Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.

Therapeutic efficacy of Nano-formulation of lactoperoxidase and lactoferrin via promoting immunomodulatory and apoptotic effects.

This study identifies promising potential of a novel and safer nanocombination of bovine milk lactoperoxidase (LPO) and lactoferrin (LF) to target breast cancer in vitro and in adult female albino rat model. Favorable selective anticancer effects of the prepared nanocombination were observed, in a dose-dependent manner, against both MCF-7 and MDA cell lines, sparing normal HFB-4 cells. The administration of LPO + LFNPs markedly improved the induced-breast cancer disorders, prolonged survival and reduced the values of serum TNF-α, IL1ß, CD4+, ALAT, ASAT, urea, creatinine, cholesterol and triglycerides with remarkable elevation in mammary SOD and GPx activity and GSH level. Moreover, the histopathological findings showed that LPO + LFNPs succeeded in prevention of mammary gland tumorigenesis. Superior efficacy of LPO + LFNPs was observed against pro-inflammatory cytokines through their anti-inflammatory and immunomodulatory properties. The treatment of LPO + LFNPs more significantly modulated the apoptosis and enhanced the expression of cell cycle regulator genes, which demonstrates a successful tumor therapy in vitro and in vivo. Therefore, this study provided evidence that the chemo-preventive feature of LPO + LFNPs may offer a novel alternative therapy for the treatment of breast cancer through enhances apoptosis pathway, improvement of immune response, reduction of inflammation and restoration of the impaired oxidative stress.

Augmenting apoptosis-mediated anticancer activity of lactoperoxidase and lactoferrin by nanocombination with copper and iron hybrid nanometals.

There is an urgent need in the medicinal fields to discover biocompatible nanoformulations with low cytotoxicity, which provide new strategies for promising therapies for several types of tumors. Bovine lactoperoxidase (LP) and lactoferrin (LF) have recently attracted attention in medicine for their antitumor activities with recognized safety pattern. Both LP and LF are suitable proteins to be coated or adsorbed to Cu and Fe nanometals for developing stable nanoformulations that boost immunity and strong anticancer effects. New nanometals of Cu and Fe NPs embedded in LP and LF forming novel nanocombinations of LP-CNPs and LF-FNPs had a spherical shape with an average nanosize of about 21 nm. The combination of LP-CNPs and LF-FNPs significantly exhibited the highest growth inhibitory efficacy, in terms of effectively lowering the half-maximal inhibitory concentration (IC50) values, against Caco-2, HepG2 and MCF7 cells comparing to nanometals, LP, LF and individual nanoproteins (LP-CNPs or LF-FNPs). The highest apoptotic effect of this nanocombination (LP-CNPs and LF-FNPs) was confirmed by the highest percentages of annexin-stained apoptotic cells and G0 population with the strongest alteration in the expression of two well-characterized apoptosis guards (p53 and Bcl-2) and the maximum suppression in the proliferation marker (Ki-67). Also, the in silico analysis predicted that LP-CNPs and LF-FNPs enhanced AMP-activated protein kinase (AMPK, p53 activator) activity and inhibited cancer migration-related proteases (cathepsin B and matrix metalloproteinase (MMP)-9). Our results offer for the first time that these novel nanocombinations of LP and LF were superior in their selectivity and apoptosis-mediating anticancer activity to Cu and Fe nanometals as well as the free form of these proteins or their individual nanoforms.

Epithelial chemerin-CMKLR1 signaling restricts microbiota-driven colonic neutrophilia and tumorigenesis by up-regulating lactoperoxidase.

