[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.

[The mechanism of action of selenium substitution in inflammatory diseases. Modification of the activity of antioxidative enzymes in patients with acute pancreatitis]. / Untersuchungen zum Wirkungsmechanismus der Selen-Substitution bei entzündlichen Erkrankungen. Beeinflussung der Aktivität antioxidativer Enzyme bei Patienten mit akuter Pankreatitis.

From 20 patients with acute pancreatitis the activities of the antioxidative enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were estimated in the blood serum immediately before and during therapy with sodium selenite. The results demonstrate significant (p < 0.01 respectively < 0.05) enhanced activities of GSH-Px under the selenite therapy. Serum SOD activities were not significant influenced by the selenium treatment. The results obtained were not dependent on the reference basis (units/mg protein or U/l serum) used.

Specific activities of antioxidative enzymes in the cochlea of guinea pigs at different stages of development.

Significant activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were found in the cochleas of guinea pigs of different ages. The specific activities of SOD and GSH-Px (expressed as units/mg protein) increased significantly from fetal animals to animals 2 days old and then to 6-month-old animals.

Biomphalaria glabrata: influence of calcium, lectins, and plasma factors on in vitro phagocytic behavior of hemocytes of noninfected or Schistosoma mansoni-infected snails.

In vitro phagocytosis of erythrocytes by hemocytes of B. glabrata, intermediate host of S. mansoni, is strongly influenced by calcium, several lectins, and plasma factors. Our results indicate that two different mechanisms of non-self-recognition in B. glabrata may occur: (1) In the presence of calcium, phagocytosis occurs in noninfected and in infected snails without involvement of any other substances, and hemocytes of schistosome resistant as well as those of susceptible snails are able to recognize and phagocytose the target cells. (2) In the absence of calcium, phagocytosis occurs if bridging molecules (heterologous lectins in our assays) were present for which effector and target cells possess binding sites or if target cells were plasma coated prior to the assays. In suspensions in homologous plasma, hemocytes of both snail strains, infected or noninfected, subsequently showed phagocytic activities of about 70-80%. Preincubation of target cells in homologous plasma resulted in similar high phagocytic activities of hemocytes even in the absence of plasma during the standard assay. In these assays, a significantly higher proportion of hemocytes of resistant snails phagocytosed plasma-opsonized erythrocytes, whereas hemocytes of susceptible snails internalized less erythrocytes per cell and needed 60 min to phagocytose at percentages equivalent to that of resistant hemocytes within 10 min. Preincubation of erythrocytes in resistant plasma significantly increased the subsequent phagocytic activity of susceptible hemocytes, whereas preincubation of erythrocytes in susceptible plasma decreased the phagocytosis level of resistant hemocytes.

Lectin binding to cells of Schistosoma mansoni sporocysts and surrounding Biomphalaria glabrata tissue.

The binding of different lectins to the surface of mother and daughter sporocysts of Schistosoma mansoni (Trematoda) and to cells of its intermediate host Biomphalaria glabrata (Gastropoda) was investigated. The test system consisted of several biotin-labeled lectins, an avidin-biotin-peroxidase complex and 3,3'-diaminobenzidine. The fixatives used were Formalin, Bouin's and Zenker's solutions; unfixed material was also studied. Most lectins reacted equally with host tissue and parasite tissue. However, receptors for Ulex europaeus I (most probably fucose) were only demonstrated on daughter sporocysts. Thus, a method was found to specifically mark Schistosoma mansoni daughter sporocysts in the digestive gland tissue of its intermediate host. Mother sporocysts and surrounding host tissue differed in their distribution of galactosyl groups. Both lack fucose and N-acetyl-galactosamine. The differences in lectin binding of galactosyl determinants were also observed during the in vitro development of mother to daughter sporocysts.

Postnatal development of substance P in the inner ear of the guinea pig.

