Saccharomyces cerevisiae as an eukaryotic cell model to assess cytotoxicity and genotoxicity of three anticancer anthraquinones

The toxicity of most drugs is associated with their enzymatic conversion to toxic metabolites. Bioactivation reactions occur in a range of cellular organs and organelles, including mitochondria. We have investigated different effects (i.e. growth inhibition, mortality and genotoxicity) of doxorubicin, epirubicin and mitoxantrone on the D7 strain of Saccharomyces cerevisiae and on its petite (rho degrees ) respiratory-deficient mutant at various cellular concentrations of cytochrome P450 and glutathione (GSH). The data confirmed the importance of oxygen production for doxorubicin toxicity. The complete absence, or a very low level, of cytochrome oxidase subunit IV conferred some resistance to doxorubicin. Low GSH levels decreased resistance to doxorubicin in both strains, suggesting that thiol depletion could potentiate membrane lipid peroxidation. Doxorubicin induction of petite colonies suggests that the drug is able to select rather than induce respiratory-deficient mutants. Epirubicin induced levels of cytotoxicity similar to those of doxorubicin. The effects did not appear to be significantly dependent on mitochondrial function or GSH levels, whereas cells were strongly protected by cytochrome P450. GSH did not induce an evident alteration. Neither were genotoxic effects induced. Mitoxantrone had reduced levels of both growth inhibition and cytotoxicity in comparison to anthracyclines and induced convertants, revertants and aberrants. All the effects considered were amplified at high cytochrome P450 cellular concentrations, although the drug was also shown to act without previous metabolism via cytochrome P450. Anthracenedione effectiveness was increased by metabolism via cytochrome P450 and partially reduced by GSH. However, further mechanisms were suggested, which might implicate mitochondrial function and/or production of electrophilic cytotoxic and/or genotoxic intermediates by means of GSH conjugation. The biological effectiveness of doxorubicin, epirubicin and mitoxantrone on S.cerevisiae was shown to be strictly dependent on cell-specific physiological/biochemical conditions, such as a functional respiratory chain and levels of cytochrome P450 and GSH.     

Bilateral salpingectomy does not compromise ovarian stimulation in an in-vitro fertilization/embryo transfer programme

The question whether salpingectomy has a negative influenceon ovarian function and the outcome of pregnancy in an in-vitrofertilization (IVF) and embryo transfer treatment programmeis not yet answered. We performed a retrospective case-controlstudy to investigate the possible negative effect of salpingectomyon ovarian response to human menopausal gonadotrophins (HMG)during IVF and embryo transfer. The study group was composedof 26 patients with bilateral salpingectomy. In 67 cycles weanalysed different parameters of ovulation such as the numberof days of ovarian stimulation, numbers of ampoules of HMG,pre-ovulatory oestradiol concentrations and the numbers of oocytesretrieved. These parameters were compared to a control groupof 134 cycles in 134 women with healthy Fallopian tubes. Nodifferences were found. Implantation ratio, pregnancy rate andoutcome were the same in both groups. We conclude that bilateralsalpingectomy had no detrimental effect on ovarian performanceduring IVF and embryo transfer treatment nor on the outcome.     

Monkeypox virus detection in rodents using real-time 3'-minor groove binder TaqMan assays on the Roche LightCycler

During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture. The rodent tissue samples were appropriately processed and then tested by using an integrated approach involving real-time polymerase chain reaction (PCR) assays, an antigen-detection immunoassay, and virus culture. We designed and extensively tested two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan-orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan-orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. In addition, we attempted to verify the presence of monkeypox virus by culture on multiple cell lines, by immunohistology, and by electron microscopy, with negative results. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses. In light of the recent monkeypox virus transmissions, early detection of the virus is crucial for both natural outbreaks and potential acts of bioterrorism.     

Rush desensitization in heparin hypersensitivity: a case report

Among all the known drug intolerances, adverse reactions to heparin are not very common. No desensitization in patients with heparin hypersensitivity has ever been attempted. We report the case of a 55–year-old female patient with mitral stenosis and insufficiency, and tricuspid and aortic insufficiency. The patient underwent heparin treatment, and urticaria occurred with either s.c. calcium heparin or i.v. sodium heparin. Allergy testing (skin tests and patch tests) was negative. A pseudoallergic intolerance was diagnosed. Mitral valve replacement with the extracorporeal circulation method was necessary; therefore, heparin treatment was administered. A heparin rush desensitization together with antihistamine drugs (4 mg clorpheniramine maleate for 3 d) was started: 50 UI (0.5 mg) s.c. sodium heparin were first administered; within 4 d, 5000 UI (50 mg) sodium heparin was administered i.v. with no side-effects. A full-dosage heparin treatment was then administered and heart surgery was easily performed. During the postsurgical course, i.v. sodium heparin was smoothly replaced with s.c. calcium heparin (25000 UI s.c. per day) and with oral anticoagulants (sodium warfarin).     

Age-dependent tendency to become sensitized to other classes of aeroallergens in atopic asthmatic children.

