Role of TrkA signalling and mast cells in the initiation of osteoarthritis pain in the monoiodoacetate model.

OBJECTIVE: Aiming to delineate novel neuro-immune mechanisms for NGF/TrkA signalling in osteoarthritis (OA) pain, we evaluated inflammatory changes in the knee joints following injection of monoiodoacetate (MIA) in mice carrying a TrkA receptor mutation (P782S; TrkA KI mice). METHOD: In behavioural studies we monitored mechanical hypersensitivity following intra-articular MIA and oral prostaglandin D2 (PGD2) synthase inhibitor treatments. In immunohistochemical studies we quantified joint mast cell numbers, calcitonin gene-related peptide expression in synovia and dorsal root ganglia, spinal cord neuron activation and microgliosis. We quantified joint leukocyte infiltration by flow cytometry analysis, and PGD2 generation and cyclooxygenase-2 (COX-2) expression in mast cell lines by ELISA and Western blot. RESULTS: In TrkA KI mice we observed rapid development of mechanical hypersensitivity and amplification of dorsal horn neurons and microglia activation 7 days after MIA. In TrkA KI knee joints we detected significant leukocyte infiltration and mast cells located in the vicinity of synovial nociceptive fibres. We demonstrated that mast cells exposure to NGF results in up-regulation of COX-2 and increase of PGD2 production. Finally, we observed that a PGD2 synthase inhibitor prevented MIA-mechanical hypersensitivity in TrkA KI, at doses which were ineffective in wild type (WT) mice. CONCLUSION: Using the TrkA KI mouse model, we delineated a novel neuro-immune pathway and suggest that NGF-induced production of PGD2 in joint mast cells is critical for referred mechanical hypersensitivity in OA, probably through the activation of PGD2 receptor 1 in nociceptors: TrkA blockade in mast cells constitutes a potential target for OA pain.

In vitro activity of antifungals in combination with essential oils against the oomycete Pythium insidiosum.

AIMS: The aim of this study was to investigate the in vitro susceptibility of Pythium insidiosum to combinations of the antifungal drugs terbinafine or itraconazole with Melaleuca alternifolia, Mentha piperita and Origanum vulgare essential oils (EOs). METHODS AND RESULTS: In vitro combinations of antifungal drugs with EOs were evaluated by checkerboard microdilution method against 20 Brazilian isolates of P. insidiosum. The tests were performed according to protocol M38-A2, and the interpretation of each combination result was based on the values of the fractional inhibitory concentration index. The combinations of itraconazole with EOs presented prominent synergistic effects against P. insidiosum isolates, and no antagonism was observed with these combinations. However, the combinations of terbinafine with EOs resulted in indifferent or antagonistic effects. CONCLUSIONS: The combination of plant-derived bioactive compounds with antifungal drugs may be an alternative therapy for the control of infections caused by P. insidiosum. Studies of new therapeutic protocols involving these proposed combinations are needed. SIGNIFICANCE AND IMPACT OF THE STUDY: The antimicrobial combinations using EOs with terbinafine or itraconazole can be an attractive therapeutic option for controlling P. insidiosum infections.

In vitro susceptibility of Brazilian Pythium insidiosum isolates to essential oils of some Lamiaceae family species.

The present study aimed to evaluate the in vitro antimicrobial action of Origanum vulgare, Origanum majorana, Mentha piperita and Rosmarinus officinalis on Pythium insidiosum oomycete zoospores. The antimicrobial activity evaluation was performed by the broth microdilution method according to CSLI M38-A2 documentation adapted to phytopharmaceuticals. Twenty-two P. insidiosum isolates were evaluated, and the minimum inhibitory concentration was determined at 100% growth inhibition. All P. insidiosum isolates evaluated showed a minimum inhibitory concentration ranging from 0.05 to 1.75 mg/mL when O. vulgare oil was used and from 0.11 to 3.5 mg/mL for O. majorana, M. piperita and R. officinalis oils. The results obtained indicate that the essential oils tested showed antimicrobial activity on P. insidiosum, with O. vulgare essential oil showing the best performance. These findings emphasize the potential use of plant essential oils as control agents in P. insidiosum infections; further research, however, is needed so as the in vivo activity of these oils can also be evaluated.

In vitro susceptibility of zoospores and hyphae of Pythium insidiosum to antifungals.