Appreciable amounts of substance P (SP) were found in guinea pig cochleas. The highest values were found in the postnatal period. Data presented favor the assumption of SP acting as a neuromodulator or neurotransmitter in the inner ear.

Intracellular distribution of calcium in smooth muscle: facts and artifacts. A correlation of cytochemical, biochemical and X-ray microanalytical findings.

Cytochemical methods, based on the use of oxalate and pyroantimonate as Ca markers, show Ca accumulation in the sarcoplasmic reticulum (SR), mitochondria and nuclei of smooth muscle cells from the guinea-pig taenia coli. A good correlation was found between the Ca concentrations measured in subcellular components in situ by X-ray microanalysis and the endogenous Ca concentrations estimated in subcellular fractions by atomic absorption spectrophotometry. Our findings suggest that the in situ Ca concentrations (mmol/kg dry weight) are: about 100 in the SR and mitochondria, and about 60 inside the nucleus, significantly higher than in cytoplasm.

Effect of histamine and stress on the gastric mucosal Ca2+ content during the development of gastric ulcers.

The influence of histamine, its triazole derivative (3-beta-aminoethyl-1,2,4-triazole) and immobilization stress on the gastric mucosal Ca2+ content during the development of gastric ulcers in guinea pigs and rats was investigated. A considerable fall in the concentration of Ca2+ in gastric tissues of guinea pigs after administration of histamine 0.25 mg/kg, down to 80% (8.0 mumol/g), its derivative (1 mg/kg) to 72% (7.2 mucol/g) and stress to 76% (7.6 mumol/g) was recorded by atomic absorption-spectrophotometric techniques, while the calcium level in the controls stood at 9.9 mumole/g. Similar changes (90-65%) were seen in the blood plasma. In rats, the Ca2+-decreasing effects of stress ran closely parallel with increasing ulceration, and depended on the duration of stress. The immobilization of rats evoked a slight rise in the Na+ content of the gastric tissues. After 4 h immobilization, the tissue concentration of Na+ was increased to 116% of control levels. Cimetidine (100 mumol kg-1 more than 50% inhibited the development of gastric ulcers and prevented the change in Ca2+ concentration ions. Thus, the data suggest that Ca2+ ions take part not only in the regulation of secretion, but also in stress tissue dystrophy.

Estimation of the Ca2+ binding by microsomal membranes from pig coronary artery using a calcium ion selective electrode.

To estimate the Ca2+ binding of microsomal membranes from pig coronary artery a modified potentiometric method was used. With this method it was possible to follow directly changes in the Ca2+ ion activity of the incubation medium down to 10(-8) moles. Compared with the Ca45-filter technique the electrometrically measuring device built up was beside the obvious advantages of a directly indicating method more fast, simple and hightly sensitive.

Calcium uptake and calcium release by subcellular fractions of smooth muscle. II. Kinetics of calcium uptake by microsomes and mitochondria from pig coronary artery and guinea pig ileum.

Calcium uptake by the microsomal and mitochondrial fractions of pig coronary artery and guinea pig ileum was studied in the presence of ATP, ATP plus oxalate and without ATP and oxalate. Microsomes and mitochondria of both smooth muscles were found to be unable to accumulate appreciable amounts of calcium in the absence of ATP. Oxalate noticeably stimulated the calcium uptake of the mitochondrial fraction from pig coronary artery but had little effect on calcium uptake by the microsomal fraction of this smooth muscle. The calcium uptake of microsomes and mitochondria from guinea pig ileum was not or only slightly enhanced by oxalate. There are typical kinetics regarding the time course and the extent of calcium uptake by microsomes and mitochondria from pig coronary artery and guinea pig ileum. In comparison, considerable qualitative and quantitative differences between both smooth muscles are observed. The high ATP-dependent calcium uptake capacity of the mitochondria from pig coronary artery and guinea pig ileum are a further argument for the hypothesis that these organelles may play an important role in the contraction-relaxation mechanism of smooth muscle.