Intestinal barrier immunity is essential for controlling gut microbiota without eliciting harmful immune responses, while its defect contributes to the breakdown of intestinal homeostasis and colitis development. Chemerin, which is abundantly expressed in barrier tissues, has been demonstrated to regulate tissue inflammation via CMKLR1, its functional receptor. Several studies have reported the association between increased expression of chemerin-CMKLR1 and disease severity and immunotherapy resistance in inflammatory bowel disease (IBD) patients. However, the pathophysiological role of endogenous chemerin-CMKLR1 signaling in intestinal homeostasis remains elusive. We herein demonstrated that deficiency of chemerin or intestinal epithelial cell (IEC)-specific CMKLR1 conferred high susceptibility to microbiota-driven neutrophilic colon inflammation and subsequent tumorigenesis in mice following epithelial injury. Unexpectedly, we found that lack of chemerin-CMKLR1 signaling specifically reduced expression of lactoperoxidase (LPO), a peroxidase that is predominantly expressed in colonic ECs and utilizes H2O2 to oxidize thiocyanates to the antibiotic compound, thereby leading to the outgrowth and mucosal invasion of gram-negative bacteria and dysregulated CXCL1/2-mediated neutrophilia. Importantly, decreased LPO expression was causally linked to aggravated microbiota-driven colitis and associated tumorigenesis, as LPO supplementation could completely rescue such phenotypes in mice deficient in epithelial chemerin-CMKLR1 signaling. Moreover, epithelial chemerin-CMKLR1 signaling is necessary for early host defense against bacterial infection in an LPO-dependent manner. Collectively, our study reveals that the chemerin-CMKLR1/LPO axis represents an unrecognized immune mechanism that potentiates epithelial antimicrobial defense and restricts harmful colonic neutrophilia and suggests that LPO supplementation may be beneficial for microbiota dysbiosis in IBD patients with a defective innate antimicrobial mechanism.

Glutathione utilization protects Streptococcus pneumoniae against lactoperoxidase-derived hypothiocyanous acid.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia, resulting in more than one million deaths each year worldwide. This pathogen generates large amounts of hydrogen peroxide (H2O2), which will be converted to hypothiocyanous acid (HOSCN) by lactoperoxidase (LPO) in the human respiratory tract. S. pneumoniae has been shown to be more resistant to HOSCN than some bacteria, and sensitizing S. pneumoniae to HOSCN may be a novel treatment strategy for combating this deadly pathogen. In this study we investigated the role of the low molecular weight thiol glutathione in HOSCN resistance. S. pneumoniae does not synthesize glutathione but imports it from the environment via an ABC transporter. Upon treatment of S. pneumoniae with HOSCN, bacterial glutathione was reversibly oxidized in a time- and dose-dependent manner, and intracellular proteins became glutathionylated. Bacterial death was observed when the reduced glutathione pool dropped below 20%. A S. pneumoniae mutant unable to import glutathione (ΔgshT) was more readily killed by exogenous HOSCN. Furthermore, bacterial growth in the presence of LPO converting bacterial H2O2 to HOSCN was significantly impeded in mutants that were unable to import glutathione, or mutants unable to recycle oxidized glutathione (Δgor). This research highlights the importance of glutathione in protecting S. pneumoniae from HOSCN. Limiting glutathione utilization by S. pneumoniae may be a way to limit colonization and pathogenicity.

Milk Proteins-Their Biological Activities and Use in Cosmetics and Dermatology.

Milk and colostrum have high biological potential, and due to their natural origin and non-toxicity, they have many uses in cosmetics and dermatology. Research is ongoing on their potential application in other fields of medicine, but there are still few results; most of the published ones are included in this review. These natural products are especially rich in proteins, such as casein, ß-lactoglobulin, α-lactalbumin, lactoferrin, immunoglobulins, lactoperoxidase, lysozyme, and growth factors, and possess various antibacterial, antifungal, antiviral, anticancer, antioxidant, immunomodulatory properties, etc. This review describes the physico-chemical properties of milk and colostrum proteins and the natural functions they perform in the body and compares their composition between animal species (cows, goats, and sheep). The milk- and colostrum-based products can be used in dietary supplementation and for performing immunomodulatory functions; they can enhance the effects of certain drugs and can have a lethal effect on pathogenic microorganisms. Milk products are widely used in the treatment of dermatological diseases for promoting the healing of chronic wounds, hastening tissue regeneration, and the treatment of acne vulgaris or plaque psoriasis. They are also increasingly regarded as active ingredients that can improve the condition of the skin by reducing the number of acne lesions and blackheads, regulating sebum secretion, ameliorating inflammatory changes as well as bestowing a range of moisturizing, protective, toning, smoothing, anti-irritation, whitening, soothing, and antiaging effects.

Dual oxidase 1 promotes antiviral innate immunity.