OBJECTIVES: The purpose of this study was to compare the in vitro susceptibilities of 22 Brazilian isolates of Pythium insidiosum to antifungals using a standardized inoculum of zoospores and a proposed novel inoculum prepared from cultured mycelia (hyphae) of P. insidiosum. METHODS: A zoospore suspension of P. insidiosum was obtained by the zoosporogenesis technique. The hyphal inoculum was prepared from a suspension of P. insidiosum mycelium. Susceptibility to each drug was evaluated using the CLSI M38-A2 method. RESULTS: Of the 88 MIC comparisons performed, 36 (41%) showed the same MIC value for the two inocula. The agreement (differences not greater than one dilution) between MICs obtained with both types of inocula was 39.8% (35/88). In other MIC comparisons analysed, 17 (19.3%) showed differences of two or three dilutions. CONCLUSIONS: We conclude that the use of hyphal inocula of P. insidiosum for in vitro susceptibility tests could be a suitable method for evaluating antimicrobial susceptibility, particularly when it is not possible to obtain a standardized zoospore inoculum.

Selective purification of supercoiled p53-encoding pDNA with L-methionine-agarose matrix.

The p53 tumor suppressor gene has been widely explored for gene therapy as an alternative to the common treatments. Recently, the supercoiled conformation of a p53-encoding plasmid proved to be more efficient in cell transfection and protein expression than the open circular conformation. To successfully isolate this isoform, several chromatographic techniques have been used, namely affinity chromatography with amino acids as ligands. However, the study of new matrices and ligands with higher specificity and robustness for supercoiled plasmid purification is still required. The present work explores for the first time a new matrix of l-methionine-agarose to efficiently purify the supercoiled p53-encoding plasmid. The binding/elution conditions, such as salt concentration and temperature, were manipulated and combined to attain the best strategy. Therefore, the supercoiled plasmid isoform was purified from a clarified lysate by using a decreasing stepwise gradient comprising 2.35 and 1.7M ammonium sulfate in 10mM Tris-HCl, pH 8.0, and finally 10mM Tris-HCl, pH 8.0, at 5°C. After accomplishing the purification process, we performed several tests to assess the quality of the supercoiled plasmid, revealing that the amounts of proteins, gDNA, RNA, and endotoxins were significantly reduced or undetectable in the final formulation.

A large community outbreak of Legionnaires disease in Vila Franca de Xira, Portugal, October to November 2014.

An outbreak of Legionnaires' disease with 334 confirmed cases was identified on 7 November 2014 in Vila Franca de Xira, Portugal and declared controlled by 21 November. Epidemiological, environmental and microbiological analysis identified industrial wet cooling systems to be the probable source of infection. Preliminary results from sequence-based typing of clinical specimens and environmental isolates confirmed this link. A series of meteorological phenomena are likely to have contributed to the scale of this outbreak.

Airways changes related to air pollution exposure in wheezing children.

In this study, we aimed to evaluate the relationship between individual total exposure to air pollution and airway changes in a group of 51 wheezing children. Respiratory status was assessed four times (January 2006, June 2006, January 2007 and June 2007) during a 1-week period through a standardised questionnaire, spirometry, exhaled nitric oxide fraction and pH in exhaled breath condensate (EBC). Concentrations of particles with a 50% cut-off aerodynamic diameter of 10 µm (PM(10)), O(3), NO(2) and volatile organic compounds were estimated through direct measurements with an ad hoc device or air pollution modelling in the children's schools and at their homes in the same 4 weeks of the study. For each child, total exposure to the different air pollutants was estimated as a function of pollutant concentrations and daily activity patterns. Increasing total exposure to PM(10), NO(2), benzene, toluene and ethylbenzene was significantly associated with a decrease of forced expiratory volume in 1 s (FEV(1)) and with an increase of change in FEV(1). Increasing exposure to NO(2) and benzene was also related to a significant decrease of FEV(1)/forced vital capacity. Increasing exposure to PM(10), NO(2), benzene and ethylbenzene was associated with acidity of EBC. This study suggests an association in wheezing children between airway changes and total exposure to air pollutants, as estimated by taking into account the concentration in the various microenvironments attended by the children.

Additive Manufacturing Tools to Improve the Performance of Chromatographic Approaches.

Chromatography is widely applied industrially. However, some limitations are associated with its common supports, and the impossibility to fully control their structural features is particularly restrictive. Additive manufacturing (AM) is emerging as a fast, highly precise, and reproducible technology for producing chromatographic supports that can improve its performance.

Dilemma on plasmid DNA purification: binding capacity vs selectivity.

Plasmid DNA chromatography is a powerful field in constant development and evolution. The use of this technique is considered mandatory in the production of an efficient and safe formulation to be applied for plasmid-mediated gene therapy. Concerning this, the search for an ideal chromatographic support/ligand combination motivated scientist to pursue a continuous improvement on the plasmid chromatography performance, looking for a progression on the ligands and supports used. The present review explores the different approaches used over time to purify plasmid DNA, ambitioning both high recovery and high purity levels. Overall, it is presented a critical discussion relying on the relevance of the binding capacity versus selectivity of the supports.

