Assessment of Efficacy and Safety Using PPAR-γ Agonist-Loaded Nanocarriers for Inflammatory Eye Diseases.

Drug-loaded nanocarriers (NCs) are new systems that can greatly improve the delivery and targeting of drugs to specific tissues and organs. In our work, a PPAR-γ agonist loaded into polymeric NCs was prepared, stabilized by spray-drying, and tested in vitro, ex vivo, and in vivo (animal models) to provide a safe formulation for optical anti-inflammatory treatments. The NCs were shown to be well tolerated, and no signs of irritancy or alterations of the eye properties were detected by the in vitro HET-CAM test and in vivo Draize test. Furthermore, no signs of cytotoxicity were found in the NC formulations on retinoblastoma cells (Y-79) analyzed using the alamarBlue assay, and the transmittance experiments evidenced good corneal transparency with the formulations tested. The ocular anti-inflammatory study confirmed the significant prevention efficacy using the NCs, and these systems did not affect the corneal tissue structure. Moreover, the animal corneal structure treated with the NCs was analyzed using X-ray diffraction using synchrotron light. Small-angle X-ray scattering (SAXS) analysis did not show a significant difference in corneal collagen interfibrillar spacing after the treatment with freshly prepared NCs or NCs after the drying process compared to the corresponding negative control when inflammation was induced. Considering these results, the PPAR-γ agonist NCs could be a safe and effective alternative for the treatment of inflammatory ocular processes.

Development and Validation of an HPLC-MS/MS Method for Pioglitazone from Nanocarriers Quantitation in Ex Vivo and In Vivo Ocular Tissues.

Pioglitazone (PGZ) is an oral anti-hyperglycemic agent, belongs to the class of thiazolidinediones, and is used for the treatment of diabetes mellitus type 2. In recent years, its anti-inflammatory activity has also been demonstrated in the literature for different diseases, including ocular inflammatory processes. Additionally, this drug belongs to Class II of the Biopharmaceutical Classification System, i.e., slightly soluble and highly permeable. The main objective of this study was to validate a new analytical HPLC-MS/MS method to quantify free-PGZ and PGZ from polymeric NPs to conduct nanoparticle application studies loaded with this active ingredient to transport it within ocular tissues. An accurate, sensitive, selective, reproducible and high throughput HPLC-MS/MS method was validated to quantify PGZ in cornea, sclera, lens, aqueous humor, and vitreous humor. The chromatographic separation was achieved in 10 min on a Kinetex C18 column. Linear response of PGZ was observed over the range of 5-100 ng/mL. The recovery of free-PGZ or PGZ from NPs was in the range of 85-110% in all tissues and levels tested. The intra-day and inter-day precision were <5% and <10%, respectively. The extracts were shown to be stable in various experimental conditions in all matrices studied. The range of concentrations covered by this validation is 80-1600 µg/kg of PGZ in ocular tissues. It is concluded that this method can be applied to quantify PGZ for in vivo and ex vivo biodistribution studies related to the ocular administration of free-PGZ and PGZ from nanoparticles.

Post-translational regulation of retinal IMPDH1 in vivo to adjust GTP synthesis to illumination conditions.

We report the in vivo regulation of Inosine-5´-monophosphate dehydrogenase 1 (IMPDH1) in the retina. IMPDH1 catalyzes the rate-limiting step in the de novo synthesis of guanine nucleotides, impacting the cellular pools of GMP, GDP and GTP. Guanine nucleotide homeostasis is central to photoreceptor cells, where cGMP is the signal transducing molecule in the light response. Mutations in IMPDH1 lead to inherited blindness. We unveil a light-dependent phosphorylation of retinal IMPDH1 at Thr159/Ser160 in the Bateman domain that desensitizes the enzyme to allosteric inhibition by GDP/GTP. When exposed to bright light, living mice increase the rate of GTP and ATP synthesis in their retinas; concomitant with IMPDH1 aggregate formation at the outer segment layer. Inhibiting IMPDH activity in living mice delays rod mass recovery. We unveil a novel mechanism of regulation of IMPDH1 in vivo, important for understanding GTP homeostasis in the retina and the pathogenesis of adRP10 IMPDH1 mutations.

