Solid Lipid Nanoparticles For Hydrophilic Biotech Drugs: Optimization And Cell Viability Studies (caco-2 & Hepg-2 Cell Lines).

Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

Autologous Platelet Gel: Five Cases Illustrating Use On Sickle Cell Disease Ulcers.

Leg ulcers represent a particularly disabling complication in patients with sickle cell disease (SCD). Platelet gel (PG) is a novel therapeutic strategy used for accelerating wound healing of a wide range of tissues through the continuous release of platelet growth factors. Here, we describe the use of PG preparation according to Anitua's PRGF (preparations rich in growth factors) protocol for treating chronic nonhealing ulcers in patients with SCD. A positive response occurred in 3 patients with an area reduction of 85.7% to 100%, which occurred within 7 to 10 weeks, and a 35.2% and 20.5% of area reduction in 2 other patients, who however, had large ulcers. After calcium chloride addition, the platelet-rich plasmas demonstrated enhanced platelet-derived growth factors-BB (P < .001), transforming growth factor-β1 (P = .015), vascular endothelial growth factors (P = .03), and hepatocyte growth factors (nonsignificant) secretion. Furthermore, calcium chloride addition induced a significant decrease in platelet number (P = .0134) and there was no leukocyte detection in the PG product. These results demonstrate that PG treatment might impact the healing of leg ulcers in sickle cell disease, especially in patients with small ulcers.

Profile Of The Use Of Disease Modifying Drugs In The Brazilian Registry Of Spondyloarthritides.

Few studies have evaluated the profile of use of disease modifying drugs (DMD) in Brazilian patients with spondyloarthritis (SpA). A common research protocol was applied prospectively in 1505 patients classified as SpA by criteria of the European Spondyloarthropathies Study Group (ESSG), followed at 29 referral centers in Rheumatology in Brazil. Demographic and clinical variables were obtained and evaluated, by analyzing their correlation with the use of DMDs methotrexate (MTX) and sulfasalazine (SSZ). At least one DMD was used by 73.6% of patients: MTX by 29.2% and SSZ by 21.7%, while 22.7% used both drugs. The use of MTX was significantly associated with peripheral involvement, and SSZ was associated with axial involvement, and the two drugs were more administered, separately or in combination, in the mixed involvement (p < 0.001). The use of a DMD was significantly associated with Caucasian ethnicity (MTX , p = 0.014), inflammatory back pain (SSZ, p = 0.002) , buttock pain (SSZ, p = 0.030), neck pain (MTX, p = 0.042), arthritis of the lower limbs (MTX, p < 0.001), arthritis of the upper limbs (MTX, p < 0.001), enthesitis (p = 0.007), dactylitis (MTX, p < 0.001), inflammatory bowel disease (SSZ, p < 0.001) and nail involvement (MTX, p < 0.001). The use of at least one DMD was reported by more than 70% of patients in a large cohort of Brazilian patients with SpA, with MTX use more associated with peripheral involvement and the use of SSZ more associated with axial involvement.

[the Use Of Fish On Buccal Smear To Investigate Mosaicism With A 45,x Cell Line: Study On Healthy Men And Patients With Disorders Of Sex Development].

To verify whether fluorescence in situ hybridization (FISH) of cells from the buccal epithelium could be employed to detect cryptomosaicism with a 45,X lineage in 46,XY patients. Samples of nineteen 46,XY healthy young men and five patients with disorders of sex development (DSD), four 45,X/46,XY and one 46,XY were used. FISH analysis with X and Y specific probes on interphase nuclei from blood lymphocytes and buccal epithelium were analyzed to investigate the proportion of nuclei containing only the signal of the X chromosome. The frequency of nuclei containing only the X signal in the two tissues of healthy men did not differ (p = 0.69). In all patients with DSD this frequency was significantly higher, and there was no difference between the two tissues (p = 0.38), either. Investigation of mosaicism with a 45,X cell line in patients with 46,XY DSD or sterility can be done by FISH directly using cells from the buccal epithelium.