Effect of Plasmid DNA Size on Chitosan or Polyethyleneimine Polyplexes Formulation.

Gene therapy could be simply defined as a strategy for the introduction of a functional copy of desired genes in patients, to correct some specific mutation and potentially treat the respective disorder. However, this straightforward definition hides very complex processes related to the design and preparation of the therapeutic genes, as well as the development of suitable gene delivery systems. Within non-viral vectors, polymeric nanocarriers have offered an ideal platform to be applied as gene delivery systems. Concerning this, the main goal of the study was to do a systematic evaluation on the formulation of pDNA delivery systems based on the complexation of different sized plasmids with chitosan (CH) or polyethyleneimine (PEI) polymers to search for the best option regarding encapsulation efficiency, surface charge, size, and delivery ability. The cytotoxicity and the transfection efficiency of these systems were accessed and, for the best p53 encoding pDNA nanosystems, the ability to promote protein expression was also evaluated. Overall, it was showed that CH polyplexes are more efficient on transfection when compared with the PEI polyplexes, resulting in higher P53 protein expression. Cells transfected with CH/p53-pDNA polyplexes presented an increase of around 54.2% on P53 expression, while the transfection with the PEI/p53-pDNA polyplexes resulted in a 32% increase.

Undoped InOx films deposited by radio frequency plasma enhanced reactive thermal evaporation at room temperature: importance of substrate.

Conductive and transparent undoped thin films of indium oxide (InOx ), 120 nm average thick, were deposited by radio frequency plasma enhanced reactive thermal evaporation (rf-PERTE) of indium in the presence of oxygen at room temperature. Several substrates were used in order to study their influence on the main properties of these films: alkali free (AF) glass, fused silica, crystalline silicon and polyethylene terephthalate (PET). Surface morphology of the InOx films as a function of the substrates was observed by SEM and showed that the undoped InOx films obtained are nanostructured. For the c-Si substrate, InOx films with increased grain size are obtained, induced by the crystalline substrate. Films deposited on fused silica and AF glass substrates show a nano-grainy surface with similar surface morphologies. The InOx films deposited on AF glass show the highest values of both: electrical conductivity of about 1100 (omega cm)(-1) and visible transmittance of 85%. The substrate has a greater influence on the surface morphology of the films when a polymer (PET) is used. InOx films deposited on PET show a decrease in the electrical conductivity (90 (omega cm)(-1)) and a slight decrease in the average visible transmittance (78%).

Perfectionism and eating behaviour in Portuguese adolescents.

OBJECTIVES: The main objective was to investigate the association between perfectionism and eating behaviour in a non-clinical sample of adolescents of both genders. METHOD: 997 middle and high school students completed the Portuguese versions of the child-adolescent perfectionism scale (CAPS) and of the eating attitudes test -25 (EAT-25). RESULTS: In both genders, the perfectionism total score and the sociallyprescribed perfectionism (SPP) score were positive and significantly correlated with the EAT total score and with all EAT dimensions: Drive for Thinness (DT), Bulimic Related Behaviour (BRB), Social Pressure to Eat (SPE). In girls, self-oriented perfectionism (SOP) was also associated with the EAT total score and its dimensions, whereas in boys it was only associated with EAT total score and DT. In both genders SPP was a useful predictor of the EAT-25 total score and of all its dimensions. In which respects SOP, there were some gender differences showing that in boys this dimension should not be considered a predictor of eating behaviours. CONCLUSION: These results confirm that high levels of perfectionism (SOP and SPP) are associated with abnormal eating behaviour in both genders.

Purification of supercoiled p53-encoding plasmid using an arginine-modified macroporous support.

p53 is a tumour suppressor gene that has been explored for cancer gene therapy as a possible alternative to the common treatments. The use of plasmid DNA (pDNA) to carry the therapeutic gene has been considered, but it is requisite to preserve its supercoiled (sc) structure, for eliciting a more effective gene expression and therapeutic action. The purification of the sc pDNA using amino acids-based affinity chromatography has been successfully applied, exploring different amino acids and supports. From these studies, it stood out the selectivity of arginine for the recognition of sc pDNA. However, some limitation on the binding capacity was found in the arginine-agarose support, and in the case of monoliths, some fouling and clogging can limit sequential runs. By using macroporous support modified with arginine it was expected to take advantage of the selectivity of the ligand combined with the flow properties and binding capacity offered by the support. The arginine-modified macroporous support was characterized by SEM, EDX and FTIR also to verify the correct immobilization of arginine, and then used for pDNA purification. The support showed to be effective on the sc p53-pDNA isolation, and the robustness was also achieved by accomplishing the purification of plasmids with different sizes, only by slightly adjusting the experimental conditions. Regarding the dynamic binding capacity of the arginine-modified macroporous support, it was achieved an improvement of more than 50% in the pDNA binding capacity when compared with their homologous arginine-agarose commercial matrix, suggesting potential economic feasibility in case of scale-up.

