Suspensions Of Human Erythrocytes Research Articles

IN VITRO PROTECTIVE EFFECT OF LEAF EXTRACT OF FICUS DELTOIDEA JACK (MORACEAE) ON BIOMARKERS OF OXIDATIVE STRESS IN THE HUMAN ERYTHROCYTES

Purpose: to investigate the antioxidant properties of the aqueous extracts derived from leaves of Ficus deltoidea using the model of human blood. For this purpose, oxidative stress biomarkers [2-thiobarbituric acid reactive substances (TBARS), content of aldehydic and ketonic derivatives of oxidatively modified proteins, total antioxidant capacity (TAC)] in the human erythrocytes after in vitro incubation with aqueous extracts derived from leaves of F. deltoidea (at a final concentration of 5 mg/mL and 0.5 mg/mL) were used. Resistance of human erythrocytes after in vitro treatment by aqueous extracts derived from leaves of F. deltoidea (at a final concentration of 5 mg/mL and 0.5 mg/mL) was evaluated by HCl-induced hemolysis using a percentage of hemolyzed erythrocytes per each 30 sec. and a total time of hemolysis. Methodology. The leaves of F. deltoidea were collected in M.M. Gryshko National Botanic Garden (Kyiv, Ukraine). Blood (10-20 mL) was obtained from normal volunteers via venipuncture (4 males and 5 females aged 28-53 years old). An erythrocyte suspension at 1% hematocrit was incubated with 4 mM phosphate buffer (pH 7.4) (control) and pre-incubated with the extract of F. deltoidea (at a final concentration of 5 mg/mL and 0.5 mg/mL, respectively) at 37 °C for 60 min. The level of lipid peroxidation was determined by quantifying the concentration of 2-thiobarbituric acid reacting substances (TBARS). The rate of protein oxidative destruction was estimated from the reaction of the resultant carbonyl derivatives of amino acid reaction with 2,4-dinitrophenylhydrazine (DNFH). The total antioxidant capacity (TAC) in the samples was estimated by measuring the TBARS level after Tween 80 oxidation. The HCl-induced resistance of erythrocytes was measured spectrophotometrically with 0.1M HCl. The significance of differences (significance level, p < 0.05) was examined using the Mann-Whitney U test. Scientific novelty. The treatment of human erythrocytes by extract derived from leaves of F. deltoidea at the final concentration of 5 mg/mL resulted in an increase of TBARS as biomarkers of lipid peroxidation and aldehydic derivatives of oxidatively modified proteins with a simultaneous decrease in the total antioxidant capacity compared to the untreated samples. On the other hand, in vitro treatment of human erythrocytes with an extract derived from leaves of F. deltoidea at the final concentration of 0.5 mg/mL resulted in the same values of TBARS level, aldehydic and ketonic derivatives of OMP, and total antioxidant capacity as the untreated samples. Extract of F. deltoidea at the final concentrations of 5 mg/mL and 0.5 mg/mL possess hemolytic properties to human erythrocyte suspension after 1-h incubation in vitro. Conclusions. The changes in the oxidative stress biomarkers using the in vitro model of human erythrocytes to evaluate the antioxidant activities of the aqueous extract derived from the leaves of F. deltoidea at the two final concentrations (5 mg/mL and 0.5 mg/mL) revealed that high concentration of extract (5 mg/mL) resulted in the increase of lipid peroxidation and protein oxidation with a simultaneous decline in the total antioxidant capacity. HCl-induced hemolysis was activated after the treatment by extract derived from the leaves of F. deltoidea at the two final concentrations. More studies are warranted in the future, to illustrate the potential and mechanisms of F. deltoidea in preventing oxidative stress using different cell models in vitro and different final concentrations of the extract. Also, further studies are warranted to identify the bioactive components that contribute to this protective effect.