Treadmill Training Increases Sirt-1 And Pgc-1 α Protein Levels And Ampk Phosphorylation In Quadriceps Of Middle-aged Rats In An Intensity-dependent Manner.

The present study investigated the effects of running at 0.8 or 1.2 km/h on inflammatory proteins (i.e., protein levels of TNF- α , IL-1 β , and NF- κ B) and metabolic proteins (i.e., protein levels of SIRT-1 and PGC-1 α , and AMPK phosphorylation) in quadriceps of rats. Male Wistar rats at 3 (young) and 18 months (middle-aged rats) of age were divided into nonexercised (NE) and exercised at 0.8 or 1.2 km/h. The rats were trained on treadmill, 50 min per day, 5 days per week, during 8 weeks. Forty-eight hours after the last training session, muscles were removed, homogenized, and analyzed using biochemical and western blot techniques. Our results showed that: (a) running at 0.8 km/h decreased the inflammatory proteins and increased the metabolic proteins compared with NE rats; (b) these responses were lower for the inflammatory proteins and higher for the metabolic proteins in young rats compared with middle-aged rats; (c) running at 1.2 km/h decreased the inflammatory proteins and increased the metabolic proteins compared with 0.8 km/h; (d) these responses were similar between young and middle-aged rats when trained at 1.2 km. In summary, the age-related increases in inflammatory proteins, and the age-related declines in metabolic proteins can be reversed and largely improved by treadmill training.

Use Of Electrocautery For Coagulation And Wound Complications In Caesarean Sections.

To evaluate the safety of electrocautery for coagulation during Caesarean sections. A randomized, controlled, clinical pilot study was performed at a university maternity hospital. After admission for delivery and decision to perform a C-section, volunteers were randomized to either the intervention group (use of electrocautery for coagulation) or nonintervention group. The women were examined at the time of postpartum discharge (day 3), at days 7 to 10, and again at days 30 to 40 for signs of infection, hematoma, seroma, or dehiscence. Data were analyzed using an intention-to-treat analysis, and risk ratios were calculated. No significant differences were found between the two groups. Only 2.8% of patients in the intervention group developed surgical wound complications during hospitalization. However, 7 to 10 days following discharge, these rates reached 23.0% and 15.4% in the intervention and nonintervention groups, respectively (RR = 1.50, 95% CI = 0.84-2.60). Further studies should confirm whether the use of electrocautery for coagulation does not increase the risk of surgical wound complications in patients undergoing Caesarean sections.

Islet Neogenesis-associated Protein (ingap): The Role Of Its Endogenous Production As A Positive Modulator Of Insulin Secretion.

Islet neogenesis-associated protein (INGAP) is a peptide found in pancreatic exocrine-, duct- and islet- non-β-cells from normal hamsters. Its increase induced by either its exogenous administration or by the overexpression of its gene enhances β-cell secretory function and increases β-cell mass by a combination of stimulation of cell replication and islet neogenesis and reduction of β-cell apoptosis. We studied the potential modulatory role of endogenous INGAP in insulin secretion using two different experimental approaches. Hamster islets transfected with INGAP-small interfering RNA (INGAP-siRNA) were used to study glucose-stimulated insulin secretion (GSIS). In parallel, freshly isolated islets were incubated with high glucose and the same concentration of either a specific anti-INGAP rabbit serum or normal rabbit serum. INGAP-siRNA transfected islets reduced their INGAP mRNA and protein content by 35.1% and 47.2%, respectively whereas GSIS decreased by 25.8%. GSIS by transfected islets attained levels comparable to those recorded in control islets when INGAP pentadecapeptide (INGAP-PP) was added to the culture medium. INGAP antibody in the medium decreased significantly GSIS in a dose-dependent manner. These results indicate that endogenous INGAP plays a physiological positive modulatory role in insulin secretion, supporting its possible use in the treatment of prediabetes and Type 2 diabetes.

Toll-like Receptor 4 Activation Promotes Cardiac Arrhythmias By Decreasing The Transient Outward Potassium Current (ito) Through An Irf3-dependent And Myd88-independent Pathway.

