Evaluation of antitumor potential of cashew gum extracted from Anacardium occidentale Linn.

This study aims to determine the antitumor potential of cashew gum in vitro and in vivo. The cashew gum (CG) structure is similar to already showed in literature. The cytotoxicity effect of CG was performed by MTT assay, and B16-F10 melanoma model was used to evaluate antitumor effect. The tumor inhibition was calculated based on tumor weight. Hematological, histopathological, FTIR, oxidative stress and Western Blot analysis were performed to elucidate the mechanism of inhibition and toxic effects. As results, CG did not demonstrate cytotoxicity in vitro, however showed a significant tumor inhibition in vivo, with about 36.9 to 43% of reduction in tumor mass, with no toxicity to organs. Animals treated with CG did not show toxicity in normal tissues, FTIR spectrum and oxidative stress analysis of the tumor tissue indicated that CG cause tumor inhibition with the presence of apoptosis morphotype cells, without alterations in the levels of antioxidants components. In addition, it was observed that CG reduced the expression of γH2AX without changing the expression of caspase-3. With this, we can suggest that this polymer can assist in the anticancer activity and/or decrease the side effects of standard drugs used in treatment of cancer.

Evaluation of filter paper as a means to transport inactivated bacteria for identification using the MALDI-TOF MS system.

A novel approach to transport inactivated bacteria in filter paper for identification in the MALDI-TOF MS was evaluated. Seventy four bacterial isolates were evaluated and the approach presented sensitivity of 97.3% and specificity of 100%. Inactivated bacteria in filter paper are a safer alternative to transport bacteria for MALDI-TOF MS identification.

Vitamin D levels reverberate in monocytes modulation in hemodialysis patients.

BACKGROUND: Patients with chronic kidney disease (CKD) have a severe vitamin D deficiency and increasing epidemiological data suggesting that this deficiency may play a role in overall morbidity and mortality associated with CKD. It is known that vitamin D regulates the immune system, however, in dialysis patients this deficiency and the modulation of proinflammatory cells is unclear. Among these, monocytes arouse interest considering they constitutively express vitamin D receptors. AIM: This study aimed the evaluation of monocytic profile in CKD patients according to vitamin D levels. METHODS: Patients in hemodialysis (HD) were divided into two groups, regarding vitamin D levels: Group 1, vitamin D <26 ng/ml (n = 15) and Group 2, vitamin D ≥26 ng/ml (n = 18). Whole blood was collected aiming evaluation of (a) monocytic populations through CD14 and CD16 expression, (b) reactive oxygen species (ROS) generation, and (c) apoptosis. RESULTS: We observed that in Group 1, when compared to Group 2, there was a significant increase in intermediate monocytes (CD14++ CD16 + ; 34.7 ± 31.6 vs. 12.1 ± 6.3; p = 0.006, respectively) and decrease in classical ones (CD14 ++ CD16 - ; 45.3 ± 31.8 vs. 70.4 ± 25.1; p = 0.017, respectively). There was no difference between groups regarding nonclassical monocytes (CD14 + CD16 ++ ), as well as to apoptosis and to ROS generation. CONCLUSION: This study suggests that HD patients with lower vitamin D levels might have an intensified inflammatory outline as intermediate monocytes with an inflammatory pattern are increased in this population, when compared with patients with higher levels of vitamin D.

Sensitive analytical method to quantify clindamycin in plasma and microdialysate samples: Application in a preclinical pharmacokinetic study.

Clindamycin is widely used in antimicrobial prophylaxis to prevent surgical site infections. Adequate subcutaneous free tissue concentrations should reach therapeutic levels, which have to be maintained throughout the surgical procedure for antibiotic prophylaxis to be efficient. A method was developed and validated using high performance liquid chromatography coupled to a mass spectrometry to determine clindamycin concentrations in two biological matrices: plasma, for drug monitoring, and subcutaneous microdialysate, to determine free concentrations at the incision site. Gradient separation of clindamycin was carried out using a reverse phase C18 column eluted with a mixture of mobile phases (1% formic acid in water and 1% formic acid in acetonitrile). The monitored transitions were m/z 425.3 > 377.3 for clindamycin, and m/z 407 > 359 for lincomycin, used as the internal standard for plasma samples. Linearity was reached in the 0.5-100 µg/mL range for plasma and 25-1000 ng/mL for microdialysate samples. The method was selective, precise, and accurate, and was successfully employed in a preliminary pharmacokinetics study to investigate plasma and subcutaneous clindamycin penetration, determined by microdialysis, after intravenous administration of a 50 mg/kg dose to Wistar rats.

