In vivo studies on mouse erythrocytes linked to transferrin

Transferrin (Tf), a plasma protein with numerous, highly specific receptors in proliferating and differentiating cells was already discussed as a targeting ligand for drugs and liposomes in previous studies. In this paper, we deal with erythrocytes linked to Tf as possible physiological targeting carrier systems for delivering anticancer drugs. For that purpose we have used glutaraldehyde (0.1%) as a coupling agent between Tf and erythrocytes. The highest amount of Tf linked to erythrocytes turned out to be 0.76 +/- 0.13 microg Tf/10(6) cells, while reaching 65% of cell recovery. After 13 days, the Tf-erythrocytes hemolysis reached 50%, with transferrin still coupled to erythrocytes. The in vivo kinetic behaviour of intravenously injected 51Cr-Tf-erythrocytes showed a reduced half-life to hours as compared to days of controls. However, a considerable percentage of Tf-erythrocytes (close to 20%) remained circulating for a relatively long period (around 2 days), which made possible the specific targeting by these carrier systems. In vivo biodistribution studies indicated that 51Cr-Tf-erythrocytes rapidly accumulated in the different studied organs (liver, spleen, lungs, kidneys, femur-tibia, and heart), suggesting a selective removal of Tf-erythrocytes by the cells of the mononuclear phagocytic system present mainly in liver and spleen. On the other hand, Tf-erythrocytes showed a poor targeting of heart tissue, therefore a reduced cardiac toxicity should be expected after administration of erythrocyte-encapsulated drugs. The presence of Tf-erythrocytes in femur-tibia and spleen could be related to the Tf-specific binding to the hematopoietic cells containing Tf receptors. The final results of this study encourage additional research on Tf-erythrocyte to investigate the relationship between transferrin-mediated targeting by carrier erythrocytes and uptake of different erythrocyte-encapsulated drugs. Consequently, the current study showed possible use of these carriers as a potential therapeutic tool for drug targeting in animal models with alterations affecting mononuclear phagocytic system or carcinomas of various origins whose cells show elevated number of Tf receptors.     

Mannosomes: a molluscan intracellular tubular membrane system related to heavy metal stress?

Amongst animals, several hydrogen peroxide-generating oxidases are apparently restricted to molluscs. One of these, D-mannitol oxidase, is concentrated in the alimentary system, where it is associated with its own subcellular membrane system of unique tubular morphology, most likely representing a structural modification of the ER. These structures can be purified by subcellular fractionation and have been termed 'mannosomes'. Little is known about the functions of mannitol oxidase or of mannosomes, but the previously reported molluscicide-induced increase in mannosomes implies their involvement in a general stress reaction. In this study, we examined the effects of heavy metal stress in the terrestrial gastropod Arion lusitanicus. The activity of mannitol oxidase and mannosome abundance were monitored, together with metal effects on heat-shock protein level, and these parameters were compared to heavy metal accumulation in the digestive gland. We found that mannitol oxidase is inhibited by heavy metals more than other oxidases. On the other hand, hsp70 levels and mannosomal protein were increased with enhanced heavy metal stress, the latter indicating a probable increase in the number of mannosome organelles. Thus, stress protein (hsp70) and mannosomal protein were positively correlated with heavy metal accumulation, whereas the enzyme activity showed a negative correlation with increasing heavy metal content of the slugs.     

Evaluation of a Western blot test,Helico blot 2.1, in the diagnosis of Helicobacter pylori infection in a pediatric population

