Deployment of response surface methodology to optimize microencapsulation of peroxidases from turnip roots (Brassica rapa L.) by double emulsion in PLA polymer.

In order to improve the preservation conditions and stability of peroxidase catalytic properties, a number of immobilization techniques have been widely developed. In this context, we set as objective, the optimization of synthesis and stability of microcapsules of peroxidases (POD) from turnip using polylactic acid (PLA) polymer with the double emulsion technique. The surfactant, polymer, and peroxidase concentrations were the optimized parameters. According to the results obtained using the Box-Behnken design, the optimal parameters found were 1.55% of PVA, 55 mg/mL of peroxidases, and 30 mg/mL of PLA polymer with an encapsulation efficiency of 57.29%. The scanning electron microscopy morphological characterization of the optimized microcapsules showed a regular spherical structure. Fourier transform infrared spectroscopy identified the specific functional groups and chemical bonds before and after microencapsulation. The elaborated microcapsules were characterized by an average size of 200 µm (mainly from 150 to 500 µm) with a low residual moisture content (2.26%) and the encapsulated peroxidases showed better thermal stability. The in vitro release of peroxidases confirmed that the microcapsules have an excellent sustained release in simulated gastric digestion. Encapsulated peroxidases' storage under 25 and 4 °C displays a good residual POD activity with about 60% of initial activities during 80 days of storage, whereas free POD losses its initial activity within 15 and 30 days, respectively. The obtained results are promising for the development of effective therapeutic treatment of some intestinal troubles due to oxidative stress. PRACTICAL APPLICATION: Brassica rapa L. root is well known for its richness on peroxidases and thus presents an interesting potential for developing high added value products. In order to preserve the activity of extracted peroxidases (POD) from turnip roots, microencapsulation was optimized using a polylactic acid polymer. The encapsulated POD showed the maintenance of its activity under the effect of different storage conditions (time and temperature) and demonstrated resistance to gastric acidity. According to the obtained results, the encapsulation of peroxidases opens up medicine and pharmaceutical applications such as intestinal and colic protection against inflammations.

Recombinant Manganese Peroxidase Reduces A2E Burden in Age-Related and Stargardt's Macular Degeneration Models.

Macular degeneration is hallmarked by retinal accumulation of toxic retinoid species (e.g., A2E) for which there is no endogenous mechanism to eliminate it. This ultimately results in progressive dysfunction and loss of vision either in advanced age for genetically normal patients (age-related macular degeneration) or in adolescence for those with inherited genetic mutations (Stargardt's disease). In this article, we present a proof-of-concept study for an enzyme-based therapy to remove these retinoids, modeled on traditional enzyme replacement therapy. Recombinant manganese peroxidase (rMnP) is produced in Pichia pastoris. In vitro, we demonstrate that rMnP breaks down A2E and other lipofuscin fluorophores with limited cellular toxicity, and as this enzyme is mannosylated, it can be taken up into cells through mannose receptor-dependent endocytosis. In vivo, we demonstrate that rMnP can significantly reduce the A2E burden when administered by intravitreal injections. Together, these data provide encouraging results toward the development of an enzyme-based therapy for macular degeneration and indicate the need for additional work to characterize the molecular mechanism of A2E breakdown and to improve the pharmacological parameters of the enzyme.

A novel antibacterial agent based on AgNPs and Fe3O4 loaded chitin microspheres with peroxidase-like activity for synergistic antibacterial activity and wound-healing.

In this study, a novel antibacterial agent was developed based on chitin nanofibrous microspheres loaded with AgNPs and Fe3O4 nanoparticles (Ag-Fe3O4-NMs) for synergistic antibacterial activity and wound healing. Ag-Fe3O4-NMs was prepared via an in situ synthetic method which showed an excellent porosity and wettability. Moreover, Ag-Fe3O4-NMs were capable of sustained release of Ag+ and catalysed the decomposition of low H2O2 concentrations to generate hydroxyl radical (OH). The OH and Ag+ showed higher antibacterial activity and inhibited the toxicity with high dose of AgNPs and H2O2. In vitro biocompatibility results suggested that Ag-Fe3O4-NMs have low toxicity and low hemolysis. Thus, a novel antibacterial agent with enhanced synergistic antibacterial activity was obtained by combination of Ag-Fe3O4-NMs and H2O2 at a low and biologlically safe dosage, which could facilitate fibroblast growth, accelerate epithelialization, and promote the healing rate of infected wounds.