Dual oxidase 1 (DUOX1) is an NADPH oxidase that is highly expre-ssed in respiratory epithelial cells and produces H2O2 in the airway lumen. While a line of prior in vitro observations suggested that DUOX1 works in partnership with an airway peroxidase, lactoperoxidase (LPO), to produce antimicrobial hypothiocyanite (OSCN-) in the airways, the in vivo role of DUOX1 in mammalian organisms has remained unproven to date. Here, we show that Duox1 promotes antiviral innate immunity in vivo. Upon influenza airway challenge, Duox1-/- mice have enhanced mortality, morbidity, and impaired lung viral clearance. Duox1 increases the airway levels of several cytokines (IL-1ß, IL-2, CCL1, CCL3, CCL11, CCL19, CCL20, CCL27, CXCL5, and CXCL11), contributes to innate immune cell recruitment, and affects epithelial apoptosis in the airways. In primary human tracheobronchial epithelial cells, OSCN- is generated by LPO using DUOX1-derived H2O2 and inactivates several influenza strains in vitro. We also show that OSCN- diminishes influenza replication and viral RNA synthesis in infected host cells that is inhibited by the H2O2 scavenger catalase. Binding of the influenza virus to host cells and viral entry are both reduced by OSCN- in an H2O2-dependent manner in vitro. OSCN- does not affect the neuraminidase activity or morphology of the influenza virus. Overall, this antiviral function of Duox1 identifies an in vivo role of this gene, defines the steps in the infection cycle targeted by OSCN-, and proposes that boosting this mechanism in vivo can have therapeutic potential in treating viral infections.

Deletion of the lactoperoxidase gene causes multisystem inflammation and tumors in mice.

Strongly oxidative H2O2 is biologically important, but if uncontrolled, would lead to tissue injuries. Lactoperoxidase (LPO) catalyzes the redox reaction of reducing highly reactive H2O2 to H2O while oxidizing thiocyanate (SCN-) to relatively tissue-innocuous hypothiocyanite (OSCN-). SCN- is the only known natural, effective reducing-substrate of LPO; humans normally derive SCN- solely from food. While its enzymatic mechanism is understood, the actual biological role of the LPO-SCN- system in mammals remains unestablished. Our group previously showed that this system protected cultured human cells from H2O2-caused injuries, a basis for the hypothesis that general deficiency of such an antioxidative mechanism would lead to multisystem inflammation and tumors. To test this hypothesis, we globally deleted the Lpo gene in mice. The mutant mice exhibited inflammation and lesions in the cardiovascular, respiratory, digestive or excretory systems, neuropathology, and tumors, with high incidence. Thus, this understudied LPO-SCN- system is an essential protective mechanism in vivo.

Duration of effect of Biotène spray in patients with symptomatic dry mouth: A pilot study.

OBJECTIVES: The objective of this study was to assess the duration of effect of a single dose of Biotène Moisturizing Spray on xerostomia compared to water spray. STUDY DESIGN: This double-blind randomized controlled crossover trial compared the duration of effect of 2 agents on relieving xerostomia in adult patients recruited through convenience sampling. Following a xerostomia questionnaire, qualifying patients with an unstimulated whole saliva flow rate of ≤0.20 mL/min rated their baseline level of discomfort from oral dryness and received a single dose (3 sprays) of Biotène Moisturizing Spray or water (active control). Patients indicated their level of oral discomfort every 15 min and the precise time when relief ceased. After a minimum 48-h washout, patients repeated the exercise with the alternative product. RESULTS: The baseline severity of discomfort from oral dryness among qualifying patients was significantly related to their level of hyposalivation (P = .001). The mean duration of effect of Biotène Moisturizing Spray was 27 ± 25 min, which was not significantly different from that for water (26 ± 25 min; P = .88; n = 25). CONCLUSION: Biotène Moisturizing Spray and water spray had variable durations of effect averaging approximately 30 min. The results of this pilot study provide guidance regarding anticipated usage and dispensing needs for patients with objective xerostomia. ClinicalTrials.gov NCT03663231.

Effects of oral administration of colostrum whey in peripartum goat on antimicrobial peptides in postpartum milk.