Ex-Vivo and In-Vivo Assessment of Cyclamen europaeum Extract After Nasal Administration.

Rhinosinusitis is a prevalent disorder with a severe impact on the health-related quality of life. Saponins of Cyclamen europaeum exert a clinically proven curative effect on rhinosinusitis symptoms when instilled into the nasal cavity, however, more extensive preclinical assessment is required to better characterize the efficacy of this botanical extract. This work evaluates the potential use of a natural freeze-dried extract of C. europaeum given as topical nasal administration. Permeation experiment on porcine nasal mucosa was performed with Franz diffusion cells. Experiments in rabbits were performed to test for any toxicological, hematological, biochemical or histological evidence of systemic action. No theoretical levels of saponins were found in the receptor chamber of Franz diffusion cells. Hematological data did not show significant differences between control and experimental animals (p > 0.05). Histological studies also showed that enhanced secretory activity in response to intranasal administration was not accompanied by any visible signs of injury. An examination of the brain, lungs, liver, kidneys, spleen, and gastrointestinal organs did not reveal any abnormality. The absence of mucosal permeation of saponins and negligible probability of C. europaeum saponins absorption in the course of a therapeutic application was demonstrated.

Soluble CD40 ligand in morbidly obese patients: effect of body mass index on recovery to normal levels after gastric bypass surgery.

IMPORTANCE: In recent years, the CD40/CD40L system has been implicated in the pathophysiology of severe chronic inflammatory diseases. Recently, obesity has been described as a low chronic inflammatory disease, so this system could also be involved in the inflammatory process. OBJECTIVE: To study soluble CD40 ligand (sCD40L) and other factors implicated in coagulation (plasminogen activator inhibitor 1, antithrombin III, and fibrinogen) and inflammation (C-reactive protein) in patients with morbid obesity and different body mass indexes (BMIs) (calculated as weight in kilograms divided by height in meters squared), before and after weight loss induced by bariatric surgery. DESIGN: Plasma samples were obtained before and after a bariatric surgery intervention. Several inflammatory markers were then studied (sCD40L, plasminogen activator inhibitor 1, antithrombin III, and C-reactive protein). The values obtained were compared with a control group of nonobese persons. PARTICIPANTS: Thirty-four morbidly obese patients undergoing gastric bypass surgery and 22 normal-weight controls matched for age and sex. INTERVENTIONS: A Roux-en-Y gastric bypass was performed in morbidly obese patients. MAIN OUTCOME MEASURES: Levels of sCD40L, plasminogen activator inhibitor 1, antithrombin III, fibrinogen, and C-reactive protein 12 months after bariatric surgery. RESULTS: Obese men showed a tendency for decreased plasma sCD40L levels 1 year after surgery (mean [SEM], 246.5 [70.4] pg/mL before vs 82.2 [23.2] pg/mL after surgery; P < .05), whereas there were not any significant changes in obese women (285.9 [67.5] pg/mL before vs 287.0 [56.9] pg/mL after surgery). Levels of the other markers studied decreased significantly with weight loss in both sexes. However, all other studied markers tend to have higher concentrations in patients with higher BMIs, except for sCD40L, which tended to have lower concentrations in patients with BMIs higher than 55. The decreases with weight loss were lower with higher BMIs for all measurements, except for antithrombin III. CONCLUSIONS AND RELEVANCE: Increased BMI, but not sex, influences recovery to normal levels for the markers studied, possibly indicating a worse prognosis.

Assessment of protein entrapment in hydroxyapatite scaffolds by size exclusion chromatography.

