Carbon dots, a powerful non-toxic support for bioimaging by fluorescence nanoscopy and eradication of bacteria by photothermia.

Carbon Dots (CDs) are innovative materials which have potential applications in many fields, including nanomedicine, energy and catalysis. Here CDs were produced by the alkali-assisted ultrasonic route and characterized by several techniques to determine their composition and properties. Fluorescence nanoscopy using single-molecule localization microscopy shows that they have very good photophysical properties and a remarkable blinking behaviour at 405 nm. Moreover, these CDs are a safe material, non-toxic towards different cell lines (cancer and non-cancer cells) even at very high concentration, reflecting an excellent biocompatibility. Photothermia, i.e. their heating capacity under laser irradiation, was evaluated at two wavelengths and at several power densities. The resulting temperature increment was high (5 < ΔT < 45 °C) and appropriate for biomedical applications. Bioimaging and photothermia were then performed on E. coli, a Gram(-) bacterium, incubated with CDs. Remarkably, by photothermia at 680 nm (0.3, 1 and 1.9 W cm-2) or 808 nm (1.9 W cm-2), CDs are able to eradicate bacteria in their exponential and stationary phases. Images obtained by 3D super-resolution microscopy clearly show the different CD distributions in surviving bacteria after mild photothermal treatment. These results confirm that CDs are multifunctional materials with a wide range of biomedical applications.

A pragmatic approach for mortality prediction after surgery in infective endocarditis: optimizing and refining EuroSCORE.

OBJECTIVE: To simplify and optimize the ability of EuroSCORE I and II to predict early mortality after surgery for infective endocarditis (IE). METHODS: Multicentre retrospective study (n = 775). Simplified scores, eliminating irrelevant variables, and new specific scores, adding specific IE variables, were created. The performance of the original, recalibrated and specific EuroSCOREs was assessed by Brier score, C-statistic and calibration plot in bootstrap samples. The Net Reclassification Index was quantified. RESULTS: Recalibrated scores including age, previous cardiac surgery, critical preoperative state, New York Heart Association >I, and emergent surgery (EuroSCORE I and II); renal failure and pulmonary hypertension (EuroSCORE I); and urgent surgery (EuroSCORE II) performed better than the original EuroSCOREs (Brier original and recalibrated: EuroSCORE I: 0.1770 and 0.1667; EuroSCORE II: 0.2307 and 0.1680). Performance improved with the addition of fistula, staphylococci and mitral location (EuroSCORE I and II) (Brier specific: EuroSCORE I 0.1587, EuroSCORE II 0.1592). Discrimination improved in specific models (C-statistic original, recalibrated and specific: EuroSCORE I: 0.7340, 0.7471 and 0.7728; EuroSCORE II: 0.7442, 0.7423 and 0.7700). Calibration improved in both EuroSCORE I models (intercept 0.295, slope 0.829 (original); intercept -0.094, slope 0.888 (recalibrated); intercept -0.059, slope 0.925 (specific)) but only in specific EuroSCORE II model (intercept 2.554, slope 1.114 (original); intercept -0.260, slope 0.703 (recalibrated); intercept -0.053, slope 0.930 (specific)). Net Reclassification Index was 5.1% and 20.3% for the specific EuroSCORE I and II. CONCLUSIONS: The use of simplified EuroSCORE I and EuroSCORE II models in IE with the addition of specific variables may lead to simpler and more accurate models.

Maghemite nanoparticles coated with human serum albumin: combining targeting by the iron-acquisition pathway and potential in photothermal therapies.