Horseradish and radish peroxidases eaten with fish could help explain observed associations between fish consumption and protection from age-related dementia.

A juxtaposition of regional cuisines and recent prospective studies of fish consumption in China and Japan points to fresh horseradish and/or radish (HRR) as possible contributors to delaying age-related dementia. The hypothesis is that the inverse association found sometimes between fish intake and cognitive decline is partially due to exposure of the oral cavity to active peroxidases from HRR served in conjunction with fish. This hypothesis can be tested by specifically looking at whether HRR is consumed with fish and whether such HRR is prepared in a way that preserves activity of HRR peroxidases. It is possible that by putting active HRR peroxidases in their mouths, elderly people supplement their age-diminished salivary antioxidant capacity and break down additional hydrogen peroxide (H2O2) in the oral cavity before it can migrate into the brain, thus decreasing the incidence of brain cell death induction by chronically-elevated H2O2. Intentional exposure of the oral cavity to active HRR peroxidases could be a prophylactic for delaying dementia. Because vegetable peroxidases are inactivated by gastric juices, it will be difficult to obtain benefit from HRR peroxidases' antioxidant effect via ingestion in encapsulated dietary supplements.

A randomized and placebo-controlled study to compare the skin-lightening efficacy and safety of lignin peroxidase cream vs. 2% hydroquinone cream.

BACKGROUND: Historically, the most effective treatments for skin lightening have contained hydroquinone. However, there is a need for an effective alternative. AIMS: The purpose of this study was to evaluate the skin-lightening efficacy and safety of lignin peroxidase (LIP) creams using a regimen of both day and night products compared with twice-daily application of 2% hydroquinone cream and placebo in Asian women. PATIENTS/METHODS: This was a randomized, double-blind, placebo-controlled, split-face, single-center study of 51 patients. Patients were randomized to receive day and night LIP cream on one randomly selected side of their face and either 2% hydroquinone cream or placebo on the other. RESULTS: A statistically significant change from baseline in the melanin index was observed in LIP-treated skin, with a mean reduction of 7.6% (P < 0.001) on Day 31. Conversely, hydroquinone and placebo did not provide a statistically significant lightening effect when instrumentally measured. Dermatologist scoring demonstrated a significant improvement in overall fairness as early as 8 days after treatment initiation in the LIP-treated group, which was not observed in the other groups. Overall, patients preferred the LIP creams. CONCLUSIONS: The application of day/night LIP cream provided a significantly more rapid and observable skin-lightening effect than hydroquinone 2% cream or placebo.

Leish-111f, a recombinant polyprotein vaccine that protects against visceral Leishmaniasis by elicitation of CD4+ T cells.

The Leishmania-derived recombinant polyprotein Leish-111f or its three component proteins, thiol-specific antioxidant (TSA), Leishmania major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF), have previously been demonstrated to be efficacious against cutaneous or mucosal leishmaniasis in mice, nonhuman primates, and humans. In this study we demonstrate that Leish-111f is also a vaccine antigen candidate against visceral leishmaniasis (VL) caused by Leishmania infantum. We evaluated the immune response and protection induced by Leish-111f formulated with monophosphoryl lipid A in a stable emulsion (Leish-111f+MPL-SE) and demonstrated that mice developed strong humoral and T-cell responses to the vaccine antigen. Analysis of the cellular immune responses of immunized, uninfected mice demonstrated that the vaccine induced a significant increase in CD4(+) T cells producing gamma interferon, interleukin 2, and tumor necrosis factor cytokines, indicating a Th1-type immune response. Experimental infection of immunized mice and hamsters demonstrated that Leish-111f+MPL-SE induced significant protection against L. infantum infection, with reductions in parasite loads of 99.6%, a level of protection greater than that reported for other vaccine candidates in animal models of VL. Taken together, our results suggest that this vaccine represents a good candidate for use against several Leishmania species. The Leish-111f+MPL-SE product we report here is the first defined vaccine for leishmaniasis in human clinical trials and has completed phase 1 and 2 safety and immunogenicity testing in normal, healthy human subjects.