Effects of biomechanical properties of blood on surface tension-driven flows in superhydrophilic channels

Surface tension-driven microfluidic flows offer low-cost solutions for blood diagnostics due to the pump-less flow handling. Knowledge of the influence of the biomechanical properties of blood on such flows is key to design such devices; however, a systematic examination of that influence is lacking in the literature. We report on the effects of specific hemorheological factors for flows in a superhydrophilic microchannel. Whole human blood and erythrocyte suspensions in phosphate buffer and dextran solutions were tested. Heat-treated counterparts of the aforementioned samples were produced to alter the deformability of the cells. The flow of the samples was imaged and characterized using micro-particle image velocimetry and tracking techniques to probe the effects of hematocrit, and erythrocyte aggregation and deformability. Meniscus velocities, velocity profiles in the channel, and local and bulk shear rates were derived. The mean velocity of blood was affected by the increasing sample viscosity and the reduced erythrocyte deformability as expected. The increased erythrocyte aggregation appeared to affect more the shape of the velocity profiles in the normal, compared to the heat-treated samples. Very high shear rates are observed in the early stages of the flow, suggesting high erythrocyte disaggregation, persisting sufficiently strong until the flow reaches the end of the channel.

Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy.

Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4–10), osmolarity (50–1000 mOsm/L), temperature (15–60 °C), and surfactant Triton X-100 (10–500 μM)). Their stability was evaluated by LS by considering the hydrodynamic radius (Rh), intensity of scattered light (I), and the shape parameter (ρ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS–citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50–1000 mOsm/L, pH 4–10) had no significant effect on the Rh (=100–130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.

Paracoccidioides restrepiensis B339 (PS3) and P. lutzii LDR2 yeast cells and soluble components display in vitro hemolytic and hemagglutinating activities on human erythrocytes.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by thermodimorfic fungi of Paracoccidioides species complex. Several pathogenic fungi produce hemagglutinins and hemolysins, which are virulence factors involved in adhesion of pathogens to host tissues or cells and in destruction of erythrocytes. The present research investigated hemolytic and hemagglutinating activities of yeast cells and soluble components from P. restrepiensis (PS3; former P. brasiliensis B339) and P. lutzii (LDR2). Different concentrations of live and heat-killed yeast cells and soluble components from cell free antigen preparation (CFA) (native or heated - 56 and 100 °C, 30 min) were mixed with 1% human erythrocyte suspension. Yeast cells from both species caused hemolysis, but P. lutzii LDR2 was more hemolytic than P. restrepiensis B339, while the opposite phenomena occurred with soluble components in most conditions. Live or heat-killed yeast cells of both fungi agglutinated erythrocytes, but only heated soluble components from P. restrepiensis B339 showed hemagglutinating activity. In conclusion, yeast cells of P. restrepiensis B339 and P. lutzii LDR2 produce hemolysins and hemagglutinins, which most likely are more restricted to yeast cells in P. lutzii LDR2 and are more released in soluble form byP. restrepiensis B339, requiring further study.

Bisthiourea Derivatives of Dipeptide Conjugated Benzo[d]isoxazole as a New Class of Therapeutics: Synthesis and Molecular Docking Studies.