Although it is well known that the textural properties of scaffolds play an important role in the process of tissue regeneration, the investigation of such effects remain difficult especially at the micro/nano level. Texture confers the material the additional ability to entrap/concentrate molecules circulating in the body fluid regardless of their binding affinity to the material. The goal of the present work is to isolate protein entrapment from protein adsorption phenomena in two macroporous hydroxyapatite scaffolds with identical chemical structure, similar macroporosity but different micro/nanoporosity using proteins of different sizes. This was achieved implementing size exclusion chromatography and using the scaffolds as chromatographic columns. The results showed that the larger the crystal size and the lower the packing density of the crystals composing the scaffold increased protein retention but decreased the protein dwelling time in the column. Differences in the amount of protein retained depended on the protein type.

An inter-laboratory validation of methods of lipid peroxidation measurement in UVA-treated human plasma samples.

Lipid peroxidation products like malondialdehyde, 4-hydroxynonenal and F(2)-isoprostanes are widely used as markers of oxidative stress in vitro and in vivo. This study reports the results of a multi-laboratory validation study by COST Action B35 to assess inter-laboratory and intra-laboratory variation in the measurement of lipid peroxidation. Human plasma samples were exposed to UVA irradiation at different doses (0, 15 J, 20 J), encoded and shipped to 15 laboratories, where analyses of malondialdehyde, 4-hydroxynonenal and isoprostanes were conducted. The results demonstrate a low within-day-variation and a good correlation of results observed on two different days. However, high coefficients of variation were observed between the laboratories. Malondialdehyde determined by HPLC was found to be the most sensitive and reproducible lipid peroxidation product in plasma upon UVA treatment. It is concluded that measurement of malondialdehyde by HPLC has good analytical validity for inter-laboratory studies on lipid peroxidation in human EDTA-plasma samples, although it is acknowledged that this may not translate to biological validity.

Study of the humoral immunological response after vaccination with a Staphylococcus aureus biofilm-embedded bacterin in dairy cows: possible role of the exopolysaccharide specific antibody production in the protection from Staphylococcus aureus induced mastitis.

The objective of the present study was to analyze an extracellular component from Staphylococcus aureus (S. aureus), which we refer to as slime associated antigenic complex (SAAC), and to investigate the role of SAAC-specific antibody production in protection from S. aureus bovine mastitis. Twelve primiparous gestating cows were randomly assigned to one of the three groups: Group 1 was vaccinated with a S. aureus bacterin with very limited SAAC content; Group 2 received a S. aureus bacterin with high SAAC content and Group 3 served as unvaccinated controls. Animals were vaccinated at 45 days before the expected parturition date and revaccinated 35 days later. All groups were challenged by intramammary infusion with a virulent heterologous strain of S. aureus 23 days after calving. Antibody response against SAAC in serum and in milk, general clinical signs, mastitis score, somatic cell count (SCC) and count of S. aureus in milk were evaluated before and after challenge. Immunization with a high SAAC content in the S. aureus bacterin (Group 2) significantly enhanced antibody titers against SAAC (in serum and milk) and reduced the S. aureus concentration in milk during the post-challenge period compared to Group 1 and Group 3. Moreover, a significant negative correlation was observed between SAAC antibody production on the day of the challenge and the S. aureus count in milk by 1 day after challenge. However, there was no evidence of a difference between vaccinated and control groups with regard to clinical signs of mastitis following the challenge. Nevertheless, the SAAC antibody concentration on the day of the challenge negatively correlated with the mastitis score in quarters infected with S. aureus at 2 days post-challenge. These results indicate that the vaccines did not prevent S. aureus intramammary infection (IMI) after the experimental challenge, but immunization with a S. aureus bacterin with high SAAC content was able to reduce S. aureus multiplication in the mammary gland after challenge and suggests that the SAAC-specific antibody response could be involved in the protection against S. aureus intramammary infection. Although further studies should be performed to confirm the efficacy (under experimental conditions and in field trials), we propose bacterins from strong biofilm-producing bacteria and with high SAAC content, rather than with limited SAAC content, as a cost-efficient vaccine design against S. aureus bovine mastitis.

Alternative methods for sampling and preservation of photosynthetic pigments and tocopherols in plant material from remote locations.