Endocytosis of microperoxidase in the marginal cells of stria vascularis.

OBJECTIVE: Endocytosis has been thought to control entry into the cell and play a crucial role in the development, immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. We investigated the basic properties of endocytosis in the marginal cells of stria vascularis (SV) to discuss whether marginal cells have a potential to maintain the endolymph homeostasis. METHODS: We perfused microperoxidase (MPO), an endocytosis tracer, into the cochlear duct. After 5-60 min of endolymphatic perfusion, the tissues were fixed and the distribution of MPO within the marginal cell was observed by transmission electron microscopy. RESULTS: Endocytosis started already at 5 min after MPO perfusion. Small MPO-loaded endosomes were observed up to 30 min after MPO perfusion. The small tubulovesicular endosomes and the plasma membrane invagination were not decorated by an electron dense bristle structure. After endocytosis, MPO labeled preendosomes were quickly transported to the large vacuolar endosomes that connected with tubular endosomes. At 60 min after MPO perfusion, MPO-loaded large vesicles that have small vesicles in the lumen were observed. CONCLUSION: The time-course of MPO-loaded endosomes was similar to that of CF-loaded endosomes in the marginal cells of SV. The strial marginal cells have vigorous endocytotic activity both in clathrin-independent and clathrin-dependent endocytosis. This high activity of endocytosis in SV seems to be needed to maintain the homeostasis of endolymph via membranous channels and/or receptors regulations.

IL-10 from regulatory T cells determines vaccine efficacy in murine Leishmania major infection.

Leishmaniasis affects 12 million people, but there are no vaccines. Immunological correlates of vaccine efficacy are unclear. Polarized Th1 vs Th2 responses in Leishmania major-infected mice suggested that a shift in balance from IL-4 to IFN-gamma was the key to vaccine success. Recently, a role for IL-10 and regulatory T cells in parasite persistence was demonstrated, prompting re-evaluation of vaccine-induced immunity. We compared DNA/modified vaccinia virus Ankara heterologous prime-boost with Leishmania homolog of the receptor for activated C kinase (LACK) or tryparedoxin peroxidase (TRYP). Both induced low IL-4 and high IFN-gamma prechallenge. Strikingly, high prechallenge CD4 T cell-derived IL-10 predicted vaccine failure using LACK, whereas low IL-10 predicted protection with TRYP. The ratio of IFN-gamma:IL-10 was thus a clear prechallenge indicator of vaccine success. Challenge infection caused further polarization to high IL-10/low IFN-gamma with LACK and low IL-10/high IFN-gamma with TRYP. Ex vivo quantitative RT-PCR and in vitro depletion and suppression experiments demonstrated that Ag-driven CD4+ CD25+ T regulatory 1-like cells were the primary source of IL-10 in LACK-vaccinated mice. Anti-IL-10R treatment in vivo demonstrated that IL-10 was functional in determining vaccine failure, rendering LACK protective in the presence of high IFN-gamma/low IL-5 responses.

Loading the multilayer dextran sulfate/protamine microsized capsules with peroxidase.

Stable polyelectrolyte capsules were produced by the layer-by-layer (LbL) assembling of biodegradable polyelectrolytes, dextran sulfate and protamine, on melamine formaldehyde (MF) microcores followed by the cores decomposition at low pH. The mean diameter of the capsules at pH 3-5 was 8.0 +/- 0.2 microm, which is more than that diameter of the templates (5.12 +/- 0.15 microm). With pH growing up to 7-8, the capsules enlarged, swelling up to the diameter 9-10 microm. The microcapsules were loaded with horseradish peroxidase. Seemingly, peroxidase is embedded in the gellike structure in the microcapsule interior formed by MF residues in the complex with polymers used for LbL coating as proved by Raman confocal spectroscopy. The amount of finally incorporated peroxidase increased from 0.2 x 10(8) to 2.2 x 10(8) peroxidase molecules per capsule with pH growing from 5 to 8. The pH shifts causing changes in capsule swelling and the replacement of solutions without pH shifts lead to the protein loss. The encapsulated peroxidase showed a high activity (57%), which remained stable for 12 months.

Effect of dietary restriction on participation in faecal occult blood test screening for colorectal cancer.

