Cristina Luxo

LIPID COMPOSITION CHANGES INDUCED BY TAMOXIFEN IN A BACTERIAL MODEL SYSTEM

A putative relationship between growth impairment of Bacillus stearothermophilus by tamoxifen (TAM) and TAM-induced perturbation of the physical properties of bacterial membrane lipids has been observed. The supplementation of the growth medium with Ca2+ (a membrane stabilizer) partially relieves growth inhibition by TAM, allowing growth at TAM concentrations that fully impair growth in the basal medium. B. stearothermophilus modifies the membrane lipid composition in response to the addition of TAM to the growth medium and the response is sensitive to Ca2+. Changes in lipid composition are observed in the acyl chains and in the polar head groups of phospholipids. The physical effects of alteration in these lipids was studied by fluorescence polarization of DPH and DPH-PA. Polar lipid dispersions from TAM-adapted cells grown in a Ca2+ medium show a shift of Tm to higher temperatures and a significant increase of the structural order as compared to lipids from control cells, suggesting that TAM-induced lipid composition changes compensate for the destabilizing effects of the cytostatic on membrane organization. The polar lipids from cells grown in the basal medium containing tamoxifen are also altered, but these alterations do not promote order increase of the bilayer in spite of a deviation of Tm to higher temperatures as detected by DPH. Data indicate that B. stearothermophilus controls the membrane lipid composition in response to tamoxifen, to compensate for TAM-promoted disordering in membranes and to provide an appropriate packing of phospholipid molecules in a stable bilayer, putatively disturbed by TAM incorporation.

Helicobacter pylori antimicrobial resistance rates in the central region of Portugal

Helicobacter pylori resistance to antimicrobial agents is steadily increasing. It is extremely important to be aware of the local prevalence of antibiotic resistance so as to adjust treatment strategies. During this single-centre, prospective study, we aimed to determine primary and secondary resistance rates of H. pylori to antibiotics as well as host and bacterial factors associated with this problem. Overall, 180 patients (131 female; mean age 43.4±13.5 years; primary resistance 103; secondary resistance 77) with positive (13) C-urea breath test were submitted to upper endoscopy with gastric biopsies. Helicobacter pylori was cultured and antimicrobial susceptibility was determined by Etest and molecular methods. Clinical and microbiological characteristics associated with resistance were evaluated by logistic regression analysis. Among the 180 isolates 50% were resistant to clarithromycin (primary 21.4%; secondary 88.3%), 34.4% to metronidazole (primary 29.1%; secondary 41.6%), 33.9% to levofloxacin (primary 26.2%; secondary 44.2%), 0.6% to tetracycline and 0.6% to amoxicillin. Being female was an independent predictor of resistance to clarithromycin and metronidazole. Previous, failed, eradication treatments were also associated with a decrease in susceptibility to clarithromycin. History of frequent infections, first-degree relatives with gastric carcinoma and low education levels determined increased resistance to levofloxacin. Mutations in the 23S rRNA and gyrA genes were frequently found in isolates with resistance to clarithromycin and levofloxacin, respectively. This study revealed that resistance rates to clarithromycin, metronidazole and levofloxacin are very high and may compromise H. pylori eradication with first-line and second-line empiric triple treatments in Portugal.

Triple Therapy with High-Dose Proton-Pump Inhibitor, Amoxicillin, and Doxycycline Is Useless for Helicobacter pylori Eradication: A Proof-of-Concept Study

Helicobacter pylori resistance to antibiotics is steadily increasing and multidrug‐resistant strains are common and difficult to eliminate, mainly in countries where bismuth, tetracycline, furazolidone, and rifabutin are unavailable.

