Cloning of a metallothionein gene and characterization of two other cDNA sequences in the Pacific oyster Crassostrea gigas (CgMT1)

Metallothionein (MT) genes encode essential metal-binding proteins involved in metallic homeostasis and detoxification in living organisms. Here, we describe the structure of the first Pacific oyster Crassostrea gigas metallothionein (CgMT1) gene and the sequences of two other MT cDNA. The CgMT1 gene sequence contains three coding exons plus a 5' entirely non-coding exon, and the predicted protein contains 21 cysteine residues organized in Cys-X-Cys motifs as classically described for MTs. The three cDNA sequences present few substitutions in either coding sequence or UTRs. Induction of these MT-mRNA in heavy metal-treated oysters (i.e. cadmium) was confirmed by Northern blot analysis and RT-PCR and suggests a potential specific tissue expression rate. Southern blot analysis suggested the presence of multiple CgMT genes, and allowed the detection of restriction fragment length polymorphisms (RFLPs). Although the CgMT1 coding sequence showed 30-73% nucleotide identities with known sequences in other mollusks, it included the specific motif Cys-X-Cys-X(3)-Cys-Thr-Gly-X-X-X-Cys-X-Cys-X(5)-Cys-X-Cys-Lys found in Mollusk family 2. Marine bivalves are commonly used as pollution bioindicators, thus the development of genetic markers based on CgMT1 polymorphism will allow a monitoring of heavy metal exposure in anthropogenically disturbed ecosystems.     

Microspheres for colonic delivery of ketoprofen-hydroxypropyl-β-cyclodextrin complex

A new multiparticulate system, with the potential for site-specific delivery to the colon, has been developed using ketoprofen as model drug. The system simultaneously exploits cyclodextrin complexation, to improve drug solubility, and vectorization in microspheres (MS) based on Ca-pectinate and chitosan. The effect of complexation with hydroxypropyl-β-cyclodextrin (HPβCyd) and of chitosan presence on drug entrapment efficiency and release properties, as well on the drug permeation rate across Caco-2 cells has been investigated. Solid-state interactions between the components have been investigated by FTIR spectroscopy, differential scanning calorimetry and X-ray powder diffractometry. The morphology of MS was examined by scanning electron microscopy. Release studies revealed a different behaviour for MS containing drug alone or as complex: drug alone was released faster than in the presence of cyclodextrin from MS without chitosan, due to a reservoir effect. The opposite was found for MS containing chitosan, due to a competition effect between polymer and drug for the cyclodextrin. Cytotoxicity tests demonstrated the safety of these formulations. Permeation studies showed an increased permeation of the drug formulated as MS, particularly marked when it was used as complex, thus revealing an enhancing power of both cyclodextrin and chitosan with a synergistic effect in improving drug permeation.     

Immunotherapy (recombinant interleukin 2), hormone therapy (medroxyprogesterone acetate) and antioxidant agents as combined maintenance treatment of responders to previous chemotherapy

An open, non-randomized phase II study was carried out including all patients treated with whatever chemotherapy or combined modality regimen for whatever cancer who were in clinical objective response or stable disease (SD) for more than three months, to receive maintenance treatment with recombinant interleukin-2 (rIL-2) plus medroxyprogesterone acetate (MPA) plus antioxidant agents alpha-lipoic acid (ALA) and N-acetyl cysteine (NAC). The main study endpoints were clinical outcome and toxicity. The secondary endpoints were effects of treatment on cancer-related anorexia/cachexia syndrome (CACS) symptoms, on serum levels of proinflammatory cytokines, IL-2, C-reactive protein (CRP) and leptin as well as the evaluation of quality of life (QL). rIL-2 was administered at a dose of 1.8 MIU subcutaneously three times/week on alternate days for the first two weeks of every month and MPA was given orally at a dose of 500 mg once a day at alternate days without interruption. ALA 300 mg/day orally and NAC 1800 mg/day orally were also administered. The treatment was administered until progression of disease or appearance of toxicity. From July 1998 to May 2000, 16 patients were enrolled in the study (M/F ratio: 15/1; mean age: 62 years, range 45-71). The median duration of maintenance treatment was 10 months (range 5-22). The response to maintenance treatment at September 2000 was: CR (persistent throughout the treatment) 4 patients (25%); SD 1 patient (6.2%); PD 11 patients (68.8%). The median duration of response was 9.8 months (range: 5-22+). The median follow-up duration was 19 months (range: 8-102). The median OS was not reached. The median PFS was 14 months (range 1-29). The 1-year survival rate was 25%. At September 2000, 9 patients are still surviving. No grade 3/4 toxicity was observed. One Grade 2 skin toxicity was observed and Grade 1: 2 fever, 2 thrombocytopenia, 1 neutropenia and 1 skin were observed. The ECOG PS did worsen significantly, the body weight and BMI increased significantly after treatment, whereas the appetite did not change significantly. The QL evaluation showed a significant amelioration of cognitive functions and a borderline significant amelioration of emotional functions after treatment, whereas a borderline worsening of dyspnea was observed. The absolute lymphocyte count increased significantly after the maintenance treatment, as well as the serum IL-2, TNFalpha decreased at borderline statistical significance; the serum levels of leptin did not change significantly. The evaluation of patient subgroups showed that responders/survivors had a pattern superimposable to that of whole patient population, the patients who rapidly progressed and died exhibited no significant changes of these parameters during treatment. The results of the present study suggest that the host immune response, evaluated by several parameters, after IL-2 administration, (e.g. lymphocytosis), are worth further study as potential markers for the major end points of cancer treatment, i.e. OS and QL, in an adequate number of patients.     