Human serum albumin (HSA), the most abundant plasma protein in human blood, is a natural transport vehicle with multiple ligand binding sites. It, therefore, constitutes an attractive candidate for drug delivery. Targeting may occur via the most known interaction of the protein with the neonatal Fc receptor (FcRn). Here, we investigate another HSA delivery path, involving the transferrin receptor, and we elaborate a maghemite-HSA nanohybrid, opening up new opportunities for medical applications. Fluorescence spectrophotometric titration and size-exclusion chromatography were used to substantiate, in cell-free assays, an interaction between HSA and the transferrin receptor R1. This occurs with a dissociation constant, KD of 6.7 nM. This interaction was confirmed in HeLa cell culture where, by confocal microscopy, rhodamine-labeled HSA is shown to be internalized. HSA was then covalently conjugated onto maghemite nanoparticles (NPs) to give a NP-HSA nanohybrid. The therapeutic potential of this hybrid was demonstrated through its heating capacity in magnetic hyperthermia (MH) and near-infrared (NIR) photothermia (PT). In particular, the Specific Absorption Rate (SAR) in the PT Therapy (PTT) mode, using a 808 nm NIR-LASER (1 W cm-2) and at iron concentration as low as 2.5 mM, was found to be very high, equal to 1870 W g-1 with a temperature increment of 9.2 °C. The nanohybrids incubated with HeLa cells were mainly localized at the cell surface. When the PTT mode was applied under the same conditions as in vitro, mortality was higher in HeLa cells than in fibroblasts (non-malignant cells). Cytotoxicity was checked in both cell lines without the PTT mode; the nanohybrids do not seem to affect cell viability. These results make the nanohybrids very promising agents for NIR-PT and for targeting in cancer therapy, since non-malignant cells were not damaged.

Surgical treatment for infective endocarditis in elderly patients.

OBJECTIVES: We evaluate the clinical, echographic and prognostic characteristics of infective endocarditis (IE) in a large population of elderly patients, and the results of surgical approach. METHODS: Multicentric, prospective, observational cohort study with 961 consecutive left-sided IE: 356 patients aged ≥65 years were compared with 605 younger. Indications for cardiac surgery, potential surgical risk, time and outcome, were compared. RESULTS: Hospital-acquired endocarditis, comorbidity, renal failure and septic shock were more frequent in elderly, but embolisms were less. Intracardiac destruction and ventricular failure were similar in both groups, but significantly fewer elderly patients underwent cardiac surgery (36% vs 51%; p < 0.01), and this group showed a worse outcome (43.2% of mortality vs 27% in younger; p < 0.01), resulting age as an independent predictor of mortality (OR: 1.02 CI95%: 1.01-1.03). Compared with medical treatment, surgery showed lower percentages of mortality compared with medical treatment (23.3% vs 31.3%; p = 0.03) in younger group, but a high mortality was observed with both procedures (47.6% vs 40.3%; p = 0.1) in the elderly. CONCLUSIONS: Although similar percentages of heart failure and intracardiac complications, increasing age is associated with higher mortality in IE. Lower rates of surgical treatment and a worse outcome after operation are common features in elderly patients.

Streptococcus agalactiae left-sided infective endocarditis. Analysis of 27 cases from a multicentric cohort.

SUMMARY OBJECTIVE: To evaluate the current trends in the clinical characteristics and the prognosis of Streptococcus agalactiae infective endocarditis (IE), uncommon disease associated with high mortality. METHODS: Descriptive analysis of 27 cases of a large cohort (961 episodes) of infective endocarditis collected in seven hospitals of Andalusia (Spain) between 1984 and 2008. RESULTS: Native valves were affected in most cases (85. 2%), multiple valves were frequently involved (22.2%). The median age of the patients was 65 (51-76) years (59.3% men), with a comorbidity, according to the Charlson index, of 2.6+/-2.3. The most frequent underlying diseases were diabetes mellitus (25.9%), chronic obstructive pulmonary disease (14.8%), neoplasms (14.8%), urological disorders (11%) and chronic liver disease (11%). Clinical presentation was characterized by rapid worsening (median of 9 (5.7-15) days from onset of symptoms until diagnosis), a high rate of embolisms (37%) and cardiac complications (abscesses, fistulas or valve rupture) - 37% of cases. Surgery was performed in 12 patients (44.4%) and a high mortality (40.7%) was observed. CONCLUSION: S. agalactiae IE is a serious disease with aggressive course and high mortality rate and affects patients with debilitating diseases. We must be alert of the development of complications and consider early valve surgery when it is necessary.

Healthcare-associated infective endocarditis: an undesirable effect of healthcare universalization.