Review article: effects of type 2 diabetes therapies on bone metabolism.

Diabetes complications and osteoporotic fractures are two of the most important causes of morbidity and mortality in older patients, and they share many features, including genetic susceptibility, molecular mechanisms, and environmental factors. Type 2 diabetes mellitus (T2DM) compromises bone microarchitecture by inducing abnormal bone cell function and matrix structure with increased osteoblast apoptosis, diminished osteoblast differentiation, and enhanced osteoclast-mediated bone resorption. The linkage between these two chronic diseases creates a possibility that certain antidiabetic therapies may affect bone function. The treatment of T2DM has been improved in the past two decades with the development of new therapeutic drugs. Each class has a pathophysiologic target related to the regulation of the energy metabolism and insulin secretion. However, both glycemic homeostasis and bone homeostasis are under the control of common regulatory factors. This background allows the individual pharmacological targets of antidiabetic therapies to affect bone quality due to their indirect effects on bone cell differentiation and the bone remodeling process. With a greater number of diabetic patients and antidiabetic agents being launched, it is critical to highlight the consequences of this disease and its pharmacological agents on bone health and fracture risk. Currently, there is little scientific knowledge approaching the impact of most anti-diabetic treatments on bone quality and fracture risk. Thus, this review aims to explore the pros and cons of the available pharmacologic treatments for T2DM on bone mineral density and risk fractures in humans.

Application of a LC-MS/MS method for evaluating lung penetration of tobramycin in rats by microdialysis.

A bioanalytical LC-MS/MS method was developed and validated to determine tobramycin in plasma and lung microdialysate samples. Tobramycin was separated using a C18 column and a mobile phase consisting of water and acetonitrile, both with 10mM of heptafluorobutyric acid, eluted as gradient. Apramycin was used as internal standard (IS) for plasma samples. Drugs were monitored using electrospray ionization operating on positive mode (ESI+) monitoring the transitions 468.2>163.3 m/z for tobramycin and 540.3>217.2 m/z for IS. The method showed linearity in the concentration range of 0.1-50µgmL-1 for microdialysate and 0.5-100µgmL-1 for plasma with coefficients of determination ≥0.991. The inter- and intra-day precision, the accuracy and the stability of the drug in different conditions were in accordance with the limits established by US Food and Drug Administration guideline. The concentrations of tobramycin in plasma and lung microdialysate, determined using CMA/20 probes and a Ringer solution at a flow rate of 1µLmin-1, were evaluated in healthy Wistar rats after a 10mgkg-1 i.v. bolus dose. Samples were harvested up to 12h post-dose. Before animal's experiments, probe recovery was determined by dialysis and retrodialysis in vitro and by retrodialysis in vivo. Probes recovery was independent of drug concentration and method of determination. In vivo recovery was 27.74±7.70%. Pharmacokinetic parameters were estimated by non-compartmental analysis using the software Phoenix®. The estimated area under the curve (AUC0-∞) was 128±19µghmL-1 and 105±12µghmL-1 for plasma and lung, respectively. Tobramycin plasma clearance was 0.07±0.01L/h/kg and the volume of distribution was 0.49±0.09L/kg. Elimination half-life in plasma was 4.4±0.7h and in lung, 4.2±0.56h. The free tissue/free plasma AUC0-∞ ratio was 0.94. This is the first study showing a validated method to quantify tobramycin in microdialysate samples and to evaluate the lung interstitial concentration of the drug.

p-Cresyl sulfate affects the oxidative burst, phagocytosis process, and antigen presentation of monocyte-derived macrophages.

Immune system dysfunction is a common condition in chronic kidney disease (CKD). The present study investigated the effect of p-Cresyl sulfate (pCS) on human cell line U937 monocyte-derived macrophages (MDM) activity. MDM (1×106 cells/mL) were incubated with pCS (10, 25, or 50µg/mL), with or without lipopolysaccharide (LPS; 25ng/mL) and then evaluated NO production, phagocytosis and antigen-presenting molecules expression (HLA-ABC, HLA-DR, CD80 and CD86). All analyses were performed by flow cytometry. All pCS concentrations were able to increase NO production (49±12.1%, 39.8±7.75%, 43.7±11.9%, respectively) compared to untreated cells (4.35±3.34%) after 6h incubation but only the lowest concentration increased this production after 12h (82.9±8.6%, 61±7.2%, 40.8±11.7%). Combined with LPS, the same results were observed. Regarding to phagocytosis, all concentrations were able to induce bead engulfment (35.4±2.71%, 30±3.04%, 23.28±4.58%). In addition, pCS (50µg/mL) was able to increase HLA-ABC and CD80 expression, showed a slight effect on HLA-DR expression and, no difference in basal CD86 levels. pCS can induce an increased oxidative burst and phagocytosis by human macrophages while no modulation of HLA-DR or CD86 expression was induced. Together, these results suggest that pCS induces macrophage activation but interfere in antigen processing, leading to a failure in adaptive immune response in CKD.