Classification, treatment, and outcome of osteochondritis dissecans of the humeral capitellum

BACKGROUND: Indications for the treatment of osteochondritis dissecans of the humeral capitellum have remained unclear. The aims of this study were to analyze the outcomes and to determine the most useful classification for the choice of treatment. METHODS: The cases of 106 patients with osteochondritis dissecans of the capitellum were studied retrospectively. At the time of the initial presentation, the mean age of the patients was 15.3 years. The capitellar growth plate was open in eighteen patients and closed in eighty-eight. Thirty-six patients were treated nonoperatively. Fifty-five patients underwent fragment removal alone, twelve underwent fragment fixation with a bone graft, and three underwent reconstruction of the articular surface with use of osteochondral plug grafts from the lateral femoral condyle. The mean follow-up period was 7.2 years. The outcomes in terms of pain in the elbow, return to sports, and radiographic findings were analyzed and compared. RESULTS: An osteochondritis dissecans lesion with an open capitellar physis and a good range of elbow motion resulted in a good outcome. Continued elbow stress resulted in the worst outcome in terms of pain and radiographic findings. In patients with a closed capitellar physis, surgery provided significantly better results than elbow rest (p < 0.01). Fragment fixation or reconstruction provided significantly better results than fragment removal alone (p < 0.05). The results of removal alone were dependent on the size of the defect in the capitellum. The outcome in terms of pain was closely associated with sports activity and radiographic findings. CONCLUSIONS: We believe that osteochondritis dissecans of the capitellum can be classified as stable or unstable. Stable lesions that healed completely with elbow rest had all of the following findings at the time of the initial presentation: an open capitellar growth plate, localized flattening or radiolucency of the subchondral bone, and good elbow motion. Unstable lesions, for which surgery provided significantly better results, had one of the following findings: a capitellum with a closed growth plate, fragmentation, or restriction of elbow motion of > or =20 degrees . For large unstable lesions, fragment fixation or reconstruction of the articular surface leads to better results than simple excision. LEVEL OF EVIDENCE: Prognostic Level II.     

Hepatitis C viremia in HIV/HCV-coinfected patients: lower levels in presence of chronic hepatitis B

Complex reciprocal interactions between hepatitis C (HCV) and hepatitis B (HBV) viruses (HBV) have been reported. We examined the influence of HBV on HCV RNA titers in 376 HCV/HIV-coinfected patients (30 were also HBsAg positive). Regression analyses identified negative HBsAg and male sex as factors associated with HCV RNA values >500,000 IU/mL.     

Effects of morphine treatment and withdrawal on striatal and limbic monoaminergic activity and ascorbic acid oxidation in the rat

Since ascorbic acid (AA) reportedly suppresses tolerance to and dependence on morphine in humans and rodents, levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), AA, dehydroascorbic acid (DHAA), uric acid, xanthine, hypoxanthine, glutamate and γ-amino-butyric acid (GABA) were determined by high-pressure liquid chromatography (HPLC) in the striatum and in the limbic forebrain of the rat following morphine treatment (single or repeated) and withdrawal. Single morphine administration (20 mg/kg s.c.) increased DOPAC + HVA/DA, 5-HIAA/5-HT and DHAA/AA ratios, uric acid levels, and decreased xanthine, hypoxanthine, glutamate and GABA levels in both regions. 3-MT levels were decreased in the striatum and increased in the limbic forebrain. After 7 days of morphine treatment, striatal DOPAC + HVA/DA and DHAA/AA ratios and uric acid levels were still higher and striatal and limbic xanthine levels still lower than in controls, while all other parameters were in the range of control values in both regions. Morphine treatment also increased the glutamate/GABA ratio in the striatum. In all morphine-treated rats, individual striatal DOPAC + HVA/DA and DHAA/AA ratio values were directly correlated. After a 48 h withdrawal period, both striatal AA oxidation and glutamate/GABA ratio further increased; limbic 3-MT levels further decreased, while all other parameters did not differ from control values. We conclude that: (i) tolerance to morphine-induced increase in hypoxanthine, xanthine and AA oxidation develops in the limbic forebrain faster than in the striatum; (ii) the morphine-induced increase in striatal and limbic AA oxidation may be considered a consequence of increased formation of reactive oxygen species due to increased DA, hypoxanthine and xanthine oxidative metabolism; (iii) a striatal excitotoxic imbalance characterizes the withdrawal state and may be taken into account to explain the further increase in striatal AA oxidation.     