Invasive medical technology has led to an increase in the incidence of healthcare-associated infective endocarditis (HAIE). A prospective multicentre cohort study was conducted at seven hospitals in Andalusia, Spain, to establish the characteristics of HAIE and to compare them with those of community-acquired infective endocarditis (CAIE). HAIE was defined as either infective endocarditis (IE) manifesting >48 h after admission to hospital, or IE associated with a significant invasive procedure performed in the 6 months before diagnosis. Seven hundred and ninety-three cases of IE were investigated, and HAIE accounted for 127 (16%). As compared with patients with CAIE, patients with HAIE were older (60.1 ± 14.4 years vs. 53.6 ± 17.5 years) and had more comorbidities (Charlson index 3.3 ± 2.3 vs. 1.8 ± 2.3) and staphylococcal infections (58.3% vs. 24.8%). Vascular manipulation was the main cause of bacteraemia responsible for HAIE (63%). Peripheral vein catheter-associated bacteraemia accounted for 32.8% of the catheter-related bacteraemias. In-hospital mortality (44.9% vs. 24.2%) was higher in the HAIE group. Septic shock (OR 2.2, 95% CI 2.9-30.2) and surgery not performed because of high surgical risk (OR 1.6, 95% CI 1.2-20) were independent predictors of mortality in HAIE. The present study demonstrates that HAIE is a growing health problem associated with high mortality. Careful management of vascular devices is essential to minimize the risk of bacteraemias leading to HAIE.

Evaluation of the in-vitro cidal activity and toxicity of a novel peroxygen biocide: 2-butanone peroxide.

The monomer of 2-butanone peroxide is a novel peroxygen derivative with potential use as biocide in the hospital environment. The aim of this study was to test the biocidal activity of different concentrations of the compound against American Tissue Culture Collection strains from 11 different micro-organisms, including bacteria, mycobacteria, spores, fungi and virus, following the European Standard guidelines. Toxicity tests were also carried out following United States Environmental Protection Agency Standards. 2-Butanone peroxide exhibited biocidal activity at 0.12% against Legionella pneumophila, at 0.5% against Escherichia coli, Pseudomonas aeruginosa and Enterococcus hirae, and at 1% against Staphylococcus aureus after 5 min contact at room temperature. Mycobactericidal activity was obtained at 0.5% after 60 min contact at 20 degrees C, and sporicidal activity was obtained at 4% after 60 min at 40 degrees C. Good fungicidal (against yeasts and moulds) and virucidal (adenovirus and poliovirus) activities were obtained at 0.5% after 60 min contact. Toxicity assessment showed negative results in the acute dermal irritation test, acute eye irritation test and acute oral toxicity test. The skin sensitisation test was negative. The safety profile in the toxicity tests and the basic cidal activity against the strains tested suggest that 2-butanone peroxide in the control of hospital infections.

Quinolone resistance in female outpatient urinary tract isolates of Escherichia coli: age-related differences.

During a 1-year period, from November 2003 to October 2004, urinary Escherichia coli isolates were collected from 20 clinical microbiology laboratories across Spain. The main objective was to assess the resistance of E. coli to the antimicrobials most commonly prescribed for community-acquired urinary tract infections depending on the patient's age. A total of 2,230 valid E. coli strains from female outpatients were isolated and sent to a single central reference laboratory for confirmation and susceptibility testing using an agar dilution method. A two-sided chi-squared test was used to assess the differences in resistance between age groups (< or =65 and >65 years). E. coli resistance was found to be more common to ampicillin (52.1%), cotrimoxazole (26%) and quinolones (18%), whereas resistance to amoxicillin-clavulanic acid, cefuroxime axetil and fosfomycin were below 3%. In women older than 65 years, resistance to ciprofloxacin reached up to 29% compared with 13% of those in the under 65 age group (p <0.001). For cotrimozaxole, rates were 32% vs. 23% (p <0.001) and for ampicillin 56% vs. 50% (p=0.02), respectively. It was concluded that fosfomycin, amoxicillin-clavulanic acid and cefuroxime axetil are the most suitable antimicrobials for empirical treatment in Spain given the high 18% and 26% resistance rates to quinolones and cotrimoxazole, respectively. Being older than 65 years of age was associated with higher resistance rates to ciprofloxacin (29%). These results should be considered when recommending empirical therapy for acute cystitis in women.