Composite Central Face Design-An Approach to Achieve Efficient Alginate Microcarriers.

Microparticulated drug delivery systems have been used as promising encapsulation systems for protecting drugs for in vitro and in vivo applications, enhancing its stability, providing an increased surface to volume ratio, reducing adverse effects, and hence an improvement in bioavailability. Among the studied microparticles, there is a rising interest in the research of alginate microparticles for pharmaceutical and biomedical fields confirming its potential to be used as an effective matrix for drug and cell delivery. Moreover, calcium alginate has been one of the most extensively forming microparticles in the presence of divalent cations providing prolonged drug release and suitable mucoadhesive properties. Regarding the above mentioned, in this research work, we intended to produce Ca-alginate micro-vehicles through electrospraying, presenting high encapsulation efficiency (EE%), reduced protein release across the time, reduced swelling effect, and high sphericity coefficient. To quickly achieve these characteristics and to perform an optimal combination among the percentage of alginate and CaCl2, design of Experiments was applied. The obtained model presented to be statistically significant (p-value < 0.05), with a coefficient of determination of 0.9207, 0.9197, 0.9499, and 0.9637 for each output (EE%, release, swelling, and sphericity, respectively). Moreover, the optimal point (4% of alginate and 6.6% of CaCl2) was successfully validated.

DoE to improve supercoiled p53-pDNA purification by O-phospho-l-tyrosine chromatography.

P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O-phospho-l-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chromatographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-encoding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was applied. The dynamic binding capacity of the O-phospho-l-tyrosine agarose column was 0.35 ±â€¯0.02 mg pDNA/mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of endotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agencies.

Bioelectric impedance vector analysis (BIVA) in hospitalised children; predictors and associations with clinical outcomes.

BACKGROUND: Clinical use of bioelectric impedance is limited by variability in hydration. Analysis of raw bioelectric impedance vectors (BIVA), resistance (R), reactance (Xc) and phase angle (PA) may be an alternative for monitoring disease progression/treatment. Clinical experience of BIVA in children is limited. We investigated predictors of BIVA and their ability to predict clinical outcomes in children with complex diagnoses. METHODS: R, Xc and PA were measured (BODYSTAT Quadscan 4000) on admission in 108 patients (4.6-16.8 years, mean 10.0). R and Xc were indexed by height (H) and BIVA-SDS for age and sex calculated using data from healthy children. Potential predictors and clinical outcomes (greater-than-expected length-of-stay (LOS), complications) were recorded. RESULTS: Mean R/H-SDS was significantly higher (0.99 (SD 1.32)) and PA-SDS lower (-1.22 (1.68))) than expected, with a wide range for all parameters. In multivariate models, the Strongkids risk category predicted R/H-SDS (adjusted mean for low, medium and high risk = 0.49, 1.28, 2.17, p = 0.009) and PA-SDS (adjusted mean -0.52, -1.53, -2.36, p = 0.01). BIVA-SDS were not significantly different in patients with or without adverse outcomes. CONCLUSIONS: These complex patients had abnormal mean BIVA-SDS suggestive of reduced hydration and poor cellular health according to conventional interpretation. R/H-SDS was higher and PA-SDS lower in those classified as higher malnutrition risk by the StrongKids tool. Further investigation in specific patient groups, including those with acute fluid shifts and using disease-specific outcomes, may better define the clinical role of BIV.

[Aspects of tobacco attributable mortality: systematic review]. / Aspectos da mortalidade atribuível ao tabaco: revisão sistemática.

The objective of the article was to assess methodologies published and applied in calculating mortality attributable to smoking. A review of the literature was made for the period 1990 to 2006, in the electronic databases MEDLINE and LILACS. A total of 186 studies were found, which measured mortality based on calculating the smoking-attributable risk. Of these, a total of 41 were selected. The studies that were carried out in the United States and Canada presented a more standard methodology and reported smoking attributable mortality to be 18%-23%, with male mortality being 25%-29% and female mortality 14%-17%. The variations can be attributed to methodological differences and to different estimates of the main tobacco-related illnesses.