Background. Noninvasive diagnostic tests are useful as screening tools for Helicobacter pylori infection in pediatric populations. The aim of this study was to evaluate performance of the immunoblot assay, Helico Blot 2.1, for the diagnosis of H. pylori infection in symptomatic children. Materials and Methods. Immunoblot assay was used for detection of IgG antibodies to specific H. pylori proteins and to a recombinant H. pylori antigen, CIM marker. The study was performed on sera collected from 134 symptomatic, untreated children (mean age, 9.1 ± 3.2 years; range, 1–14 years). H. pylori infection status was determined by culture, histology and rapid urease test. Results. Immunoblot assay yielded a positive result in 71 of the 72 infected patients (sensitivity 98.6%) and in eight of the 62 noninfected ones (specificity 87.1%). The predictive values for a positive and a negative result were 89.9% and 98.2%, respectively. The performance of the CIM band alone, as a marker for H. pylori infection status, was also evaluated. This band was present on the blot of 71 infected patients and on four of the 62 H. pylori‐negative patients. The sensitivity, specificity, PPV and NPV of the CIM antigen were 98.6%, 93.5%, 94.7% and 98.3%, respectively. Conclusions. The immunoblot assay Helico Blot 2.1 is a suitable noninvasive test for the serodiagnosis of H. pylori infection in children. The good level of performance demonstrated by the novel recombinant antigen CIM suggests it may be a useful contribution to the qualitative and quantitative performance of the Helico Blot 2.1 in pediatric populations.     

Comparative study of two culture media for the detection of phospholipase activity of Candida albicans and Cryptococcus neoformans strains

Phospholipase activity (PHA) is considered a virulence factor related to pathogenicity of Candida albicans and Cryptococcus neoformans. The aim of this work was to compare the ability of two culture media: malt egg-yolk agar (MEA) and Sabouraud-egg yolk agar (SEA), for the detection of phospholipase activity. Forty four strains of C. neoformans and 54 of C. albicans isolated from different clinical specimens of human origin were studied. The phospholipase production was determined as a ratio between the diameter of each colony and the corresponding lysis halo. The values ranged between 0 and 1, and the highest level of enzymatic activity was the nearest to 0. Enzymatic activity was observed in 34 C. neoformans strains, grown either in MEA or SEA media; 59% of enzyme producers were detected in SEA only, while five strains (15% of producers) were detected just in MEA medium. Phospholipase activity was observed in both media only in nine of 34 enzyme producer strains. Forty two out of 54 strains of C. albicans were detected as enzyme producers; 31 of them (73.8%) were detected in MEA medium only. On the other hand 10 strains (23.8% of the enzyme producers) showed phospholipase activity just in SEA medium. Detection of PHA could be done by both media in one case only. In order to evaluate the time needed to detect PHA, 41 C. albicans strains were incubated 72 h. They were read at 24 h intervals. No enzyme activity was detected at 24 h, 15 enzyme producer strains remain negative at 48 h and the halos of all strains with PHA were better distinguished after 72 h. It was possible to conclude that neither MEA nor SEA media were good enough as the unique medium to detect phospholipase activity. Nevertheless, MEA was better than SEA to detect PHA of C. albicans after 72 h incubation. The opposite situation was seen when we studied PHA in C. neoformans strains. In this case, greater sensibility was observed with SEA medium compared with MEA medium. Six days incubation, but not longer incubation times, were necessary to detect phospholipase activity in C. neoformans strains.     

Epidemiology of candidemia in Spain - Multicenter study

The results of the epidemiological study on candidemias with the highest number of cases carried out in Spain is presented. This study is included in the Epidemiological Survey of Candidemia in Europe supported by the ECMM in which another five countries take part. In the Spanish study, 19 hospitals participated, 290 candidemia episodes were analysed (80 in children under 15 years and 210 in adults), 293 strains of yeasts being isolated. Both in children and in adults, the risks factors more frequently observed were the intravenous catheter and previous antibiotic therapy. In adults, the most habitual underlying disease was the solid tumor and, in children, hematological diseases. Candida albicans was the most prevalent species isolated in adults (46.1%) and Candida parapsilosis in children (50%). As part of the therapy, the intravenous line was removed and antifungal treatment was prescribed to 74% and 92.5% of children, respectively and to 43.8% and 73.8% of adults. The antifungal agent of election in adults was fluconazole (54.8%) and liposomal amphotericin B (58.1%) in children. The global mortality of the study was 38.9%, which for ages was major in adults (41.4%) than in children (38.7%). The geographical distribution of the isolated species was homogeneous, C. albicans being the predominant species, with the exception of Galicia and Extremadura where C. parapsilosis was the most frequent.     