Development and validation of a bioanalytical method by LC-MS/MS for the quantification of the LAFIS 10 - an antimalarial candidate - and its pharmacokinetics first evaluation.

A rapid and highly sensitive method by LC-MS/MS was developed and validated for the quantification of an antimalarial candidate (LAFIS10) in rat plasma using dexamethasone as internal standard (IS). The chromatographic separation was performed with a Poroshell 120 EC-C18 column. The mobile phase consisted of water (A) and acetonitrile (B), both containing 10 m m of ammonium formate and 0.1% formic acid, delivered in the form of elution gradient. The LAFIS10 was monitored using an electrospray ionization interface operating in the positive mode in multiple reaction monitoring mode, monitoring the transitions 681.47 → 538.2 for LAFIS10 and 393.20 → 355.30 for the IS. The flow rate was 500 µL/min. The column temperature was kept at 40 °C and the injection volume was 2 µL. The lower limit of quantification was of 10 ng/mL and linearity between 10 and 1000 ng/mL was observed, with an R(2) > 0.99. The accuracy of the method was >90%. The relative standard deviations intra- and interday were <8.80 and <6.37%, respectively. The method showed sensitivity, linearity, precision, accuracy and selectivity required to quantify LAFIS 10 in preclinical pharmacokinetic studies according to criteria established by the US Food and Drug Administration and European Medicines Agency.

Activity of cycloartane-type triterpenes and sterols isolated from Musa paradisiaca fruit peel against Leishmania infantum chagasi.

UNLABELLED: The aim of the study was to evaluate in vitro the antileishmanial activity of triterpenes and sterols isolated from Musa paradisiaca (banana) fruit peel used traditionally to treat leishmaniasis. The compounds were isolated from the ethanolic extract of the peel of the banana fruit by column chromatography. The chemical structure of compounds was determined by (1)H and (13)C - nuclear magnetic resonance spectroscopy. The cytotoxicity was measured in RAW 264.7 cells and LLC-MK2. Leishmanicidal activity against L. infantum chagasi promastigotes was performed by the MTT colorimetric method and activity against amastigotes was assayed in mammalian cells using in situ ELISA method. Five compounds were identified, consisting of three triterpenes: cycloeucalenone, 31-norcyclolaudenone and 24-methylene-cicloartanol and a mixture of two sterols: beta-sitosterol and stigmasterol. With the exception of cycloeucalenone, all compounds showed statistically similar activity against promastigote to pentamidine. While, acting against amastigotes, excluding 31-norcyclolaudenone, other compounds showed activity similar to amphotericin B. All compounds showed low cytotoxicity in mammalian cells. CONCLUSION: This study partially confirms the use of Musa paradisiaca in folk medicine against leishmaniasis. Further in vivo studies are necessary to evaluate the efficacy.

Detection and confirmation of milk adulteration with cheese whey using proteomic-like sample preparation and liquid chromatography-electrospray-tandem mass spectrometry analysis.

Caseinomacropeptide (CMP) is a peptide released by chymosin in cheese production, remaining in whey. Thus, CMP can be used as a biomarker to fluid milk adulteration through whey addition. Commonly, CMP is analyzed by reversed phase (RP-HPLC) or size-exclusion chromatography (SEC). However, some psychrotropic microorganisms - specially Pseudomonas fluorescens - when present in storaged milk, can produce, by enzymatic pathway, a CMP-like peptide generally called pseudo-CMP. These two peptides differ from each other only by one amino acid. RP-HPLC and SEC methods are unable to distinguish these two peptides, which demand development of a confirmatory method with high selectivity. Considering the several degrees of glycosilation and phosphorylation sites in CMP, allied with possible genetic variation (CMP A and CMP B), analytical methods able to differentiate these peptides are extremely complex. In the present work, we developed a proteomic-like technique for separation and characterization of these peptides, using liquid chromatography coupled to mass spectrometry with electrospray ionization able to differentiate and subsequently quantify CMP and pseudo-CMP in milk samples in order to identify adulteration or contamination of these products. The method shows satisfactory precision (<11%) with a detection limit of 1.0 µg mL(-1) and quantification limit of 5.0 µg mL(-1). Specificity, matrix effects and applicability to real samples analysis were also performed and discussed.