Studies on anti-inflammatory and antimicrobial agents remains a challenging and important area in medicinal chemistry research due to more toxic and rapid development of resistance against first effective drugs. In search of novel anti-inflammatory and antimicrobials agents, bisthiourea derivatives of dipeptide conjugated to 6-fluoro-3- (piperidin-4-yl)benzo[d]isoxazole were synthesized. The peptides were synthesized by solution phase method and conjugated to 6- fluoro-3-(piperidin-4-yl)benzo[d]isoxazole using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDCI)/hydroxybenzotriazole (HOBt) as a coupling agent and N-methylmorpholine (NMM) as a base. The protecting group, 2-chlorobenzyloxycarbonyl (2-ClZ) and tertbutyloxycarbonyl (Boc) were deblocked and further reacted with substituted phenyl isothiocyanates to obtain bisthiourea derivatives. The molecules 1-24 were examined for their in vitro anti-inflammatory activity by employing human erythrocyte suspension test and it was found that the IC50 values of 9, 12, 21 and 24 were lower than the IC50 of standard references indomethacin and ibuprofen. Further, all the molecules were also evaluated for their in vitro antibacterial and antifungal activities against various pathogens of human origin by agar well diffusion method. In addition, binding interaction of active molecules (7-12 and 19-24) was performed on active site of cyclooxygenase-2 (COX-2) and Staphylococcus aureus (SA) TyrRS showing good binding profile. Molecular docking result, along with the biological assay data, revealed that the compounds containing electron withdrawing group (F) on phenyl ring of thiourea moiety were potential anti-inflammatory and antimicrobial agents.

Electrochemical activity and morphology of human erythrocytes at optically transparent ITO electrode

The electrochemical activity of a suspension of human erythrocytes was found at potentials above +0.4 V. The limiting current of electrooxidation was found to linearly depend on the cell concentration in the suspension. The electrochemical activity of an erythrocyte suspension is accompanied by reversible changes in the cell morphology only in non-buffered media.

On evaluation of electric conductivity by mean of non equilibrium thermodynamic approach with internal variables. An application to human erythrocyte suspension for metabolic characterizations

In this paper we will approach a new method to obtain electrical conductivity by means of dielectric measurements. After a remark on non-equilibrium thermodynamics with internal variables formulated by Kluitenberg, we introduce complex conductivity and the relationship with the complex dielectric constant. Taking into account the dielectric model by us introduced we determine the real and imaginary part of conductivity as function of the frequency of perturbation. Also we determine phenomenological and state coefficients that lead to the determination of the two components in which is splitted the polarization, and to the entropy production due to electrical conductivity. These results are applied to an human erythrocyte suspension for metabolic deductions.

NMR of 133Cs+ in stretched hydrogels: One-dimensional, z- and NOESY spectra, and probing the ion’s environment in erythrocytes

133Cs nuclear magnetic resonance (NMR) spectroscopy was conducted on 133Cs+ in gelatin hydrogels that were either relaxed or stretched. Stretching generated a septet from this spin-7/2 nucleus, and its nuclear magnetic relaxation was studied via z-spectra, and two-dimensional nuclear Overhauser (NOESY) spectroscopy. Various spectral features were well simulated by using Mathematica and the software package SpinDynamica. Spectra of CsCl in suspensions of human erythrocytes embedded in gelatin gel showed separation of the resonances from the cation inside and outside the cells. Upon stretching the sample, the extracellular 133Cs+ signal split into a septet, while the intracellular peak was unchanged, revealing different alignment/ordering properties of the environment inside and around the cells. Differential interference contrast light microscopy confirmed that the cells were stretched when the overall sample was elongated. Analysis of the various spectral features of 133Cs+ reported here opens up applications of this K+ congener for studies of cation-handling by metabolically-active cells and tissues in aligned states.

Lectins of <i>Erythrina poeppigiana</i> and <i>Erythrina steyermarkii</i> (Leguminosae): charaderization and mitogenic effect