Current methods for the study of pigments involve freezing in liquid nitrogen and storage at -80 degrees C or lyophilization until HPLC analysis. These requirements greatly restrict ecophysiological research in remote areas where such resources are hardly available. We aimed to overcome such limitations by developing several techniques not requiring freezing or lyophilization. Two species with contrasting foliar characteristics (Olea europaea and Taraxacum officinale) were chosen. Seven preservation methods were designed, optimized and tested in a field trial. These protocols were compared with a control immediately frozen after collection. Pigments and tocopherols were analysed by HPLC. Main artefacts were chlorophyll epimerization or phaeophytinization, carotenoid isomerization, altered de-epoxidation index and tocopherol degradation. Among all methods, sample desiccation in silica gel provides robust samples (pigment composition was unaffected by storage time or temperature) and almost unaltered pigment profiles, except for a shift in epoxidation state. Although liquid nitrogen freezing and subsequent lyophilization or freezer storage were preferred, when these facilities are either not available or not suitable for long-distance transport, desiccation with silica gel, passive extraction in acetone and/or storage of fresh samples in water vapour saturated atmospheres enable a complete pigment characterization. Silica gel is advisable for long-term sample conservation.

The production of a new extracellular putative long-chain saturated polyester by smooth variants of Mycobacterium vaccae interferes with Th1-cytokine production.

Mycobacterium vaccae is of major pharmaceutical interest as an immunotherapeutic agent. Although M. vaccae 15483 ATCC(T) strain displays smooth and rough colonial morphologies on solid culture media, it is not known in which conditions M. vaccae switches from one colonial morphotype to the other or whether there are biological differences, especially immunological, between them. We have found that the change from a smooth to rough stable variant occurs spontaneously at 30 degrees C. The analysis of the composition of the cell wall in both variants showed that the smooth morphotype presents an extracellular material that has never previously been described and was identified as a long-chain saturated polyester that, interestingly, is not produced by the rough morphotype. Our results also indicate that this compound could be implicated in the spreading ability of smooth colonies. Proliferation, IFN-gamma and IL-12(p40) production by splenocyte cultures was significantly higher in mice immunised with the rough variant compared with those immunised with the smooth one. This latter finding suggests that the different colonial morphology of M. vaccae may affect the immunomodulatory effects induced from M. vaccae preparations.

Rapid high-performance liquid chromatography-electrospray ionization tandem mass spectrometry method for qualitative and quantitative analysis of virgin olive oil phenolic metabolites in human low-density lipoproteins.

A rapid method for detection and quantification of metabolites of specific olive oil phenolic compounds (hydroxytyrosol monoglucuronide, hydroxytyrosol monosulfate, tyrosol glucuronide, tyrosol sulfate and homovanillic acid sulfate) in low-density lipoprotein (LDL) fractions by solid-phase extraction (SPE) and high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) is described. A 3 microm particle size fast C18 Luna column, 5 cm x 2.0 mm I.D., was used at a flow rate of 0.6 mL/min with a mobile phase consisting of 0.1% (v/v) formic acid (A) and acetonitrile (B). A linear gradient profile was used for separation at column temperature 40 degrees C. The proposed chromatographic procedure is rapid without loosing its separation efficiency and sensitivity. Validation proofs were carried out for the method described, showing a linear system (r>0.99) and a recovery of 81.9 and 101.3% for hydroxytyrosol and homovanillic acid, respectively. The results show that this method is effective and can be used in routine analysis.

Liquid chromatographic/electrospray ionization tandem mass spectrometric study of the phenolic composition of cocoa (Theobroma cacao).