ß-lactam antibiotics residues analysis in bovine milk by LC-ESI-MS/MS: a simple and fast liquid-liquid extraction method.

This study presents the development and validation of a simple method for the detection and quantification of six ß-lactam antibiotics residues (ceftiofur, penicillin G, penicillin V, oxacillin, cloxacillin and dicloxacillin) in bovine milk using a fast liquid-liquid extraction (LLE) for sample preparation, followed by liquid chromatography-electrospray-tandem mass spectrometry (LC-MS/MS). LLE consisted of the addition of acetonitrile to the sample, followed by addition of sodium chloride, centrifugation and direct injection of an aliquot into the LC-MS/MS system. Separation was performed in a C(18) column, using acetonitrile and water, both with 0.1% of formic acid, as mobile phase. Method validation was performed according to the criteria of Commission Decision 2002/657/EC. Limits of detection ranged from 0.4 (penicillin G and penicillin V) to 10.0 ng ml(-1) (ceftiofur), and linearity was achieved. The decision limit (CCα), detection capability (CCß), accuracy, inter- and intra-day repeatability of the method are reported.

High-throughput multiclass screening method for antibiotic residue analysis in meat using liquid chromatography-tandem mass spectrometry: a novel minimum sample preparation procedure.

A multiresidue and multiclass method based on liquid chromatography-tandem mass spectrometry for the determination of antibacterials was developed and validated for screening purposes. This method can be applied to commonly used drugs in veterinary medicine such as tetracyclines, quinolones and sulfonamides. Sample preparation consists in cell disruption with sand (previously purified and washed with EDTA 100 mM) followed by protein precipitation with acidified acetonitrile. Validation was conducted in accordance to European Union requirements (2002/657/EC) for qualitative methods covering detection capability (CCß), selectivity, specificity and stability. The method enabled the detection of 21 different drugs and had a false-compliant rate of <5% (ß error) at between 25% and 50% of the maximum residue levels established by legal authorities. The methodology was successfully applied to incurred poultry samples.

SU-E-I-64: 1H-MRS Quality Assurance in Short and Long Term Using a Phantom Object.

PURPOSE: To evaluate intrinsic variability of 1H-MRS single voxel technique for short and long periods of time using a metabolic phantom in a clinical protocol (hippocampi). METHODS: A solution with several metabolites (NAA, Cre, Cho, Lac, M-Ins, Glu, Gln) was placed in a spherical round glass with 300 mL. MRS data were acquired in a 3 T MRI scanner, with a PRESS sequence of TE/TR=35/1500 ms, 2048 data points, 1725 Hz of bandwidth, 6 cm3 VOI centered in the phantom and NSA of 128. A total of 130 spectra were obtained from 27 acquisition dates (5 spectra/date without removing the phantom) over 15 months. Spectra were processed with LCModel software to reduce the human variability during spectra processing. SNR and FWHM outputs from LCModel are mean calculated values from identified resonances. RESULTS: Spectra from each acquisition date showed no mean deviations superior to 4% for all metabolites except for Lac (14 %). During 15 months all metabolites showed inferior deviations to 12%, once again except for Lac (20.5%). The worst estimated concentration was from Gln, which represent 60% of the expected value. The mean SNR was 16.3±2.1. There was no correlation between SNR and mean FWHM from resonances, thus wrong water suppression must be the main factor for variable SNR. CONCLUSIONS: NAA was the most stable metabolite in the whole analysis period with a variation of 6.8%, and Gln was the less stable with a 25.4% variation. FWHM was more variable than SNR, possible due to shimming variability, although it showed no influence in SNR. It was observed two types of artifacts in some spectra generated by unbalanced gradients, B0 inhomogeneity and also insufficient amplitude from crusher gradients. Temporal analysis demonstrated the feasibility of compare results obtained on measurements of the same date and for long periods due to low deviations provided by the technique.

Modelling the dynamics of dengue real epidemics.

In this work, we use a mathematical model for dengue transmission with the aim of analysing and comparing two dengue epidemics that occurred in Salvador, Brazil, in 1995-1996 and 2002. Using real data, we obtain the force of infection, Λ, and the basic reproductive number, R(0), for both epidemics. We also obtain the time evolution of the effective reproduction number, R(t), which results in a very suitable measure to compare the patterns of both epidemics. Based on the analysis of the behaviour of R(0) and R(t) in relation to the adult mosquito control parameter of the model, we show that the control applied only to the adult stage of the mosquito population is not sufficient to stop dengue transmission, emphasizing the importance of applying the control to the aquatic phase of the mosquito.