Toxicity assessment of tamoxifen by means of a bacterial model

A strain of Bacillus stearothermophilus was used as a model to study physical perturbations induced in the membrane by the cytostatic tamoxifen (TAM). This study was carried out using two lines of criteria: (1) bacterial growth, and temperature growth range, with determination of growth parameters as a function of TAM concentration; and (2) biophysical studies by differential scanning calorimetry (DSC) and by means of two fluorescent probes to evaluate perturbations promoted by the drug on the structural order of bacterial lipid membranes. The inhibition of growth induced by TAM, the structural bilayer disordering, and the shift in the phase transition temperature to a lower range were also determined in the presence of Ca2+, i.e., a natural membrane stabilizer, to elucidate further perturbing effects of TAM on membranes with putative implications in cell toxicity. Growth inhibition promoted by TAM is potentiated by an increase in growth temperature above the optimal range, but attenuated or relieved by the addition of 2.5 mM Ca2+ to the culture medium. Consistently, fluorescence polarization and DSC studies showed that Ca2+ ions (2.5 mM) effectively compensated for the destabilizing effects promoted by TAM in bacterial lipid membranes.

Individuals infected with JC polyomavirus do not present detectable JC virus DNA in oropharyngeal fluids.

JC virus (JCV) is ubiquitous in the human population. Primary infection normally occurs during childhood and is followed by a lifelong persistent infection. The main mode of transmission remains unknown. Several authors have hypothesized that JCV transmission occurs through the respiratory route, and that respiratory secretions could represent a possible source of viral particles. The present study intended to evaluate oropharyngeal fluids from patients infected with JCV, in order to ascertain if respiratory secretions could indeed constitute a source of exposure to this polyomavirus. Oropharyngeal washing samples from 25 patients co-infected with JCV and human immunodeficiency virus type 1 were evaluated for the presence of JCV DNA. Regardless of the titre of antibodies or the presence of viral urinary excretion, JCV genome was not detected in oropharyngeal samples collected from any of the patients infected with JCV included in this study, which may suggest that oropharyngeal fluids are an unlikely source for JCV infection.

Tamoxifen induces ultrastructural alterations in membranes of Bacillus Stearothermophilus

Tamoxifen (TAM), a non-steroid antiestrogen, is the mostly used drug for chemotherapy and chemoprevention of breast cancer. However, the mechanisms by which TAM inhibits cell proliferation in breast cancer are not fully understood. TAM strongly incorporates in biomembranes and a variety of effects have been assigned to biophysical and biochemical interactions with membranes. Therefore, a better understanding of the physicochemical basis of interaction of TAM with biomembranes is essential to elucidate the molecular mechanisms of action. A strain of Bacillus stearothermophilus has been used as a model to clarify the interaction of TAM with the cell membrane. TAM effects on the ultrastructure of membranes of this bacterium were evaluated by electron microscopy. Important ultrastructural alterations were observed in B. stearothermophilus treated with TAM, namely change in the geometry of the membrane profile from asymmetric to symmetric, disaggregation of ribosomes, coagulation of the cytoplasmic matrix, occurrence of mesossomes, appearance of fractures in membranes and the alteration of the ultrastructure of cell wall. These ultrastructural alterations confirm that TAM is a membrane-active drug and that membrane damage may be involved in molecular mechanisms of cell death induced by this drug.

Toxic effects of tamoxifen on the growth and respiratory activity of Bacillus stearothermophilus

The anticancer drug tamoxifen (TAM) is used as first line therapy in breast cancer. Although tamoxifen is usually considered an estrogen antagonist, several studies suggest alternative mechanisms of action. Bacillus stearothermophilus has been used as a model to clarify the antiproliferative action of tamoxifen putatively related with drug-membrane interaction. According to previous data, TAM induces perturbation of membrane structure along with impairment of bacterial growth. The aim of this work was to correlate the effects of TAM on growth of intact B. stearothermophilus with the respiratory activity of isolated protoplasts of this bacteria. TAM inhibits bacterial growth and oxygen consumption of protoplasts as a function of concentration. Effects on oxygen consumption depend on the substrate used: NADH, allowing to study the full respiratory chain and ascorbate-TMPD to probe the final oxidase segment. The interaction of TAM with the respiratory components occurs at a level preceding the cytochrome oxidase segment.