Antimicrobial susceptibility of clinical Escherichia coli isolates from uncomplicated cystitis in women over a 1-year period in Spain.

High usage of antibiotics in Spain has led to an increase in resistance in urinary Escherichia coli isolates in different geographic regions. The problem of resistance in urinary E. coli in Spain was investigated by gathering a large number of isolates from 20 different sites nationwide over a 1-year period from November 2003 to October 2004 in a large population of women. The objectives of this study were to assess the resistance to the antibiotics most commonly prescribed for community-acquired urinary tract infections (UTIs), according to age and different geographical areas of Spain, and to evaluate the potential association between geographical differences in quinolone consumption and resistance to E. coli. A total of 2,292 valid E. coli strains from female outpatients were isolated and sent to a single central reference laboratory for confirmation and susceptibility testing. Of these, 2,230 isolates were available for the age analysis. A two-sided chi2 test was used to identify differences in resistance between age groups. Antibiotic units per province were purchased from IMS and consumption was expressed in units per 1,000 people per year. Univariate correlation (Pearson coefficient) between resistance to ciprofloxacin and quinolone consumption was calculated using a two-sided p-value. Resistance shown by E. coli was more common to ampicillin (52.1%) and cotrimoxazole (26%), followed by quinolones (18%), whereas resistance to amoxicillin-clavulanic acid, cefuroxime-axetil and fosfomycin was less than 3%. In the subgroup of women aged >65 years, resistance to ciprofloxacin was 29% compared to 13% for the subgroup of women <65 years (p<0.001). For these same subgroups, resistance rates were 32% vs. 23% for cotrimoxazole (p<0.001) and 56% vs. 50% for ampicillin (p=0.02), respectively. Statistically significant correlations were found between consumption of quinolones and E. coli resistance to ciprofloxacin (r=0.5; p=0.025). Resistance of E. coli isolates to quinolones varied significantly according to geographical areas, ranging from a high of 16.5% and 16.6% in the southern and eastern regions of Spain, respectively, to a low of 8% in the north in women aged <65 years. Additionally, the susceptibility to quinolones of E. coli isolates recovered from women aged >65 years was significantly lower across all regions of Spain than that of isolates recovered from younger women. Fosfomycin, amoxicillin/clavulanic acid and cefuroxime-axetil are the most suitable antibiotics for empirical treatment in Spain given the high 18% and 26% resistance rates to quinolones and cotrimoxazole, respectively. Higher resistance rates to ciprofloxacin were associated with being aged 65 years and over. These data need to be considered when recommending empirical therapy for acute cystitis.

Aberrant methylation of multiple genes in neuroblastic tumours. relationship with MYCN amplification and allelic status at 1p.

Aberrant hypermethylation occurs in tumour cell CpG islands and is an important pathway for the repression of gene transcription in cancers. We investigated aberrant hypermethylation of 11 genes by methylation-specific polymerase chain reaction (PCR), after treatment of the DNA with bisulphite, and correlated the findings with MYCN amplification and allelic status at 1p in a series of 44 neuroblastic tumours. This tumour series includes five ganglioneuromas (G), one ganglioneuroblastoma (GN) and 38 neuroblastomas (six stage 1 tumours; five stage 2 tumours; six stage 3 cases; 19 stage 4 tumours, and two stage 4S cases). Aberrant methylation of at least one of the 11 genes studied was detected in 95% (42 of 44) of the cases. The frequencies of aberrant methylation were: 64% for thrombospondin-1 (THBS1); 30% for tissue inhibitor of metalloproteinase 3 (TIMP-3); 27% for O6-methylguanine-DNA methyltransferase (MGMT); 25% for p73; 18% for RB1; 14% for death-associated protein kinase (DAPK), p14ARF, p16INK4a and caspase 8, and 0% for TP53 and glutathione S-transferase P1 (GSTP1). No aberrant methylation was observed in four control normal tissue samples (brain and adrenal medulla). MYCN amplification was found in 11 cases (all stage 4 neuroblastomas), whereas allelic loss at 1p was identified in 16 samples (13 stage 4 and two stage 3 neuroblastomas, and one ganglioneuroma). All but one case with caspase 8 methylation also displayed MYCN amplification. Our results suggest that promoter hypermethylation is a frequent epigenetic event in the tumorigenesis of neuroblastic tumours, but no specific pattern of hypermethylated genes could be demonstrated.