BACKGROUND: Several longitudinal studies report that allergic sensitization increases with age from childhood to adulthood. OBJECTIVE: To evaluate whether an age-dependent tendency to become sensitized to new classes of allergens is present in atopic children, we studied retrospectively the changes in allergic sensitization in 165 asthmatic patients, monosensitized (ie, sensitized to only one class of allergens) in the first survey. METHODS: All the children (18 months to 8 years at enrollment), attended our outpatient clinics twice, at time intervals ranging from 2 to 10 years. On each visit, sensitization to house dust mites, pollens, animal danders, and molds was determined by skin prick test. RESULTS: We found that 43.6% (n = 72) of the patients became polysensitized on the second survey. According to age on first survey, the patients were further divided into two age groups: (1) group 1 = 18 months to < 5 years old (n = 98) and (2) group 2 = 5 to 8 years (n = 67). The transition from monosensitization to polysensitization observed in the entire population was present in both groups: 47 (47.9%) of the 98 children in group 1 and 25 (37.3%) of the 67 children in group 2 showed to be sensitized to more classes of allergens, as compared with first survey. Both in the whole population and in the two age subgroups, the changes in the frequency of monosensitization between the two evaluations were time-dependent (P < .05, each Chi(2)). Finally, to investigate whether monosensitization to a specific class of allergens could favor the development of polysensitization, we evaluated the frequency of polysensitization in the second survey in patients originally monosensitized to house dust mites or to pollens. We found that of the 130 patients originally monosensitized to house dust mites, 59 became polysensitized (45.4%), while of the 28 patients originally monosensitized to pollens, 9 became polysensitized (32.1%) (P > . 1). Similar results were obtained when patients were divided into age groups. CONCLUSION: These data demonstrate that (1) monosensitized children are likely to become polysensitized and (2) house dust mite sensitization and, at a lower degree, pollen sensitization, apparently seem to play a "triggering" role in the development of polysensitization, since a high proportion of children originally monosensitized to house dust mites or to pollens became polysensitized.     

Neutrophil peripheral count and human leukocyte elastase during chronic lithium carbonate therapy

Plasma levels of human polymorphonuclear elastase (PMN-E) are considered a marker of granulocyte activation and can potentially complement the peripheral neutrophil count in laboratory and pathophysiological settings. Neutrophilic leukocytosis is a well known effect of lithium therapy, but there is no information about the concomitant behaviour of PMN-E in these patients. The aim of this study was to evaluate both polymorphonuclear leukocyte count and plasma PMN-E levels in depression patients undergoing chronic lithium therapy. Absolute and differential leukocyte count in venous peripheral blood was determined by an automated method, and PMN-E evaluated by enzyme immunoassay. 39 patients (11 males, 28 females; mean age 43. +/- 6.02) with depression disorders were studied, during lithium carbonate therapy. Neutrophilia (neutrophil count > 7.500x10(9) cells per liter) was found in 7 (18%) patients and an increase in plasma PMN-E levels (PMN-E > 56 microg per liter ) in 6 (15%). No correlations were found between neutrophil count, plasma concentration of PMN-E, plasma level of lithium and duration of therapy. The results show that in these patients, not only the PMN count but also elastase levels can exceed the normal range. The absence of correlation between these two parameters suggests that the state of PMN activation is not linked to their number in peripheral blood.     

Modulation of HLA-DR antigen and ICAM-1 molecule expression on airway epithelial cells by sodium nedocromil.

OBJECTIVE: To test in vitro and in vivo the hypothesis that sodium nedocromil could modulate the expression of surface molecules on airway epithelial cells. METHODS: Human bronchial epithelial cells, obtained from surgically resected bronchi, were cultured and stimulated with recombinant IFN-gamma in the presence of sodium nedocromil. The intensity of the expression of surface molecules HLA-DR and ICAM-1 molecules on bronchial epithelial cells in vitro, was quantified by specific antibody staining and flow-cytometry analysis. Furthermore, we studied the effect of the drug on airway inflammation in vivo and on allergic rhinitis patients sensitized to house dust mites. Nasal epithelial cells were collected by brushing, at baseline and 2 to 3 weeks after treatment with sodium nedocromil. The expression of HLA-DR and ICAM-1 molecules was measured by flow-cytometry, and the proportions of neutrophils and eosinophils "contaminating" the epithelial cells evaluated by light microscopy examination of nasal brushings. RESULTS: The enhanced HLA-DR and ICAM-1 expression, induced by IFN-gamma, was effectively downregulated, in a dose-dependent manner, by sodium nedocromil. At all the concentrations tested (10(-9) to 10(-4) M), the inhibitory activity of the drug was stronger on HLA-DR than on ICAM-1 expression (P<.05, all comparisons). As compared with healthy subjects, patients with allergic rhinitis had a higher expression of HLA-DR (P<.05) but not of ICAM-1 molecules (P>.05) on nasal epithelial cells, and higher proportions of nasal eosinophils (P<.05). Treatment with sodium nedocromil downregulated the expression of HLA-DR (P<.05), but not of ICAM-1 (P>.05), and induced a mild, but not statistically significant, decrease of nasal eosinophilia (P>.05). CONCLUSION: These data demonstrate that the antiinflammatory activity of sodium nedocromil may include modulation of surface molecule expression on airway epithelial cells.