OBJECTIVES: To determine if participation in colorectal cancer screening using faecal occult blood testing (FOBT) is affected by a restrictive diet and if it is associated with certain demographic variables. PARTICIPANTS AND SETTING: 1,203 residents of South Australia aged 50-69 years, with no "currently active bowel disease", randomly selected from a database of people willing to be contacted about unspecified health issues. DESIGN: Randomised controlled trial: participants were offered screening by immunochemical FOBT by mail in 1998. Half were randomly allocated to a group instructed to follow a low-peroxidase diet, as required for guaiac FOBT, while the other group was not so restricted. MAIN OUTCOME MEASURES: Effect of diet restriction on participation (return of correctly completed FOBT sample cards within 15 weeks); time taken to return cards; relationships between participation and demographic variables. RESULTS: Participation rates were 65.9% (no-diet group) and 53.3% (diet group) (difference, 12.6%; 95% CI, 7.1%-18.1%). In the first week, rates of return as a proportion of all tests returned were 13.1% (no-diet) and 1.6% (diet) (difference, 11.5%; 95% CI, 8.6%-14.4%), increasing to 54.3% and 44.5%, respectively, after five weeks (difference, 9.8%; 95% CI, 4.2%-15.4%). Participation was significantly associated with older age (odds ratio, 1.40; 95% CI, 1.10-1.78), but not sex, Index of Social Disadvantage or rural versus urban address. CONCLUSIONS: Dietary restrictions create a barrier to FOBT-based screening for colorectal cancer. The use of immunochemical rather than guaiac FOBT removes this barrier.

Interference of plant peroxidases with guaiac-based fecal occult blood tests is avoidable.

Peroxidase-rich fruits and vegetables are reputed to interfere with guaiac-based fecal occult blood tests. We added horseradish peroxidase to fecal samples and tested them with Hemoccult, Hemoccult SENSA(R), and hydrated Hemoccult. Positivity rates with Hemoccult and Hemoccult SENSA decreased rapidly as the time between smearing (preparation) and development increased, whereas they remained high with hydrated Hemoccult. For samples with added blood, positivity rates did not decrease with time. When 61 volunteers were tested on a standard restriction and on a challenge diet high in plant peroxidase, no positive results occurred during standard restriction. During the challenge diet, one volunteer was positive with Hemoccult and Hemoccult SENSA when development was delayed 24 h, and no volunteers were positive when it was delayed 48 h and 72 h. However, with hydrated Hemoccult, positives occurred in 13 of 61 volunteers at 24 h, 8 of 61 at 48 h, and 5 of 61 at 72 h. Thus, peroxidase-rich plant foods do not need to be excluded from the diet with Hemoccult and Hemoccult SENSA if development is delayed for at least 48 h after smearing. A delay of this duration will not solve the problem of plant peroxidase interference with hydrated Hemoccult.

Induced hydrocephalus in postnatal rats following an intracerebral injection of ricin.

A single injection of peroxidase-labelled Ricinus communis agglutinin (RCA-HRP) was given intracerebrally in 5-day old postnatal rats to determine its effects on neural tissues. The rats were sacrificed at various time intervals ranging from 1 hour to 8 weeks after the injection. 5 days after the injection, the lateral ventricle ipsilateral to the injection was progressively enlarged. The size of the ventricle continued to expand so that 10-15 days after the injection the ventricle on the contralateral side was also affected. In longer surviving rats, i.e 3-8 weeks after the injection, both the ventricles were extremely dilated resulting in the thinning of the cerebral cortex. Scanning electron microscopy of the dilated ventricles showed signs of disruption of the ependyma in some regions. A number of cells including macrophages, neurons, glioblasts, astrocytes and oligodendrocytes were present on the ependyma. Their identification was confirmed by scanning- and transmission electron microscopy. Transmission electron microscopy of the cerebral cortex subjacent to the dilated ventricles showed the presence of many degenerating neurons, 2-5 hours after the injection of RCA-HRP. The neurons displayed typical features of degeneration, i.e. displacement of nucleus, dilatation of cisternae of rough endoplasmic reticulum, and swelling and disintegration of mitochondria. In conclusion, following a single intracerebral injection of RCA-HRP, drastic neuronal degeneration was elicited near the site of injection and this resulted in the dilatation of the lateral ventricles similar to hydrocephalus.