CpG island methylation status and mutation analysis of the RB1 gene essential promoter region and protein-binding pocket domain in nervous system tumours.

A series of 136 nervous system tumours were studied to determine the methylation status of the CpG island contained within the promoter region of the RB1 gene, as well as mutation analysis of the essential promoter region and exons 20-24 (and surrounding intronic regions) coding for the protein-binding pocket domain. Methylation of the RB1 CpG island was detected in 26 samples corresponding to nine glioblastomas, three anaplastic astrocytomas, one mixed oligo-astrocytoma, one ependymoma, two medulloblastomas, two primary central nervous system lymphomas, two neurofibrosarcomas, and six brain metastasis from solid tumours. No inactivating mutations were found within the RB1 promoter region, whereas one glioblastoma and one oligodendroglioma displayed similar sequence variations consisting of 12 and 8 base pair deletions at intron 21. These results suggest that RB1 CpG island hypermethylation is a common epigenetic event that is associated with the development of malignant nervous system tumours.

Absence of mutation of the p73 gene in astrocytic neoplasms.

In subgroups of astrocytic neoplasms, including glioblastoma (GBM), mutations of the p53 tumour suppressor gene lead to loss of growth-suppressive properties. A p53-related gene termed p73 has recently been identified; its gene product shows structural and functional similarities to p53. After being mapped to chromosome region 1p36, p73 was proposed to act as a tumour suppressor gene, as this region is frequently deleted in a variety of human cancers, including astrocytic tumours. To determine whether p73 is involved in astrocytoma/GBM development, we analysed 10 pilocytic astrocytomas, 15 WHO grade II astrocytomas, 15 WHO grade III anaplastic astrocytomas, and 20 GBM for p73 gene alterations. In parallel, we used six polymorphic markers to determine the allelic status of region 1p36 in this tumour series. Although loss of heterozygosity was evidenced in 12 of 60 cases (20% of samples), PCR-SSCP and direct sequencing failed to detect any gene mutation in the entire coding region and intronic sequences of p73. Eight tumours displayed five distinct polymorphic nucleotide changes, also present in the corresponding normal DNA. These variations consisted of T-->C variation, with no change in Thr173; C-->T transition, with no change in His197; exon 9 simultaneous double change C-->T and T-->C , with no variations in Ala336 and His349, respectively, and C-->T change at exon 9/-24 position of intron 8. These results suggest that, in astrocytic gliomas, p73 may not play a major role as a tumour suppressor, but the relatively high incidence of LOH confirms the presence at 1p36 of an as yet unidentified gene of this category, with a key function in astrocytoma/GBM progression.

Inhibition of Fas signaling prevents hepatic injury and improves organ blood flow during sepsis.

BACKGROUND: Fas/Fas ligand (FasL) system is one of the major pathways triggering apoptosis that has been shown to play an important role in development and pathogenesis of various diseases including liver and gastrointestinal diseases. Studies indicate that FasL deficiency provides a survival advantage in mice subjected to polymicrobial sepsis. However, the extent to which Fas/FasL contributes to organ injury during sepsis is unclear. Thus, the aim of this study was to determine whether in vivo administration of a Fas-signaling inhibitor during sepsis preserves organ function. METHODS: Male adult C3H/HeN mice were subjected to cecal ligation and puncture (CLP) or sham CLP (sham). Twelve hours after CLP, mice received either Fas-receptor fusion protein (FasFP) (200 microg/kg body weight) or the saline vehicle. Twenty-four hours after the onset of sepsis, cardiac output and organ blood flow were measured with radioactive microspheres. Plasma levels of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase were assessed as indexes of liver damage. Changes in systemic cytokines were measured by enzyme-linked immunosorbent assay. RESULTS. The data indicate that although cardiac output and organ blood flow in the liver, intestine, kidneys, spleen, and heart decreased markedly at 24 hours after CLP, treatment with FasFP maintained the measured hemodynamic parameters and improved hepatic, intestinal, and heart blood flow (P <.05) and partially restored spleen and renal blood flow. Moreover, FasFP treatment markedly attenuated the systemic rise in alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and interleukin 10 (P <.05). CONCLUSIONS: These results not only indicate that there is a role for Fas/FasL-mediated processes in the induction of organ injury but suggest that inhibition of Fas/FasL pathway may represent a novel therapeutic modality for maintaining organ perfusion and preventing liver injury during sepsis.

