted from isolated hemocytes. A number of differentially expressed fragments from resistant and susceptible snails were detected by DDRT-PCR and confirmed using single-strand conformation polymorphism. These methods proved to be sensitive enough to allow comparison and verification of differential gene expression in our system, where only small numbers of cells are available. The most interesting phenotype-specific fragments detected so far show sequence homologies to an adhesion molecule, defensin, serine/ threonine kinases, peroxidases and glycosidases.

Schistosoma mansoni: adhesion of mannan-binding lectin to surface glycoproteins of cercariae and adult worms.

Schistosoma mansoni is a blood-dwelling trematode which can persist for several years in the vessels of the human host. The schistosomal surface has been extensively characterized by lectin binding studies, revealing the carbohydrate composition of the worm's tegument. Using fluorescent and scanning electron microscopy we demonstrate that the surface carbohydrates of cercariae and adult worms are the binding ligands for mannanbinding lectin (MBL), a serum protein that is part of the innate immune system. An in vitro complement activation assay with C1q-deficient complement suggests that MBL, in association with the serine proteases MASP-1 and MASP-2, is capable of fixing complement components on the schistosomal tegument and activating the complement cascade via the "MBL pathway." MBL is constitutively expressed by hepatocytes and present in the blood at a stable level. Since it is also a weak acute-phase protein and therefore upregulated in an acute-phase response we investigated the serum MBL levels in patients infected with Schistosoma sp. and in healthy control persons. An enzyme-linked immunosorbent assay indicated no differences between the two groups. Although our results suggest an involvement of MBL activated complement in vitro, its role in vivo remains to be clarified.

Glycosidase activities in plasma of naive and schistosome-infected Biomphalaria glabrata (Gastropoda).

Activity of the following glycosidases was detected in the plasma of the freshwater snail Biomphalaria glabrata: beta-D-fucosidase, beta-D-glucosidase, beta-D-galactosidase, beta-D-mannosidase, beta-D-glucuronidase, N-acetyl-beta-D-galactosaminidase, N-acetyl-beta-D-glucosaminidase, and lysozyme. At the physiological pH (7.2-7.4) of snail haemolymph, enzymatic activity was about 10-50% of the maximum activity at each enzyme's respective acid pH-optimum. Schistosome-susceptible B. glabrata showed lower plasma protein concentration and significantly lower enzymatic activities (U/mg protein) than schistosome-resistant snails. Changes in glycosidase activity levels correlate with the progress of infection. After successful schistosome invasion, activities of plasma glycosidases but not the concentration of total plasma proteins increased significantly during the first 2 days in both snail strains. Thus, most tegumental glycoproteins of schistosome larvae can be altered by humoral host glycosidases. The detection of only very low activities of hexosaminidases leads to the hypothesis that GalNAc/GlcNAc may be involved in the process of non-self recognition. At 4 days post-infection, glycosidase activities were identical or slightly below the levels found in naive snails. At this time of infection the parasite is encapsulated and destroyed by haemocytes of resistant snails. In susceptible snails, however, the schistosomes have transformed into sporocysts and will complete their life-cycle without eliciting effective defence reactions. After > 30 days post-infection, when cercariae are fully developed in susceptible snails, plasma protein concentration decreased significantly, whereas glycosidase activities were elevated.

A comparative study of the organic acid content of the hemolymph of Schistosoma mansoni-resistant and susceptible strains of Biomphalaria glabrata.

The freshwater snail Biomphalaria glabrata is an intermediate host of the trematode Schistosoma mansoni. However, some strains of B. glabrata are resistant to successful infection by S. mansoni larvae. The present work examines the profile of organic acids present in S. mansoni-resistant and -susceptible strains of B. glabrata, in order to determine whether the type of organic acid present is related to susceptibility. The organic acids were extracted from the hemolymph of two susceptible B. glabrata strains (PR, Puerto Rico and Ba, Jacobina-Bahia from Brazil), and from the resistant strains 13-16-R1 and 10R2, using solid phase extraction procedures followed by high performance liquid chromatography. The organic acids obtained were analyzed and identified by comparison with known standards. Pyruvate, lactate, succinate, malate, fumarate, acetate, propionate, beta-hydroxybutyrate and acetoacetate were detected in all hemolymph samples. Under standard conditions, the concentration of each of these substances varied among the strains tested and appeared to be specific for each strain. An interesting variation was the low concentration of pyruvate in the hemolymph of PR-snails. Only the concentration of fumarate was consistently different (p < or = 0.05) between resistant and susceptible strains.

The plasma proteins of Biomphalaria glabrata in the presence and absence of Schistosoma mansoni.

Snail plasma serves as both a sink for metabolites and a source of nutrients for parasites developing within their intermediate hosts. It also contains molecules involved in immunological events like non-self recognition, phagocytosis and encapsulation. In this study we present improved protocols for the separation and partial characterization of plasma proteins of schistosome-susceptible and resistant strains of Biomphalaria glabrata. Within each strain, the plasma of snails 12, 24, 48 and 72 h post-exposure to Schistosoma mansoni and of non-exposed snails was compared. Protein concentrations in hemolymph of all snail strains, non-exposed or parasite-exposed, were about 29 mg/mL and were not found to differ significantly. The dominant plasma molecule (80-85%) is extracellular hemoglobin (Hb) with a native mass of > 1 M Da, and subunits of 190 kDa. It is the only protein bearing heme as shown after separation by native-PAGE and LDS-PAGE. The relatively large amounts of Hb and its large size cause problems if native plasma components are to be separated in PAGE. To obtain satisfactory separation, we used short-term ultracentrifugation to deplete Hb from plasma without qualitative loss of other proteins. Using this methodology, we have examined proteins by native polyacrylamide gel electrophoresis, in the presence of SDS or LDS only or SDS and mercaptoethanol, and by isoelectric focusing. Proteins have been detected in gels by silver stains and staining for heme groups, and, after transfer to membranes, by means of lectins and neoglycoproteins. Molecular weights of plasma proteins range between 10 and > 450 kDa, and isoelectric points are from pH 4 to 9.4. All strains show similar protein patterns, although minor inter- and intrastrain differences occur. These differences are quantitative rather than qualitative, not consistent, and cannot be correlated with the snail's ability to effectively attack and kill S. mansoni sporocysts. In all snail strains, plasma proteins remained qualitatively stable during 3 days after exposure to S. mansoni. New proteins were not evident, and none was lost as a consequence of exposure to parasites. Our new Hb-depletion technique is an excellent approach to separate and examine Biomphalaria plasma proteins in their native state. The use of lectins to probe for the presence of carbohydrates showed that the majority of plasma proteins is glycosylated. Mannose, galactose, and N-acetylgalactosamine are their major carbohydrate components; fucose was not detected. Several lectins apparently in the molecular mass range of 330-500 and 56-135 kDa with major carbohydrate-specificities for N-acetyl-galactosamine, N-acetylglucosamine, mannose, glucose, galactose and fucose were detected in the plasma of both resistant and susceptible snails by using neoglycoproteins as probes.