Las diferentes especies de Erythrina se encuentran ampliamente distribuidas en Costa Rica y se las conoce popularmente con el nombre de "poró". En el presente estudio, se seleccionaron dos especies: Erythrina poeppigiana y Erythrina steyermarkii. Se prepararon extractos de las semillas en solución tampón salina de fosfatos y se verificó la presencia de lectinas en ellos mediante la técnica de hemaglutinación, utilizando eritrocitos humanos. Se trató de demostrar un efecto selectivo de la hemaglutinación empleando eritrocitos de varias especies de mamíferos, específicamente de carnero, caballo y conejo. Solo los eritrocitos de conejo fueron aglutinados con la lectina de E. steyermarkii. El efecto hemaglutinante de las dos lectinas fue inhibido con los siguientes carbohidratos: D-galactosa, N-D-acetil galactosamina, D-Iactosa y Drafinosa. La lectina de E. steyermarkii también fue inhibida con L-rhamnosa. Las dos lectinas fueron aisladas por filtración en gel y cromatografia de afinidad, usando lactosa como ligando. Las fracciones que resultaron positivas se analizaron mediante la técnica de electroforesis en gel de poliacrilamida y duodecil sulfato de sodio (SDSPAGE). Con la filtración en gel y el SDS-PAGE, se comprobó que las dos lectinas tienen una masa molecular aparente de 50 kDa y. que están formadas por dos subunidades de 25 kDa, aproximadamente. Se buscó un efecto mitogénico en las dos lectÍnas y se encontró que sólo E. steyermarkii lo manifestaba sobre células mononucleares humanas aisladas de sangre periférica. Se determinó la estabilidad de las ¡eetinas en diferentes ámbitos de temperatura (4 a 100°C) y de pH (2 a 12 ). Las dos lectinas se mantuvieron estables en un rango de temperatura de 40 a 70°C y en un pH de 2 a 10.

In vitro optical detection of simulated blood pulse in a human tooth pulp model

Noninvasive optical methods such as photoplethysmography, established for blood pulse detection in organs, have been proposed for vitality testing of human dental pulp. However, no information is available on the mechanism of action in a closed pulp chamber and on the impairing influence of other than pulpal blood flow sources. Therefore, the aim of the present in vitro study was to develop a device for the optical detection of pulpal blood pulse and to investigate the influence of different parameters (including gingival blood flow [GBF] simulation) on the derived signals. Air, Millipore water, human erythrocyte suspensions (HES), non-particulate hemoglobin suspension (NPHS), and lysed hemoglobin suspension (LHES) were pulsed through a flexible (silicone) or a rigid (glass) tube placed within an extracted human molar in a tooth-gingiva model. HES was additionally pulsed through a rigid tube around the tooth, simulating GBF alone or combined with the flow through the tooth by two separate peristaltic pumps. Light from high-power light-emitting diodes (625 nm (red) and 940 nm (infrared [IR]); Golden Dragon, Osram, Germany) was introduced to the coronal/buccal part of the tooth, and the signal amplitude [∆U, in volts] of transmitted light was detected by a sensor at the opposite side of the tooth. Signal processing was carried out by means of a newly developed blood pulse detector. Finally, experiments were repeated with the application of rubber dam (blue, purple, pink, and black), aluminum foil, and black antistatic plastic foil. Nonparametric statistical analysis was applied (n = 5; α = 0.05). Signals were obtained for HES and LHES, but not with air, Millipore water, or NPHS. Using a flexible tube, signals for HES were higher for IR compared to red light, whereas for the rigid tube, the signals were significantly higher for red light than for IR. In general, significantly less signal amplitude was recorded for HES with the rigid glass tube than with the flexible tube, but it was still enough to be detected. ∆U from gingiva compared to tooth was significantly lower for red light and higher for IR. Shielding the gingiva was effective for 940 nm light and negligible for 625 nm light. Pulpal blood pulse can be optically detected in a rigid environment such as a pulp chamber, but GBF may interfere with the signal and the shielding effect of the rubber dam depends on the light wavelength used. The optically based recording of blood pulse may be a suitable method for pulp vitality testing, if improvements in the differentiation between different sources of blood pulse are possible.