Transmission of Hansen's disease and unscreened household contacts.

INTRODUCTION: Leprosy household contact investigation has been recommended as an epidemiological surveillance strategy for more than 50 years. OBJECTIVE: The purpose of this study was to estimate the yield that could be achieved in case detection if four contacts could be examined for every case found. METHODS: For the estimation of the number of cases not detected (lost) and yield per contact investigation in Mato Grosso, the incidence rates and yield calculations from a cohort study conducted in Rio de Janeiro by Matos et al (1999) were applied to data from the state of Mato Grosso. Also, to identify high-risk groups for leprosy, a cross-sectional study was conducted in which leprosy cases found as a result of a contact investigation were compared with index cases detected by other means. RESULTS: The lost cases among household contacts were at least 4 per every 10 new cases detected. This is the result of insufficient contact investigations--it being 0.8 instead of 4 contact investigations per each case as recommended by the Brazilian Ministry of Health. Up to 60% of the incidence of leprosy could be explained by the high number of lost cases among household contacts not examined. Women and children are more likely to be contacts. CONCLUSION: The lost cases due to insufficient contact investigation represent lost opportunities in early detection and treatment, thus losing the opportunity to reduce leprosy transmission.

Late hepatic artery thrombosis in liver allograft recipients is associated with intrahepatic biliary necrosis.

Hepatic artery thrombosis (HAT) after liver transplantation is a potentially life-threatening complication that occurs in 2-25% of patients, depending on several risk factors and the patient population studied. Arterial thrombosis occurring early after liver transplantation is associated with acute fulminant hepatic failure, biliary tract necrosis and leaks, or relapsing bacteremia and is associated with a high rate of graft loss and patient mortality. The onset of late posttransplant HAT (after 6 months) has been thought to have a more benign and often asymptomatic course. The reasons for the differences between the manifestations of early and late HAT are not well understood. We reviewed the adult liver transplant experience at the University of Cincinnati and found four patients with late HAT, three of whom developed severe intrahepatic biliary necrosis. Two patients were successfully retransplanted and 1 patient who refused retransplantation died. One patient had mild, transient graft damage due to gradual arterial stenosis and the development of arterial collaterals prior to thrombosis. Late HAT has a significant potential for irreversible graft damage requiring retransplantation. Screening for the development of hepatic artery stenosis prior to late thrombosis may be worthwhile.

Bone marrow and splenocyte coculture-generated cells enhance allograft survival.

BACKGROUND: Protocols that incorporate donor-specific cell infusions using bone marrow, spleen, or blood transfusion continue to enhance allograft survival and often lead to tolerance in experimental models. Clinical benefits from these modalities have not been as striking, leading to ongoing study in this field. We have explored culture techniques for the in vitro selection and development of cellular effectors capable of enhancing allograft survival. METHODS: Rat bone marrow or spleen cells cultured under a variety of conditions were screened for suppressor function. Bone marrow cells, nonadherent to plastic, cultured for 7 days with granulocyte-macrophage colony-stimulating factor, lipopolysaccharide, and with or without splenocytes were found to contain predominantly myeloid lineage cells and had the ability to suppress phytohemagglutinin or mixed lymphocyte reaction-induced splenocyte proliferation. Standard donor-specific peripheral blood transfusion was compared with cultured donor-specific bone marrow cells, splenocytes, or marrow cells cultured with splenocytes (cocultured) administered intravenously at 1 x 10(7) cells/kg the day before an ACI to Lewis heterotopic heart transplant. Cyclosporine was administered at 10 mg/kg on day -1 and 2.5 mg/kg on days 0-6 relative to transplantation. RESULTS: Mean allograft survival in cyclosporine-treated animals was 8.5 days without and 16.6 days with a donor-specific blood transfusion. Cocultured cells extended allograft survival (39.5 days), whereas bone marrow or splenocytes cultured alone did not. With Percoll gradient separation, two predominant culture subfractions, one with potent suppressor function and another with stimulator function, were identified. Flow cytometric analysis showed mixed populations enriched for macrophages but also including dendritic cells in both subfractions. The suppressive fraction extended allograft survival to 20.8 days and the stimulatory fraction was less effective, yet remixing of both fractions regained the full allograft survival advantage. CONCLUSIONS: In this model, the coculture of bone marrow cells and splenocytes with granulocyte-macrophage colony-stimulating factor and lipopolysaccharide produced functionally divergent subpopulations that synergistically enhanced allograft survival. The development of cellular effectors with enhanced ability to prolong allograft survival using in vitro culture techniques is possible, and provides a new therapeutic option in the use of cell infusion-based therapies.