Evidence for sublaminar organization of the cells of origin of the geniculo-striate pathway in the cat: a novel method for cortical deposition of HRP.

A novel technique for retrograde labelling was used to trace the axonal projections from the lateral geniculate nucleus (LGN) to the striate cortex in the cat. Cortical deposits of horseradish peroxidase were made in the form of long straight lines oblique to layer IV. The projections along the lengths of these linear deposits were inferred, by retinotopic correspondence, from the distributions of the retrogradely labelled cells in the LGN. The results are consistent with an earlier report, by Humphrey et al., that the depths of axon terminations in layer IV reflect the depths of the parent cell bodies in layers A and A1 of the LGN. The pattern observed in the present experiments--border cells in the A layers project mainly to cortical layers IVa and centre cells to IVb--suggests that: (1) the sublaminar segregation of the ON and OFF pathways that occurs in the cat LGN is reversed by convergent projections to IVa from the dorsal (ON-rich) and ventral (OFF-rich) borders of the A layers, and (2) the paucity of Y input to IVb is due to the near absence of Y cells in the narrow central sublaminae of the A layers that supply the main input to IVb.

Immunotoxins containing glucose oxidase and lactoperoxidase with tumoricidal properties: in vitro killing effectiveness in a mouse plasmacytoma cell model.

We have tested the tumoricidal potency of enzyme immunotoxins constructed of antibodies conjugated to glucose oxidase and to lactoperoxidase. Murine plasmacytoma cells were targeted in vitro with the use of affinity-purified rabbit anti-plasmacytoma membrane antibodies (conjugated to glucose oxidase or lactoperoxidase) or rabbit serum raised against plasmacytoma microsome membranes followed by goat anti-rabbit immunoglobulin conjugates (to glucose oxidase or lactoperoxidase). Cytotoxicity was generated subsequently by incubation of the washed cells in a medium supplemented with glucose and sodium iodide, which were the substrates of these enzymes. This resulted in the presumed metabolic release of highly toxic reduced oxygen species and iodinated derivatives. Targeting of tumor cells with both conjugates, as opposed to one of them alone, produced a synergistic killing effect. The gain of specific versus unspecific cytotoxicity was upwards of 10,000-fold. The killing rates were elevated (t10 values less than 30 min) and linear over time. The resultant reduction in tumor cell viability was in the order of 5 to 6 logs after only 20 to 90 min of incubation in the glucose/NaI medium. Cytotoxicity was enhanced by the gamma-glutamyl cysteine synthetase inhibitor buthionine-S,R-sulfoximine and by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea, while catalase was inhibitory. The results suggest that these enzyme immunotoxins may be suitable for the ex vivo purging of autologous bone marrow grafts.

[A new concept of cardioplegic protection in cardiac surgery: iron chelation]. / Un nouveau concept de la protection cardioplégique en chirurgie cardiaque: la chélation du fer.

Hydroxyl is one of the most cytotoxic of all oxygen-derived free radicals produced during the myocardial ischaemia-reperfusion sequence. The purpose of the present study was to determine the effects of various interventions aimed at diminishing the production of hydroxyl radicals by reducing either one of their precursors (hydrogen peroxide) or the metal (ferric iron) which catalyzes the reaction generating these radicals. Sixty isolated and perfused rat hearts with isovolaemic contraction were studied. Except for non-ischaemic controls, these hearts were subjected to a 3-hour cardioplegic arrest in hypothermia (15-18 degrees C) followed by a 45-min reperfusion. The following interventions were performed: pretreatment with peroxidase, a hydrogen peroxide scavenger; pretreatment with peroxidase combined with deferoxamine, an ironchelating agent; pretreatment with peroxidase followed by addition of deferoxamine to the cardioplegic solution; addition of deferoxamine to the cardioplegic solution without pretreatment with the enzyme. Judging from the post-ischaemic values of developed pressure (maximum systolic pressure--diastolic pressure), left ventricular dP/dt and diastolic pressure and coronary flow rate, it appeared that the best myocardial protection was provided by deferoxamine-enriched cardioplegia. This study confirms that hydroxyl radicals most probably play a role in the genesis of the myocardial lesions associated with global ischaemia followed by reperfusion. Moreover, our results highlight the potential value of deferoxamine added to cardioplegic protection in heart surgery performed under extracorporeal circulation.