The present study was conducted to examine whether colostrum supplementation in peripartum goats increases the antimicrobial peptides in their milk. Goats were orally administered 2 ml of colostrum whey products (colostrum group) or water (control group) daily, from 2 weeks before until 2 weeks after kidding. Body weights of mothers and kids were measured. Blood, milk, and fecal samples were collected from the mothers, and blood samples were collected from the kids. Concentrations of milk antimicrobial peptides (beta-defensin, cathelicidin, lactoferrin, S100A7, lactoperoxidase, and immunoglobulin A [IgA]) were determined. IgA and nutritional parameters (glucose, total cholesterol, triglyceride, ketone bodies, and non-esterified fatty acids) were also determined in the blood of mothers and kids. Milk IgA and lactoferrin concentrations were higher in the colostrum group than in the control group. Conversely, lower milk concentrations of S100A7 were observed in the colostrum group than that in the control group. Plasma IgA concentrations were higher for kids from the colostrum group than for those from the control group. These results suggest that oral administration of colostrum in pregnant goats increases IgA concentration in postpartum milk, which can subsequently improve the health of their kids.

Milk lactoperoxidase decreases ID1 and ID3 expression in human oral squamous cell carcinoma cell lines.

Milk consumption may modify the risk of squamous cell carcinoma. The role of milk to modulate the gene expression in oral squamous cell carcinoma cells has not been investigated so far. Here, HSC2 oral squamous carcinoma cells were exposed to an aqueous fraction of human milk and a whole-genome array was performed. Among the genes that were significantly reduced by human and cow milk were the DNA-binding protein inhibitor 1 (ID1), ID3 and Distal-Less Homeobox 2 (DLX2) in HSC2 cells. Also, in TR146 oral squamous carcinoma cells, there was a tendency towards a decreased gene expression. Upon size fractionation, lactoperoxidase but not lactoferrin and osteopontin was identified to reduce ID1 and ID3 in HSC2 cells. Dairy products and hypoallergenic infant formula failed to decrease the respective genes. These data suggest that milk can reduce the expression of transcription factors in oral squamous carcinoma cells.

The inhibition of lactoperoxidase catalytic activity through mesna (2-mercaptoethane sodium sulfonate).

Here, we show that mesna (sodium-2-mercaptoethane sulfonate), primarily used to prevent nephrotoxicity and urinary tract toxicity caused by chemotherapeutic agents such as cyclophosphamide and ifosfamide, modulates the catalytic activity of lactoperoxidase (LPO) by binding tightly to the enzyme, functioning either as a one electron substrate for LPO Compounds I and II, destabilizing Compound III. Lactoperoxidase is a hemoprotein that utilizes hydrogen peroxide (H2O2) and thiocyanate (SCN-) to produce hypothiocyanous acid (HOSCN), an antimicrobial agent also thought to be associated with carcinogenesis. Our results revealed that mesna binds stably to LPO within the SCN- binding site, dependent of the heme iron moiety, and its combination with LPO-Fe(III) is associated with a disturbance in the water molecule network in the heme cavity. At low concentrations, mesna accelerated the formation and decay of LPO compound II via its ability to serve as a one electron substrate for LPO compounds I and II. At higher concentrations, mesna also accelerated the formation of Compound II but it decays to LPO-Fe(III) directly or through the formation of an intermediate, Compound I*, that displays characteristic spectrum similar to that of LPO Compound I. Mesna inhibits LPO's halogenation activity (IC50 value of 9.08 µM) by switching the reaction from a 2e- to a 1e- pathway, allowing the enzyme to function with significant peroxidase activity (conversion of H2O2 to H2O without generation of HOSCN). Collectively, mesna interaction with LPO may serve as a potential mechanism for modulating its steady-state catalysis, impacting the regulation of local inflammatory and infectious events.

In vitro effects of standard antioxidants on lactoperoxidase enzyme-A molecular docking approach.