Stoichiometric Relationship between Na+ Ions Transported and Glucose Consumed in Human Erythrocytes: Bayesian Analysis of 23Na and 13C NMR Time Course Data

We examined the response of Na+,K+-ATPase (NKA) to monensin, a Na+ ionophore, with and without ouabain, an NKA inhibitor, in suspensions of human erythrocytes (red blood cells). A combination of 13C and 23Na NMR methods allowed the recording of intra- and extracellular Na+, and 13C-labeled glucose time courses. The net influx of Na+ and the consumption of glucose were measured with and without NKA inhibited by ouabain. A Bayesian analysis was used to determine probability distributions of the parameter values of a minimalist mathematical model of the kinetics involved, and then used to infer the rates of Na+ transported and glucose consumed. It was estimated that the numerical relationship between the number of Na+ ions transported by NKA per molecule of glucose consumed by a red blood cell was close to the ratio 6.0:1.0, agreeing with theoretical prediction.

In vitro kinetics of oxygen transport in erythrocyte suspension or unmodified hemoglobin solution from human and other animals

Oxygen transport behavior in erythrocyte suspension or in hemoglobin solution was studied as a potential therapeutic model for the clinical treatment of blood loss, and this can also provide physiological data with which to evaluate blood substitutes. In the present project, we examined the in vitro kinetics of hemoglobin binding to and releasing oxygen, to provide detailed oxygen-flux measurements for unmodified hemoglobin solutions and erythrocyte suspensions in human, as well as other vertebrates. An in vitro method was used, based on a widely used artificial system, with the oxygen saturation level being detected in real time. Results from this study indicated that the kinetic curves of human erythrocyte suspensions and hemoglobin solutions were either S-shaped or hyperbolic, respectively. Based on these curves, the significance of T(50) emerged in our investigation, where T(50) is defined as the time needed for 50% hemoglobin to be saturated with oxygen, and reflects the efficiency with which hemoglobin carries oxygen. This parameter may be used to diagnose blood diseases, and could be a standard for evaluating blood substitutes. In this study, we also compared the T(50) of 4 species of vertebrates, and found that it shows a distinct efficiency of oxygen binding related to species, and potentially reveals the evolutionary function of hemoglobin and its possible adaptation to the environment.

Influence of sodium nitroprusside on human erythrocyte membrane water permeability: an NMR study

Sodium nitroprusside (SNP) is a nitric oxide (•NO) donor in vitro and in vivo. In this paper the time variation of the intracellular water proton nuclear magnetic resonance (NMR) effective relaxation time T'(2a) in SNP-treated human erythrocyte suspensions, containing 10 mM membrane impermeable paramagnetic MnCl2, has been measured. The observed T'(2a) time-course was analyzed in terms of the two mechanisms by which released •NO affects T'(2a). These are, respectively, enhancement of the intracellular water proton intrinsic NMR relaxation rate 1/T(2a) by paramagnetism of •NO subsequently bonded to iron atoms of intracellular deoxyhemoglobin, and suppression of diffusional water permeability P(d) as a consequence of nitrosylation of aquaporin-1 (AQP1) channel Cys189, either by direct reaction with •NO or with one of the •NO oxidation products, such as N2O3. The bound •NO on the Cys189 thiol residue appears to impose a less efficient barrier to water permeation through AQP1 than the larger carboxyphenylmercuryl residue from p-chloromercuribenzoate. The effect of •NO on P(d) is discussed in terms of NO-induced vasodilation.

10.1007/s11171-008-2002-2

According to the previously reported data, the superntant of the primary culture of human erythrocytes contains 33 hemoglobin fragments. An analysis of the supernatant of a 20% (v/v) suspension of human erythrocytes allowed us to identify additionally four peptides whose precursors are cytoplasmic β-actin (two fragments), fructose diphosphate aldolase B, and an unknown protein, and amino acids tyrosine and tryptophan. The composition and the content of the components of the supernatant did not depend on the age and blood group of donors. The dynamics of accumulation in the supernatant (20–80 min of incubation) of 14 hemoglobin fragments with the most reliably reproducible contents was obtained. The content of six peptides increased more than twofold between 20 and 40 min of incubation; the maximum increase in concentration was observed between 40 and 80 min (140%). The level of peptides that had the maximum concentration at the end of incubation was about 1000 pmol/ml of sedimented erythrocytes. The biological effects of the peptides identified in the supernatant of erythrocytes involve the stimulation of proliferation and hemopoiesis, suppression of proliferation, a bactericide effect, etc. These effects indicate the physiological importance of the peptide release by erythrocytes.