A method for intracellular injection of horseradish peroxidase by pressure.

A technique for intracellular injection of horseradish peroxidase and other tracers into neurones is described. The method utilises gas pressure to force the tracer solution out of glass micropipettes and allows electrophysiological recordings to be made simultaneously with the injection process. Constructional details of the simple and inexpensive equipment are given. The method has the advantages of being equally suitable for charged and uncharged tracer molecules, and of providing reliable indication of the success of the injection at the time of the injection attempt.

Kinetics of respiratory tract absorption and plasma clearance of horseradish peroxidase in guinea pigs.

Horseradish peroxidase (HRP) absorption across the wall of the upper airway, monitored by the amount detected in the blood, is used to measure epithelial damage by toxins. Full details of kinetics of absorption and blood clearance have not been reported previously. We measured the kinetics under the experimental conditions used in testing toxins. HRP was administered to guinea pigs either by intraarterial injection of a 7.5 microgram bolus (plasma clearance) or by intratracheal instillation of 1 mg (respiratory tract absorption). Plasma concentrations were monitored for 60 min. Plasma concentrations of HRP rose linearly with time after intratracheal instillation, reaching 236 +/- 51 ng/ml (mean +/- SE) at 60 min after instillation. HRP was cleared from the plasma rapidly after bolus injection. The elimination coefficient, k2, determined from the biphasic log normal plot, was 0.322 min-1. These data were used to estimate the kinetics of absorption across the respiratory epithelium. A single 3-hr exposure to an atmosphere containing 2.5 mg/m3 of submicrometer zinc oxide particles increased plasma concentration of HRP after intratracheal deposition (407 +/- 63 ng/ml at 60 min) and had no effect on plasma clearance (k2 = 0.342 min-1). Therefore plasma concentrations of HRP measured after intratracheal deposition can be used as a sensitive indicator to evaluate the effects of inhalation of a test atmosphere on epithelial permeability, if plasma clearance kinetics are not altered by the exposure.

Permeability studies of the labyrinth in the guinea pig placenta. I. Perfusion of fixatives and tracers into the fetal circulation.

A surgical procedure was developed for cannulation of umbilical vessels in the guinea pig placenta. This approach allows an administration of electron opaque tracers, perfusion of fixatives and injection of corrosion casting compounds with minimal disturbance to the fetus and the placenta.

Intracellular labelling of rat subthalamic neurones with horseradish peroxidase: computer analysis of dendrites and characterization of axon arborization.

Neurones of the rat subthalamic nucleus were identified by their response to cortical stimulation and then intracellularly labelled with horseradish peroxidase. After fixation, the brains were cut serially in sagittal plane and processed by the cobalt chloride-diaminobenzidine procedure. The morphology of nine of the twenty stained neurones strictly located inside the subthalamic nucleus is described by means of quantitative parameters following light-microscopic examination and three-dimensional computer reconstruction. They were all identified as Golgi type I neurones. The somata were ovoidal in shape. A mean of four dendritic stems arose from the soma and gave rise to a mean of 27 tips. The dendrites were thin with long and pedunculated spines. The dendritic fields were ellipsoidal in shape (100 x 600 x 300 micrometer) and were parallel to the principal plane of the nucleus. The dimensions of the dendritic fields are very close to those of the nucleus, and some dendrites cross its limits. The axons gave off two branches, one going caudally and the other rostrally. The caudal-going branch of the axon of one neurone, followed into the substantia nigra, divided into several collaterals coursing dorsoventrally. The rostral-going branch was never followed up to its termination. An intranuclear axonal collateral was observed in only one case. The present data are compared with those obtained from the primate subthalamic neurons. In spite of slight differences in the pattern of dendritic branching, the neurones are similar in both species. However, major differences in the internal organization of the dendritic fields are observed. Dendrites mixing with other neuronal populations were never observed in the primate. Moreover, the relative sizes of the dendritic fields and of the nucleus are strikingly different. This gives to the primate subthalamic nucleus specific and more precisely organized afferent connections.