Analysis of p73 gene in meningiomas with deletion at 1p.

The p73 gene has been mapped to 1p36.33, a chromosome region that is frequently deleted in a wide variety of neoplasms including meningiomas. The protein encoded by p73 shows structural and functional similarities to p53 and may thus represent a candidate tumor suppressor gene. To determine whether p73 is involved in the development of meningiomas, we examined 30 meningioma samples with proven 1p deletion for mutations of p73. Sequence analysis of the entire coding region of the p73 gene revealed previously reported polymorphisms in eight cases. A tumor-specific missense mutation as a result of an A-to-G transition with an Asn204Ser change was found in one meningioma that nevertheless retained the normal allele. These results suggest that if p73 plays a role in meningioma carcinogenesis, it must be in a manner different from the Knudson two-hit model.

Mutation analysis of the p73 gene in nonastrocytic brain tumours.

Loss of heterozygosity (LOH) involving the distal chromosome 1 p36 region occurs frequently in nonastrocytic brain tumours, but the tumour suppressor gene targeted by this deletion is unknown. p73 is a novel gene that has high sequence homology and similar gene structure to the p53 gene; it has been mapped to 1 p36, and may thus represent a candidate for this tumour suppressor gene. To determine whether p73 is involved in nonastrocytic brain tumour development, we analysed 65 tumour samples including 26 oligodendrogliomas, 4 ependymomas, 5 medulloblastomas, 10 meningiomas, 2 meningeal haemangiopericytomas, 2 neurofibrosarcomas, 3 primary lymphomas, 8 schwannomas and 5 metastatic tumours to the brain, for p73 alterations. Characterization of allelic loss at 1 p36-p35 showed LOH in about 50% of cases, primarily involving oligodendroglial tumours (22 of 26 cases analysed; 85%) and meningiomas (4 of 10; 40%). PCR-SSCP and direct DNA sequencing of exons 2 to 14 of p73 revealed a missense mutation in one primary lymphoma: a G-to-A transition, with Glu291Lys change. 8 additional cases displayed no tumour-specific alterations, as 3 distinct polymorphic changes were identified: a double polymorphic change of exon 5 was found in one ependymoma and both samples derived from an oligodendroglioma, as follows: a G-to-A transition with no change in Pro 146, and a C-to-T variation with no change in Asn 204: a delG at exon 3/+12 position was identified in 4 samples corresponding to 2 oligodendrogliomas, 1 ependymoma and 1 meningioma, and a C-to-T change at exon 2/+10 position was present in a metastatic tumour. Although both LOH at 1 p36 and p73 sequence changes were evidenced in 4 cases, it is difficult to establish a causal role of the p73 variations and nonastrocytic brain tumours development.

Immunological diagnosis of human hydatid cyst relapse: utility of the enzyme-linked immunoelectrotransfer blot and discriminant analysis.

A discriminant technique was applied to the different serological patterns obtained by enzyme-linked immunoelectrotransfer blotting (EITB) and by conventional immunological tests, in order to differentiate the residual antibody patterns present in healed hydatidosis from the ones present in patients with active hydatidosis. For this purpose, specific antibodies against Echinococcus granulosus were detected by indirect hemagglutination, agglutination of latex particles, basophil degranulation, and EITB for 23 patients with active hydatidosis and 45 patients with surgically cured hydatidosis. Discriminant analysis of the different serological patterns obtained by EITB and conventional serology correctly classified 92.54% of patients (93.3% if the patients are differentiated according to the time elapsed since surgery). This method detected the presence of active hydatidosis in 95.6% of patients for whom abdominal ultrasonography had confirmed the presence of active hydatid cysts. The global specificity was 88.9%. The specificity was 97.1% for patients who had been operated on 3 years ago or more and 63.6% for patients with less time since surgery.