Antinociceptive effect of citronellal in mice.

Citronellal is a monoterpene reported to be a major component of the essential oils in various aromatic species of plants. The present study evaluated the central nervous system depressant and antinociceptive properties of citronellal through behavioral experimental models. Following intraperitoneal injection, citronellal induced the reduction of spontaneous activity, ataxia, analgesia, and sedation. In pentobarbital-induced hypnosis, CTL (citronellal) at 50, 100, and 200 mg/kg (i.p.) significantly increased sleeping time (88.0 +/- 11.4, 100.2 +/- 16.4, and 119.5 +/- 20.9 min) when compared to vehicle solution injections (43.0 +/- 6.1). Citronellal (100 and 200 mg/kg, i.p.) significantly reduced the number of writhes (66.4 and 81.9%) in a writhing test and the number of paw licks during phase 1 (47.0 and 66.8%) and phase 2 (71.1 and 79.2%) of a formalin test when compared to control group animals. In addition, the results of a hot plate test showed central analgesic properties for citronellal (p < 0.05). These results indicate depressant, hypnotic, and antinociceptive properties of this monoterpene.

Lack of evidence for regulation of cardiac P-type ATPases and MAP kinases in transgenic mice with cardiac-specific overexpression of constitutively active á1B-adrenoceptors

The regulatory function of á1B-adrenoceptors in mammalian heart homeostasis is controversial. The objective of the present study was to characterize the expression/activity of key proteins implicated in cardiac calcium handling (Na+/K+-ATPase and Ca2+-ATPases) and growth (ERK1/2, JNK1/2 and p38) in mice with cardiac-selective overexpression of constitutively active mutant á1B-adrenoceptor (CAMá1B-AR), which present a mild cardiac hypertrophy phenotype. Immunoblot assays showed that myocardial plasma membrane Ca2+-ATPase (PMCA) expression was increased by 30 percent in CAMá1B-AR mice (N = 6, P < 0.05), although there was no change in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) expression. Moreover, total Ca2+-ATPase activity was not modified, but a significant increase in the activity of the thapsigargin-resistant (PMCA) to thapsigargin-sensitive (SERCA) ratio was detected. Neither Na+/K+-ATPase activity nor the expression of á1 and á2 subunit isoforms was changed in CAMá1B-AR mouse hearts. Moreover, immunoblot assays did not provide evidence for an enhanced activation of the three mitogen-activated protein kinases studied in this stage of hypertrophy. Therefore, these findings indicate that chronic cardiac á1B-AR activation in vivo led to mild hypertrophy devoid of significant signs of adaptive modifications concerning primary intracellular calcium control and growth-related proteins, suggesting a minor pathophysiological role of this adrenergic receptor in mouse heart at this stage of development.

Lack of evidence for regulation of cardiac P-type ATPases and MAP kinases in transgenic mice with cardiac-specific overexpression of constitutively active alpha(1B)-adrenoceptors.

The regulatory function of alpha(1B)-adrenoceptors in mammalian heart homeostasis is controversial. The objective of the present study was to characterize the expression/activity of key proteins implicated in cardiac calcium handling (Na(+)/K(+)-ATPase and Ca(2+)-ATPases) and growth (ERK1/2, JNK1/2 and p38) in mice with cardiac-selective overexpression of constitutively active mutant alpha1B-adrenoceptor (CAMalpha(1B)-AR), which present a mild cardiac hypertrophy phenotype. Immunoblot assays showed that myocardial plasma membrane Ca(2+)-ATPase (PMCA) expression was increased by 30% in CAMalpha(1B)-AR mice (N = 6, P < 0.05), although there was no change in sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2) expression. Moreover, total Ca(2+)-ATPase activity was not modified, but a significant increase in the activity of the thapsigargin-resistant (PMCA) to thapsigargin-sensitive (SERCA) ratio was detected. Neither Na(+)/K(+)-ATPase activity nor the expression of alpha(1) and alpha(2) subunit isoforms was changed in CAMalpha(1B)-AR mouse hearts. Moreover, immunoblot assays did not provide evidence for an enhanced activation of the three mitogen-activated protein kinases studied in this stage of hypertrophy. Therefore, these findings indicate that chronic cardiac alpha(1B)-AR activation in vivo led to mild hypertrophy devoid of significant signs of adaptive modifications concerning primary intracellular calcium control and growth-related proteins, suggesting a minor pathophysiological role of this adrenergic receptor in mouse heart at this stage of development.