Influence of carbonyl stress on rheological alterations of blood materials and decarbonylation effect of glutathione

The effects of various toxic carbonyls such as malondialdehyde (MDA), a secondary product of lipid peroxidation, and other aldehydes on rheological parameters and their relationship with aging-associated alterations were studied. Both MDA and glutaraldehyde (Glu) in different concentrations significantly increase viscosity, plastic viscosity and yield stress of human plasma and erythrocyte suspensions. MDA (20 mmol/L) reduces sharply the typical fluorescence of proteins (excitation 280 nm/emission 350 nm), and produces age pigment-like fluorescence with a strong emission peak at 460 nm when excites at 395 nm by only being incubated for some hours. In contrast, Glu decreases merely the fluorescence of proteins without producing age pigment-like fluorescence. These data suggest interestingly that the MDA-induced gradual protein cross linking seems to form from different mechanisms compared to the fast rheological changes of blood materials which may take place either in acute and chronic diseases or during aging. On the other hand, MDA induces various deleterious alterations of erythrocytes whereas glutathione (GSH) inhibits the MDA-related carbonyl stress in a concentration-dependent manner. The results indicate that carbonyl-amino reaction exists in the blood widely and GSH has the ability to interrupt or reverse this reaction in a certain way. It implies that carbonyl stress may be one of the important factors in blood stasis and suggests a theoretical and practical approach in anti-stresses and anti-aging.

Fragments of functional proteins in a primary culture of human erythrocytes

According to previously reported data, the supernatant of a primary culture of human erythrocytes contains 33 hemoglobin fragments. An analysis of the supernatant of a 20% (v/v) suspension of human erythrocytes allowed us to identify additionally four peptides whose precursors are cytoplasmic beta-actin (two fragments), fructose diphosphate aldolase B, and an unknown protein, as well as the amino acids tyrosine and tryptophan. The composition and the content of the components of the supernatant did not depend on the age or blood group of donors. The dynamics of accumulation in the supernatant (20-80 min of incubation) of the 14 hemoglobin fragments with the most reliably reproducible contents was obtained. The content of six peptides increased more than twofold between 20 and 40 min of incubation: the maximum increase in concentration was observed between 40 and 80 min (140%). The level of peptides that had the maximum concentration at the end of incubation was about 1000 pmol/ml of sedimented erythrocytes. The biological effects of the peptides identified in the supernatant of erythrocytes involve the stimulation of proliferation and hemopoiesis, suppression of proliferation, a bactericide effect, etc. These effects indicate the physiological importance of peptide release by erythrocytes. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

A Modified Differential Scanning Calorimetry Method for Determining Water Transport Properties in Biological Cells During the Freezing Process

A modified analytical and experimental method using differential scanning calorimeter (DSC) was applied to determine the cell water transport properties of human erythrocytes during the freezing process. Using DSC, samples containing human erythrocyte cell suspensions of human erythrocytes with different cytocrits were cooled to −40°C at slow cooling rates (5°C/min) after nucleation. It was shown that latent heat release from one unit mass of the cell suspension was a linear function of cytocrits (cell numbers), with a temperature-dependent slope and intercept. Based on the theoretical model, cell volumes were calculated from the slope and intercept at corresponding temperatures. Cell water transport properties (lpg,Ea) were next calculated by curve fitting of the cell volume change during the freezing process. The results revealed that, for human erythrocyte, Lpg (Tr = 273.15 K) is 0.10 ± 0.01 μm/min ċ atm and Ea is 279.1 ± 0.7 kJ/mol at the cooling rate of 5°C/min. For comparison, both DSC and cryomicro...