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca(2+) transients, as well as the L-type Ca(2+) current (ICaL) and the transient outward K(+) current (Ito), were recorded in isolated myocytes after 24 h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1 μg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24 h LPS incubation, Ito amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The Ito decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca(2+) transients and ICaL were not affected by LPS; however, Na(+)/Ca(2+) exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased Ito, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca(2+) efflux via NCX, could explain the presence of arrhythmias in the LPS group.

Endocytosis Of Tight Junctions Caveolin Nitrosylation Dependent Is Improved By Cocoa Via Opioid Receptor On Rpe Cells In Diabetic Conditions.

Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions. Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively). Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation. The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation.

Effect Of Daily Use Of An Enzymatic Denture Cleanser On Candida Albicans Biofilms Formed On Polyamide And Poly(methyl Methacrylate) Resins: An In Vitro Study.

Candida biofilms on denture surfaces are substantially reduced after a single immersion in denture cleanser. However, whether this effect is maintained when dentures are immersed in cleanser daily is unclear. The purpose of this study was to evaluate the effect of the daily use of enzymatic cleanser on Candida albicans biofilms on denture base materials. The surfaces of polyamide and poly(methyl methacrylate) resin specimens (n=54) were standardized and divided into 12 groups (n=9 per group), according to study factors (material type, treatment type, and periods of treatment). Candida albicans biofilms were allowed to form over 72 hours, after which the specimens were treated with enzymatic cleanser once daily for 1, 4, or 7 days. Thereafter, residual biofilm was ultrasonically removed and analyzed for viable cells (colony forming units/mm(2)) and enzymatic activity (phospholipase, aspartyl-protease, and hemolysin). Factors that interfered with the response variables were analyzed by 3-way ANOVA with the Holm-Sidak multiple comparison method (α=.05). Polyamide resin presented more viable cells of Candida albicans (P<.001) for both the evaluated treatment types and periods. Although enzymatic cleansing significantly (P<.001) reduced viable cells, daily use did not maintain this reduction (P<.001). Phospholipase activity significantly increased with time (P<.001) for both materials and treatments. However, poly(methyl methacrylate) based resin (P<.001) and enzymatic cleansing treatment (P<.001) contributed to lower phospholipase activity. Aspartyl-protease and hemolysin activities were not influenced by study factors (P>.05). Although daily use of an enzymatic cleanser reduced the number of viable cells and phospholipase activity, this treatment was not effective against residual biofilm over time.

Impact Of Pregabalin Treatment On Synaptic Plasticity And Glial Reactivity During The Course Of Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease that affects young adults. It is characterized by generating a chronic demyelinating autoimmune inflammation in the central nervous system. An experimental model for studying MS is the experimental autoimmune encephalomyelitis (EAE), induced by immunization with antigenic proteins from myelin. The present study investigated the evolution of EAE in pregabalin treated animals up to the remission phase. The results demonstrated a delay in the onset of the disease with statistical differences at the 10th and the 16th day after immunization. Additionally, the walking track test (CatWalk) was used to evaluate different parameters related to motor function. Although no difference between groups was obtained for the foot print pressure, the regularity index was improved post treatment, indicating a better motor coordination. The immunohistochemical analysis of putative synapse preservation and glial reactivity revealed that pregabalin treatment improved the overall morphology of the spinal cord. A preservation of circuits was depicted and the glial reaction was downregulated during the course of the disease. qRT-PCR data did not show immunomodulatory effects of pregabalin, indicating that the positive effects were restricted to the CNS environment. Overall, the present data indicate that pregabalin is efficient for reducing the seriousness of EAE, delaying its course as well as reducing synaptic loss and astroglial reaction.

Avoiding Pitfalls Of Intraoperative Peripheral Endovascular Surgery With The Aid Of Osirix: Expanding The Use Of Virtual Fluoroscopy.