Topoisomerase I Improve JC virus DNA detection

Introduction: Progressive Multifocal Leukoencephalopathy (PML) is a severe and often fatal CNS disease caused by JC human polyomavirus infection. Diagnosis of PML is based upon suggestive clinical symptoms and brain images, supported by the presence of JC virus genome in CSF samples. Objective: The main objective of our study was the search for an alternative method for JC virus DNA detection in CSF samples, with sensitivity and specificity characteristics close to that of standard techniques, but feasible at any clinical laboratory. Methods: In order to evaluate the effect of topoisomerase I treatment in the detection of JC virus genome, and its feasibility in laboratory diagnosis of PML, 129 CSF samples were examined for the presence of JC virus DNA by a nested-PCR protocol, with and without previous treatment with topoisomerase I. All CSF samples were also evaluated through a real-time PCR protocol. Results: Eleven CSF samples presented detectable JC virus DNA with all used protocols. On 9 CSF samples, JC virus DNA was only detectable with topoisomerase I modified nested-PCR and real-time PCR protocols. Real-time PCR was the only protocol able to detect JC virus genome in 4 CSF samples. One CSF sample revealed the presence of the expected amplified fragment only when tested with topoisomerase I modified nested-PCR protocol. Conclusion: The results of the present study point towards the benefit of using topoisomerase I DNA treatment before amplification reactions in JC virus DNA detection on CSF samples, and confirm that topoisomerase I modified nested-PCR protocol represents a good alternative method to detect JC virus DNA in CSF samples from patients with clinical signs and brain images suggestive of PML.

Cytotoxicity and Membrane Interaction of Tamoxifen as Affected by Ca2+ and Mg2+: Use of a Bacterial Model System

A strain of Bacillus stearothermophilus was used as a model to study the interaction of tamoxifen (TAM) with the membrane and the cytostatic antiproliferative effects not related to estrogen binding. TAM inhibits the growth of B. stearothermophilus as a function of concentration. The supplementation of the growth medium with Ca(2+) or Mg(2+) partially relieves the growth inhibition by TAM, allowing growth at TAM concentrations that fully impair growth in the basal medium. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and of its propionic acid derivative (DPH-PA) reveals opposite effects induced by TAM and Ca(2+). The addition of Ca(2+) to liposomes of bacterial lipids promoted physical ordering as opposed to disordering induced by TAM. Thus, it is predictable that growth impairment induced by TAM is mediated through perturbations at the membrane level.

First Serological Evidence on Endemicity of HEV Infection in Wild Boar ( Sus scrofa ) Populations from Portugal

Hunting is a common and popular pastime in Portugal. Hunted animals are, generally, for human consumption as meat or local products that are consumed without cooking, increasing the risk of zoonotic transmission of several infectious agents. The present study intended to characterize HEV infection in hunted wild boars (species Sus scrofa) from two regions of Portugal in order to estimate its importance as reservoir for zoonotic spread of HEV to humans, and its possible implication in public health. Markers for both past and/or ongoing HEV infection were evaluated in serum, bile and stool samples of 29 wild boars. The presence of specific HEV antibodies as marker of past infection was evaluated in serum samples, while active HEV infection was evaluated through the detection of HEV genome in bile and stool samples. HEV specific antibodies were detected in 14% of the studied animals, while none of the tested bile or stool samples revealed detectable HEV genome. Despite no active HEV infection was demonstrated in the hunted animals included in the present study, serological analysis revealed the endemicity of HEV infection in Portuguese wild boars from the studied regions, corroborating its possible role as zoonotic reservoir of such virus. The proved endemicity of HEV infection among wild boars further support the importance of including HEV in national and regional surveillance programs for wild animal diseases, as well as to the awareness for thorough cook all wild boar products and to the education of occupationally exposed people in order to prevent HEV infection.