Lactoperoxidase (LPO), an antioxidant enzyme, is a natural antimicrobial system that eliminates the harmful effects of microorganisms in milk. It has a wide range of applications and is also preferred in cosmetic and clinical applications, as well as used in foods. The use of antioxidants is well recognized in the food and feed industries to improve the shelf life of products. This study aimed to determine the in vitro inhibition effects of Trolox, α-tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, and propyl gallate, which are commonly used as antioxidants in food and pharmaceutical products. For this purpose, LPO was first purified in a single step using sepharose-4B-l-tyrosine-sulfanilamide affinity gel chromatography. Also, some inhibition parameters, including half-maximal inhibitory concentration (IC50 ), Ki values, and inhibition types, were calculated for each antioxidant molecule. The IC50 values of these molecules, which exhibited competitive inhibition, varied between 377.7 and 3397.8 nM. Molecular docking studies were also performed for all compounds. According to the binding scores, α-tocopherol was shown to exhibit the most effective inhibitor property (IC50 : 377.7 nM and Ki : 635.8 ± 16.8 nM) among the standard antioxidants used in this study. Inhibiting the LPO activity by standard antioxidants results in the weakening of the immune system during lactation, which is important for metabolism.

Eficiência da pasteurização lenta do leite de cabra em diferentes binômios tempo/temperatura / Efficiency of slow pasteurization of goat milk in the different time/temperature binomials

O presente estudo analisou um binômio de tempo-temperatura alternativo para ser utilizado na pasteurização lenta sobre a inativação da fosfatase alcalina no leite caprino. Sua eficiência foi demonstrada pela contagem padrão em placas, e foi feita a comparação no processamento de leite refrigerado e congelado. Foram utilizados 18 tratamentos em leite caprino cru (nove em leite refrigerado e nove em leite congelado). Estes foram acondicionados em frascos de 300 mL, pasteurizados a 60, 63 e 65°C durante 10-20-30 minutos, e testadas às enzimas fosfatase alcalina e peroxidase. A contagem padrão em placas (CPP) e coliformes a 35 e 45°C foi feita nas amostras cruas e em cada tratamento, em duplicata. Após a pasteurização, todos os tratamentos apresentaram: não crescimento de microrganismos mesófilos, coliformes com <0,3 NMP/mL, prova de fosfatase negativa e peroxidase positiva. A pasteurização foi eficiente para melhorar a qualidade microbiológica do leite tanto refrigerado quanto congelado. Todos os binômios avaliados apresentaram resultados satisfatórios para alcançar os parâmetros preconizados em legislação, sugerindo-se o menor binômio (60°C por 10 min). Não houve diferença entre as formas de armazenamento das amostras: refrigerada ou congelada. (AU)

The objective of this study was to investigate an alternative time-temperature binomial to be used in the slow pasteurization on the alkaline phosphatase inactivation in the goat milk. Its efficiency was demonstrated with the standard counting in plates, and also refrigerated and the frozen milks processing were compared. Eighteen treatments were used in the raw goat milk (nine refrigerated milk and nine frozen milk). They were packed in 300 mL-flasks, pasteurized at 60-63-65°C for 10, 20, 30 minutes, and then tested for alkaline phosphatase and peroxidase enzymes. The standard counts in plates (CPP) and coliforms at 35°C and 45°C were performed in the raw samples and in the every treatment, in duplicate. After the pasteurization process, all of the treatments showed: no growth of mesophilic microorganisms, coliforms with <0.3 MPN / mL, negative phosphatase and positive peroxidase tests. The pasteurization was efficient to improve the microbiological quality of the milk either refrigerated or frozen. All of the evaluated binomials presented satisfactory results to reach the recommended parameters preconized in the legislation, suggesting the smaller binomial (60°C for 10 min). There was no difference between the samples storage form, either refrigerated or frozen. (AU)

Eficiência da pasteurização lenta do leite de cabra em diferentes binômios tempo/temperatura / Efficiency of slow pasteurization of goat milk in the different time/temperature binomials