Acylation of Secretory Phospholipases A2 Alter the Catalytic Activity on Erythrocyte Membrane

Secretory Phospholipases A2 (sPLA2’s) catalyze the calcium dependent hydrolysis of sn-2 linkages in phosphoglicerades Simultaneous assay of binding and hydrolysis of radiolabeled sPLA2 with a suspension of intact human erythrocytes was carried out. One of the enzymes used in these studies has been a Group IA PLA isolated from the snake venom of Naja nigricollis (NnPLA) and the Agkistrodon halys blomhofffii (AhPLA) which has been shown to display a preference for arachidonoyl containing species. In an effort to direct these sPLA2’s toward specific regions of the human erythrocyte membrane surface, this enzyme was covalently coupled to methyl arachidonyl fluorophosphonate (MAFP) and farnesyl pyrophosphate (FPP). Approximately, 60 percent of inhibition was observed when NnPLA was covalently couple to MAFP or FPP, but a significant increase in specificity toward the 20:4 fatty acids species was observed with the former. Interestingly, FFP induced a slightly increase on the activity of AhPLA but MAFP had an almost 2.4 fold increasing effect on the activity of AhPLA with a decrease specificity against 20:4 fatty acids species. The covalent modification of these sPLA2’s was confirmed by ESI-MS. The increase in sPLA2 molecular mass correspond to one equivalent of the modifying compound used in each case. Work support by UPR-RP RISE Program (2R25GM061151)

Evaluation of Phototoxic Properties of Fragrances

Fragrances are widely used in topical formulations and can cause photoallergic or phototoxic reactions. To identify phototoxic effects, 43 fragrances were evaluated in vitro with a photohaemolysis test using suspensions of human erythrocytes exposed to radiation sources rich in ultraviolet (UV) A or B in the presence of the test compounds. Haemolysis was measured by reading the absorbance values, and photohaemolysis was calculated as a percentage of total haemolysis. Oakmoss caused photohaemolysis of up to 100% with radiation rich in UVA and up to 26% with radiation rich in UVB. Moderate UVA-induced haemolysis (5-11%) was found with benzyl alcohol, bergamot oil, costus root oil, lime oil, orange oil, alpha-amyl cinnamic aldehyde and laurel leaf oil. Moderate UVB-induced haemolysis was induced by hydroxy citronellal, cinnamic alcohol, cinnamic aldehyde, alpha-amyl cinnamic aldehyde and laurel leaf oil. The phototoxic effects depended on the concentration of the compounds and the UV doses administered. We conclude that some, but not all, fragrances exert phototoxic effects in vitro. Assessment of the correlation of the clinical effects of these findings could lead to improved protection of the skin from noxious compounds.

Safety Study of an Antimicrobial Peptide Lactocin 160, Produced by the VaginalLactobacillus rhamnosus

Objective. To evaluate the safety of the antimicrobial peptide, lactocin 160. Methods. Lactocin 160, a product of vaginal probiotic Lactobacillus rhamnosus 160 was evaluated for toxicity and irritation. An in vitro human organotypic vaginal-ectocervical tissue model (EpiVaginal) was employed for the safety testing by determining the exposure time to reduce tissue viability to 50% (ET-50). Hemolytic activity of lactocin160 was tested using 8% of human erythrocyte suspension. Susceptibility of lactobacilli to lactocin160 was also studied. Rabbit vaginal irritation (RVI) model was used for an in vivo safety evaluation. Results. The ET-50 value was 17.5 hours for lactocin 160 (4.9 hours for nonoxynol 9, N9). Hemolytic activity of lactocin 160 was 8.2% (N9 caused total hemolysis). Lactobacilli resisted to high concentrations of peptide preparation. The RVI model revealed slight vaginal irritation. An average irritation index grade was evaluated as “none.” Conclusions. Lactocin 160 showed minimal irritation and has a good potential for intravaginal application.