Protective properties of butanolic extract of the Calendula officinalis L. (marigold) against lipid peroxidation of rat liver microsomes and action as free radical scavenger

Calendula officinalis (marigold) has many pharmacological properties. It is used for the treatment of skin disorders, pain and also as a bactericide, antiseptic and anti-inflammatory. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known to participate in the pathogenesis of various human diseases and may be involved in the conditions which C. officinalis is used to treat. The aim of this study was to investigate the relationship between the beneficial properties of this plant and its antioxidant action. The butanolic fraction (BF) was studied because it is non-cytotoxic and is rich in a variety of bioactive metabolites including flavonoids and terpenoids. Superoxide radicals (O(2)(*-)) and hydroxyl radicals (HO(*)) are observed in decreasing concentrations in the presence of increasing concentrations of BF with IC(50) values of 1.0 +/- 0.09 mg/ml and 0.5 +/- 0.02 mg/ml, respectively, suggesting a possible free radical scavenging effect. Lipid peroxidation in liver microsomes induced by Fe(2+)/ascorbate was 100% inhibited by 0.5 mg/ml of BF (IC(50) = 0.15 mg/ml). Its total reactive antioxidant potential (TRAP) (in microM Trolox equivalents) was 368.14 +/- 23.03 and its total antioxidant reactivity (TAR) was calculated to be 249.19 +/- 14.5 microM. The results obtained suggest that the butanolic fraction of C. officinalis possesses a significant free radical scavenging and antioxidant activity and that the proposed therapeutic efficacy of this plant could be due, in part, to these properties.     

Melatonin protects against delta-aminolevulinic acid-induced oxidative damage in male Syrian hamster Harderian glands

Effects of the prooxidant delta-aminolevulinic acid (ALA) and the antioxidant melatonin (MEL) were investigated in the male Syrian hamster Harderian gland (HG). Rodent Harderian glands are highly porphyrogenic organs, which may be used as model systems for studying damage by delta-aminolevulinic acid and its metabolites, as occurring in porphyrias. Chronic administration of delta-aminolevulinic acid (2 weeks) markedly decreased activities of the porphyrogenic enzymes delta-aminolevulinate synthase (ALA-S) and delta-aminolevulinate dehydratase (ALA-D) and of the antioxidant enzymes superoxide dismutase (SOD), glutathione reductase (GR) and catalase (CAT), whereas porphobilinogen deaminase (PBG-D) remained unaffected. This treatment led to increased lipid peroxidation (LPO) and oxidatively modified protein (protein carbonyl) as well as to morphologically apparent tissue damage. Melatonin also caused decreases in delta-aminolevulinate synthase, delta-aminolevulinate dehydratase, superoxide dismutase, glutathione reductase and catalase. Despite lower activities of antioxidant enzymes, lipid peroxidation and protein carbonyl were markedly diminished. The combination of delta-aminolevulinic acid and melatonin led to approximately normal levels of delta-aminolevulinate dehydratase, glutathione reductase, catalase and protein carbonyl, and to rises in superoxide dismutase and porphobilinogen deaminase activities; lipid peroxidation remained even lower than in controls and the appearance of the tissue revealed a protective influence of melatonin. These results suggest that melatonin may have profound effects on the oxidant status of the Harderian gland.     

Guanidinium and aminoimidazolinium derivatives of N-(4-piperidyl)propanamides as potential ligands for mu opioid and I2-imidazoline receptors: synthesis and pharmacological screening

Derivatives of N-(1-phenethyl-4-piperidyl)propanamides incorporating guanidinium and 2-aminoimidazolinium groups have been prepared by a synthetic approach involving first introduction of a spacer between the piperidine and the functional group by reductive amination of piperidinone. The formation of each of these functional groups was carried out using N-N'-di(tert-butoxycarbonyl)thiourea and 2-methylthioimidazolinium iodide, respectively. These structures have been designed to incorporate two pharmacologic goals into one entity. Radioligand binding assays have been used to study their affinity for opioid (mu, delta and kappa) and I2-imidazoline receptors. Two of them, 10 and 16, showed high affinity for mu opioid receptors and functionally they had moderate analgesic properties in the hot plate and writhing tests. The in vitro studies on guinea pig ileum (GPI) indicated that both compounds are mu opioid agonists. In what concerns I2-imidazoline receptor activity, these derivatives showed low affinity around 6 to 7 times less than idazoxan.