O presente estudo analisou um binômio de tempo-temperatura alternativo para ser utilizado na pasteurização lenta sobre a inativação da fosfatase alcalina no leite caprino. Sua eficiência foi demonstrada pela contagem padrão em placas, e foi feita a comparação no processamento de leite refrigerado e congelado. Foram utilizados 18 tratamentos em leite caprino cru (nove em leite refrigerado e nove em leite congelado). Estes foram acondicionados em frascos de 300 mL, pasteurizados a 60, 63 e 65°C durante 10-20-30 minutos, e testadas às enzimas fosfatase alcalina e peroxidase. A contagem padrão em placas (CPP) e coliformes a 35 e 45°C foi feita nas amostras cruas e em cada tratamento, em duplicata. Após a pasteurização, todos os tratamentos apresentaram: não crescimento de microrganismos mesófilos, coliformes com <0,3 NMP/mL, prova de fosfatase negativa e peroxidase positiva. A pasteurização foi eficiente para melhorar a qualidade microbiológica do leite tanto refrigerado quanto congelado. Todos os binômios avaliados apresentaram resultados satisfatórios para alcançar os parâmetros preconizados em legislação, sugerindo-se o menor binômio (60°C por 10 min). Não houve diferença entre as formas de armazenamento das amostras: refrigerada ou congelada.

The objective of this study was to investigate an alternative time-temperature binomial to be used in the slow pasteurization on the alkaline phosphatase inactivation in the goat milk. Its efficiency was demonstrated with the standard counting in plates, and also refrigerated and the frozen milks processing were compared. Eighteen treatments were used in the raw goat milk (nine refrigerated milk and nine frozen milk). They were packed in 300 mL-flasks, pasteurized at 60-63-65°C for 10, 20, 30 minutes, and then tested for alkaline phosphatase and peroxidase enzymes. The standard counts in plates (CPP) and coliforms at 35°C and 45°C were performed in the raw samples and in the every treatment, in duplicate. After the pasteurization process, all of the treatments showed: no growth of mesophilic microorganisms, coliforms with <0.3 MPN / mL, negative phosphatase and positive peroxidase tests. The pasteurization was efficient to improve the microbiological quality of the milk either refrigerated or frozen. All of the evaluated binomials presented satisfactory results to reach the recommended parameters preconized in the legislation, suggesting the smaller binomial (60°C for 10 min). There was no difference between the samples storage form, either refrigerated or frozen.

Flaminal® versus Flamazine® in the treatment of partial thickness burns: A randomized controlled trial on clinical effectiveness and scar quality (FLAM study).

Although partial thickness burns are the most frequently reported burn injuries, there is no consensus on the optimal treatment. The objective of this study was to compare the clinical effectiveness and scar quality of Flaminal® Forte to silver sulfadiazine (Flamazine®) in the treatment of partial thickness burns. In this two-arm open label multicenter randomized controlled trial, adult patients with acute partial thickness burns and an affected total body surface area of less than 30% were randomized between Flaminal® Forte and Flamazine® and followed for 12 months. Dressing changes in the Flamazine® group were performed daily, and in the Flaminal® group during the first 3 days post burn and thereafter every other day until complete wound healing or surgery. Forty-one patients were randomly allocated to Flaminal® Forte and 48 patients to Flamazine®. The primary outcome was time to wound healing, which did not differ between the groups: median 18 days with Flaminal® Forte (range 8-49 days) versus 16 days with Flamazine® (range 7-48 days; p = 0.24). Regarding the secondary outcomes during hospital admission, there were no statistically significant differences between the groups concerning need for surgery, pain scores, pruritus, or pain-related and anticipatory anxiety. More patients in the Flaminal® group developed wound colonization (78% versus 32%, p < 0.001), but the treatment groups did not differ regarding the incidence of local infections and use of systemic antibiotics. In terms of scar quality, no statistically significant differences between both treatment groups were found regarding subjective scar assessment (Patient and Observer Scar Assessment Scale (POSAS)), scar melanin and pigmentation (DermaSpectrometer®), and scar elasticity and maximal extension (Cutometer®) during 12 month postburn. In conclusion, time to wound healing did not differ, but the use of Flaminal® Forte seemed favorable because less dressing changes are needed which lowers the burden of wound care.