Liquid chromatography coupled with ionspray mass spectrometry in the tandem mode (LC/MS/MS) with negative ion detection was used for the identification of a variety of phenolic compounds in a cocoa sample. Gradient elution with water and acetonitrile, both containing 0.1% HCOOH, was used. Standard solutions of 31 phenolic compounds, including benzoic and cinnamic acids and flavonoid compounds, were studied in the negative ion mode using MS/MS product ion scans. At low collisional activation, the deprotonated molecule [M - H](-) was observed for all the compounds studied. For cinnamic and benzoic acids, losses of CO(2) or formation of [M - CH(3)](-*) in the case of methoxylated compounds were observed. However, for flavonol and flavone glycosides, the spectra present both the deprotonated molecule [M - H](-) of the glycoside and the ion corresponding to the deprotonated aglycone [A - H](-). The latter ion is formed by loss of the rhamnose, glucose, galactose or arabinose residue from the glycosides. Different fragmentation patterns were observed in MS/MS experiments for flavone-C-glycosides which showed fragmentation in the sugar part. Fragmentation of aglycones provided characteristic ions for each family of flavonoids. The optimum LC/MS/MS conditions were applied to the characterization of a cocoa sample that had been subjected to an extraction/clean-up procedure which involved chromatography on Sephadex LH20 and thin-layer chromatographic monitoring. In addition to compounds described in the literature, such as epicatechin and catechin, quercetin, isoquercitrin (quercetin-3-O-glucoside) and quercetin-3-O-arabinose, other compounds were identified for the first time in cocoa samples, such as hyperoside (quercetin-3-O-galactoside), naringenin, luteolin, apigenin and some O-glucosides and C-glucosides of these compounds.

Improved high performance liquid chromatographic determination of ginsenosides in Panax ginseng-based pharmaceuticals using a diol column.

A reversed-phase high-performance liquid chromatographic assay for the simultaneous quantitative determination of seven ginsenosides, Rb(1), Rb(2), Rc, Rd, Rg(1), Re and Rf in pharmaceutical preparations is described. Chromatographic separation was achieved in less than 20 min using a 250 x 4 mm Lichrospher, 5 microm, 100 A diol column with detection at 203 nm. The method was validated over the range of 2.5-20 ng/microL using a 20 microL sample volume. The average accuracy at five concentrations was 90-100%, and the within-day and between-day precision ranged from 1 to 7% expressed as coefficient of variation. The detection limit and the quantitation limit of the method were 20 and 50 ng injected for each ginsenoside, respectively.

Comparison of the Kjeldahl method and a combustion method for total nitrogen determination in animal feed.

The features of the Dumas combustion method (CM) and those of the Kjeldahl method (KM) were compared as they apply to total nitrogen determination in animal feed. Both methods achieved similar repeatability (S.D., 0.11-0.38 from Kjeldahl and 0.15-0.36 from combustion) and similar intra-laboratory reproducibility (S.D., 0.11-0.39 from Kjeldahl and 0.15-0.37 from combustion). R.S.D. is always below 2%. These results show that the CM is suitable for the analysis of protein content in animal feed (5-75% protein content). The CM is recommended owing to its shorter analysis time, its cost and its environmental suitability.

A straightforward method for analysis of highly phosphorylated inositols in blood cells by high-performance liquid chromatography.

Soluble inositol polyphosphates are found in many cells. The trisphosphate isomers, mainly inositol-1,4,5-trisphosphate, have been extensively studied because of their involvement in signal transduction. However, higher phosphorylated inositols are less frequently studied and their physiological role is poorly understood. Among these, only the myo-inositol-1,3,4,5,6-pentakisphosphate (Ins1,3,4,5,6P5), an important component of bird erythrocytes, has been intensively studied in comparative studies because it is a potent allosteric effector of hemoglobin and decreases its affinity to oxygen. We have developed a procedure for the analysis of inositol polyphosphates and other phosphate compounds in vertebrate blood cells based on a quick and accurate HPLC separation coupled to metal-dye detection. The procedure includes acid extraction of cellular phosphates, acid elimination and concentration of the extract, HPLC separation of phosphate compounds, and quantification by coupled highly sensitive metal-dye detection. The method is especially useful for analyses of highly phosphorylated inositols and for red cell comparative studies. Using the described method we have quantified Ins1,3,4,5,6P5 and the low quantities of InsP6 found in bird erythrocytes. We also identified traces of Ins3,4,5,6P4 and Ins1,3,4,6P4. Moreover, by applying the method in cultured murine macrophages, we have found changes of highly phosphorylated inositols when these cells are activated by lipopolysaccharide.