Formulation and characterization of triclosan sub-micron emulsions and nanocapsules

Triclosan, a non-ionic, broad spectrum anti-microbic agent, has recently demonstrated its effectiveness as an anti-malarial drug by inhibition of the growth of Plasmodium Falciparum. The aim of this work was to formulate suitable triclosan colloidal carriers with the final objective of obtaining a drug delivery system suitable for a potential anti-malarial oral treatment. Two different nanotechnological approaches were experimented with that could be suitable for developing effective triclosan formulations against this established and re-emerging infectious disease. Sub-micron emulsions were prepared by the solvent displacement method, using different oily amounts in order to vary the drug amount entrapped in the formulation. Chitosan-coated nanocapsules were obtained with chitosan hydrochloride at two different viscosity degrees (Cl 113 and Cl 213). All formulations were appropriately characterized by determining drug loading capacity and encapsulation efficiency and measuring particle size and zeta potential. Morphological characterization of the different systems was performed by TEM analysis, whereas release studies were carried out by reverse bag dialysis method. All preparations resulted stable. Cl 113-coated nanocapsules appeared particularly suitable as triclosan carriers for obtaining a systemic drug release, owing to both chitosan's good mucoadhesive and enhancer properties as well as the effectiveness shown by its coating in adequately controlling drug release rate.     

Dehydrocrotonin and its derivative, dimethylamide-crotonin induce apoptosis with lipid peroxidation and activation of caspases-2, -6 and -9 in human leukemic cells HL60

A variety of stimuli can induce cells to undergo apoptosis, with one of the most reproducible inducers being mild oxidative stress following exposure to anticancer agents. Apoptosis involves events mediated by cysteine proteases (caspases) that are classified as initiators (-8, -9 and -12) or executors (-2, -3, -6 and -7). In this study, we examined the mechanisms of apoptosis induced by dehydrocrotonin (DHC), a diterpene lactone isolated from the Amazonian plant Croton cajucara, and its synthetic derivative, dimethylamide-crotonin (DCR), in human HL60 promyelocytic leukemia cells. Flow cytometric analysis of HL60 cells after treatment for 72 h showed that DCR- and DHC-induced apoptosis, with maximum cell death at a concentration of 250 microM for both compounds. DCR and DHC were effective in triggering the activation of caspases-2, -6 and -9. The level of reduced glutathione (GSH) decreased, whereas there was an increase in thiobarbituric acid-reactive substance (TBARS) production and in mitochondrial swelling. These effects on mitochondrial swelling, GSH content and lipid peroxidation were abolished by cyclosporine A, an inhibitor of the membrane permeability transition. The cytotoxicity of DHC and DCR was prevented by a high concentration of GSH (15 mM) in the culture medium. These results indicate that DCR and DHC produced apoptosis partly by oxidative stress-induced lipid peroxidation, which triggered the caspase cascade, that lead to apoptotic cell death in HL60 cells. Based on the pattern of caspase activation, on the increase in mitochondrial swelling and on the inhibitory action of cyclosporine A, we conclude that DCR and DHC triggered apoptosis in HL60 cells probably through cytochrome c release and apoptosome formation.     

Chronic use of benzodiazepines and latent cognitive decline in the elderly: Results from the Three-city study

We aimed to examine whether long-term use of benzodiazepines is associated with an accelerated decline of cognitive performances by using a statistical model specifically adapted to multivariate longitudinal bounded quantitative outcomes. The data came from the “Three-city” study, a French population based study. All the subjects were 65 years old or older at inclusion and had been followed-up for 7 years. The use of benzodiazepines and cognitive functioning were assessed at each examination phase (baseline, 2, 4 and 7 years). Cognitive decline was analyzed using a nonlinear multivariate mixed model with a latent process. This model makes it possible to assess change over time of the latent cognitive process underlying several neuropsychological tests: Mini Mental Status Examination, Isaacs Set test, Benton Visual Retention Test, and Trail Making Test (A and B), and to describe and account for their metrological properties. Analyses were adjusted for age, center, gender, education, socio-professional status, depression, insomnia, high blood pressure, hypercholesterolemia, alcohol, tobacco consumption and physical activity. Nine hundred and sixty nine subjects who reported taking benzodiazepines for 2, 4 or 7 consecutive years were compared to 4226 subjects who were non-benzodiazepine users. Chronic use of benzodiazepine was significantly associated with a lower latent cognitive level (β=?1.79 SE=0.25 p=<0.001), but no association was found between chronic use and an acceleration of cognitive decline, neither on the latent cognitive process (β×time=0.010 SE=0.04 p=0.81), nor on specific neuropsychological tests. Our results suggest that chronic benzodiazepine use is associated with poorer cognitive performance but not with accelerated cognitive decline with age.