Effect of hyperbaric oxygenation on the expression of glutathione peroxidase 4 and lactoperoxidase genes in the lung of isogenic mice after ischemia/reperfusion injury in the small bowel

Abstract Purpose: To evaluate the effect of hyperbaric oxygenation (HBO) on the expression of the genes antioxidant glutathione peroxidase 4 (Gpx4) and lactoperoxidase (Lpo) in the lung of mice subjected to intestinal ischemia and reperfusion (IIR). Methods: Control group (CG) in which were subjected to anesthesia, laparotomy and observation for 120 minutes; an ischemia and reperfusion group (IRG) subjected to anesthesia, laparotomy, small bowel ischemia for 60 minutes and reperfusion for 60 minutes; and three groups treated with HBO during ischemia (HBOG + I), during reperfusion (HBOG + R) and during ischemia and reperfusion (HBOG + IR). Studied 84 genes of oxidative stress by the method (RT-qPCR). Genes with expression levels three times below or above the threshold cycle were considered significantly hypoexpressed or hyperexpressed, respectively (Student's t-test p<0.05). Results: Gpx4 and Lpo were hiperexpressed on IRG, showing a correlation with these genes with lung oxidative stress. Treated with HBO, there was a significant reduction on genic expression on HBOG+I. Conclusion: Hyperbaric oxygenation showed to be associated with decreased expression of these antioxidant genes, suggesting a beneficial effect on the mechanism of pulmonary oxidative stress whenever applied during the ischemia.

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines.

Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes.

Reactivation of peroxidase activity in human saliva samples by polyphenols.

OBJECTIVES: The enzyme lactoperoxidase (LPO), which is released into several body fluids like saliva, is an essential part to maintain the oral bacterial homeostasis by catalysing the oxidation of thiocyanate (SCN-) to hypo-thiocyanite (-OSCN). The formation of unreactive redox intermediates (like Compound II) leads to a decreased pseudo-halogenating enzyme activity, which is associated with a higher risk for oral infections. According to former studies with bovine LPO selected flavonoids were tested in respect to their potential to reactivate the enzymatic activity in a more physiological, human salivary system. DESIGN: Saliva samples from healthy donors were collected and characterized by using several gel staining methods and immunoblotting. Afterwards kinetic measurements were performed by applying the TNB-assay to evaluate the pseudo-halogenating salivary peroxidase (SAPX) activity. The measurements were performed in the presence of excess H2O2 to simulate pro-inflammatory conditions. Moreover selected flavonoids or an ethanolic extract of Tormentillae rhizoma were applied to test their regenerating effect on the LPO-derived -OSCN production. RESULTS: Despite the complex protein composition of the collected saliva samples, an SAPX-derived pseudo-halogenating activity could be identified. The -OSCN regenerating effects of the tested polyphenols were completely comparable to previous in vitro experiments with bovine LPO. Thus, we could show that phenolic substances are suitable to regenerate the peroxidase activity in human saliva samples after H2O2-induced inactivation. CONCLUSION: The studies provide new insights into the effect of pharmaceutical relevant polyphenols on salivary peroxidase activity and thus, suggest this enzyme as a new target for the prevention and therapy of oral inflammatory diseases.

Efficiency of novel nanocombinations of bovine milk proteins (lactoperoxidase and lactoferrin) for combating different human cancer cell lines.

Bovine lactoperoxidase (LPO) and lactoferrin (LF) are suitable proteins to be loaded or adsorbed to chitosan nanoparticles (NPs) for preparing stable nanoformulations with potent anticancer activity. In the present study, nanocombinations of LPO and LF revealed improvement in their stability and activity compared to single (free or nanoformulated) bovine proteins. The coating or loading of LPO-loaded NPs with LF resulted in the highest synergistic cytotoxicity effect against Caco-2, HepG-2, MCF-7 and PC-3 cells in comparison with other NPs and free proteins without causing toxicity toward normal cells. This synergistic improvement in the anticancer activity was apoptosis-dependent that was confirmed by severe alterations in cellular morphology, high percentage of annexin-stained cells and sub-G1 populations as well as nuclear staining with orange fluorescence of treated cancer cells. Additionally, significant alterations in the expression of well characterized cellular proliferation and apoptosis guards (NF-κB, Bcl-2 and p53) in these NPs-treated cancer cells compared to 5-fluorouracil (5-FU) treated cells. Our findings provide for the first time that these new synergistic nanoformulated forms of LPO and LF were superior in their selective apoptosis-mediating anticancer effect than free form of these proteins and 5-FU. LF coating or loading of LPO-loaded NPs present as promising therapy for cancer.