We have shown how the analysis of the angiotomography reconstruction through OsiriX program has assisted in endovascular perioperative programming. We presented its application in situations when an unexpected existence of metallic overlapping artifact (orthopedic osteosynthesis) compromised the adequate visualization of the arterial lesion during the procedure. Through manipulation upon OsiriX software, with assistance of preview under virtual fluoroscopy, it was possible to obtain the angles that would avoid this juxtaposition. These angles were reproduced in the C-arm, allowing visualization of the occluded segment, reducing the need for repeated image acquisitions and contrast overload, allowing the continuation of the procedure.

The Use Of Medication And Associated Factors Among Adults Living In Campinas, São Paulo, Brazil: Differences Between Men And Women.

The objective of this study was to verify factors associated with the use of medication by adults, with emphasis on the differences between men and women. It was a population-based, cross-sectional study with cluster sampling conducted in two stages in Campinas in the state of São Paulo in 2008. Among the 2,413 individuals aged 20 or older, the prevalence of use of at least one drug in the three days before the research was 45.4% (95% CI: 41.3 - 49.4) in men and 64.6% (95% CI: 59.8 - 69.2) in women. For adult men over 40 years old who were not working, former smokers, with one or more chronic diseases, with two or more health problems and who sought health care or a health professional in the two weeks preceding the research showed higher prevalence of medication use. Among women, a higher prevalence of use was observed in females over 40, obese, former smokers, who reported a short sleep pattern, with one or more chronic diseases and two or more health problems, and who reported seeking a health care service or professional in the past 15 days. The findings showed some differences in the determinants of drug use in relation to gender, revealing the greater importance of health-related behavior among women.

Factors Associated With The Age Of The Onset Of Diabetes In Women Aged 50 years Or More: A Population-based Study.

Investigate factors associated with the onset of diabetes in women aged more than 49 years. Cross-sectional, population-based study using self-reports with 622 women. The dependent variable was the age of occurrence of diabetes using the life table method. Cox multiple regression models were adjusted to analyse the onset of diabetes according to predictor variables. Sociodemographic, clinical and behavioural factors were evaluated. Of the 622 women interviewed, 22.7% had diabetes. The mean age at onset was 56 years. The factors associated with the age of occurrence of diabetes were self-rated health (very good, good) (coefficient=-0.792; SE of the coefficient=0.215; p=0.0001), more than two individuals living in the household (coefficient=0.656, SE of the coefficient=0.223; p=0.003), and body mass index (BMI) (kg/m(2)) at 20-30 years of age (coefficient= 0.056, SE of the coefficient=0.023; p=0.014). Self-rated health considered good or very good was associated with a higher rate of survival without diabetes. Sharing a home with two or more other people and a weight increase at 20-30 years of age was associated with the onset of type 2 diabetes.

Sci1 Is A Component Of The Auxin-dependent Control Of Cell Proliferation In Arabidopsis Upper Pistil.

To characterize the recently described SCI1 (stigma/style cell cycle inhibitor 1) gene relationship with the auxin pathway, we have taken the advantage of the Arabidopsis model system and its available tools. At first, we have analyzed the At1g79200 T-DNA insertion mutants and constructed various transgenic plants. The loss- and gain-of-function plants displayed cell number alterations in upper pistils that were controlled by the amino-terminal domain of the protein. These data also confirmed that this locus holds the functional homolog (AtSCI1) of the Nicotiana tabacum SCI1 gene. Then, we have provided some evidences the auxin synthesis/signaling pathways are required for downstream proper AtSCI1 control of cell number: (a) its expression is downregulated in yuc2yuc6 and npy1 auxin-deficient mutants, (b) triple (yuc2yuc6sci1) and double (npy1sci1) mutants mimicked the auxin-deficient phenotypes, with no synergistic interactions, and (c) the increased upper pistil phenotype in these last mutants, which is a consequence of an increased cell number, was able to be complemented by AtSCI1 overexpression. Taken together, our data strongly suggests SCI1 as a component of the auxin signaling transduction pathway to control cell proliferation/differentiation in stigma/style, representing a molecular effector of this hormone on pistil development.