Topical odorant application of the specific olfactory receptor OR2AT4 agonist, Sandalore® , improves telogen effluvium-associated parameters.

BACKGROUND: Human hair follicles (HFs) express the olfactory receptor (OR)2AT4, which is selectively stimulated by the synthetic sandalwood-like odorant, Sandalore® . In organ-cultured, human scalp HFs, Sandalore® prolongs anagen and suppresses apoptosis by up-regulating intrafollicular IGF-1 mediated signaling. AIMS: The objective of this study is to demonstrate whether effects of Sandalore® observed ex vivo translate into a clinically relevant effect in patients with telogen effluvium. PATIENTS/METHODS: In a randomized, double-blinded, placebo-controlled, clinical trial, 60 female volunteers (18-65 years) affected by telogen effluvium received over a period of 24 weeks treatment with either 1% Sandalore® solution (n = 30) or placebo (identically smelling, but non-OR2AT4 activating sandalwood oil n = 30). The study read-out parameters were the degree of hair shedding, hair volume, terminal/vellus hair ratio, anagen/catagen-telogen ratio, and patient self-assessment. RESULTS: Sandalore® 1% ameliorated clinical signs of telogen effluvium, namely it reduced hair shedding, and increased hair volume and the percentage of anagen HFs, the latter two parameters significantly more than placebo when changes were calculated to baseline. Sandalore® also increased the ratio of terminal/vellus hairs at week 8. Most of the anti-hair shedding effects were seen after 8 weeks and maintained at week 24. Patient questionnaire showed that verum group patients were more satisfied than the placebo group in regard to the overall results. CONCLUSION: This clinical trial supports previous findings of anagen-prolonging effects of Sandalore® ex vivo with similar results now reproduced in clinical practice. It also provides proof-of-principle that a topically applied cosmetic odorant acting through HF olfactory receptors can be a therapeutic alternative to treat hair loss disorders characterized by excessive hair shedding such as telogen effluvium.

A New Selective PPARγ Modulator Inhibits Triglycerides Accumulation during Murine Adipocytes' and Human Adipose-Derived Mesenchymal Stem Cells Differentiation.

Understanding the molecular basis of adipogenesis is vital to identify new therapeutic targets to improve anti-obesity drugs. The adipogenic process could be a new target in the management of this disease. Our aim was to evaluate the effect of GMG-43AC, a selective peroxisome proliferator-activated receptor γ (PPARγ) modulator, during adipose differentiation of murine pre-adipocytes and human Adipose Derived Stem Cells (hADSCs). We differentiated 3T3-L1 cells and primary hADSCs in the presence of various doses of GMG-43AC and evaluated the differentiation efficiency measuring lipid accumulation, the expression of specific differentiation markers and the quantification of accumulated triglycerides. The treatment with GMG-43AC is not toxic as shown by cell viability assessments after the treatments. Our findings demonstrate the inhibition of lipid accumulation and the significant decrease in the expression of adipocyte-specific genes, such as PPARγ, FABP-4, and leptin. This effect was long lasting, as the removal of GMG-43AC from culture medium did not allow the restoration of adipogenic process. The above actions were confirmed in hADSCs exposed to adipogenic stimuli. Together, these results indicate that GMG-43AC efficiently inhibits adipocytes differentiation in murine and human cells, suggesting its possible function in the reversal of adipogenesis and modulation of lipolysis.

Scalp bacterial shift in Alopecia areata.

The role of microbial dysbiosis in scalp disease has been recently hypothesized. However, little information is available with regards to the association between microbial population on the scalp and hair diseases related to hair growth. Here we investigated bacterial communities in healthy and Alopecia areata (AA) subjects. The analysis of bacterial distribution at the genus level highlighted an increase of Propionibacterium in AA subjects alongside a general decrease of Staphylococcus. Analysis of log Relative abundance of main bacterial species inhabiting the scalp showed a significant increase of Propionibacterium acnes in AA subjects compared to control ones. AA scalp condition is also associated with a significant decrease of Staphylococcus epidermidis relative abundance. No significant changes were found for Staphylococcus aureus. Therefore, data from sequencing profiling of the bacterial population strongly support a different microbial composition of the different area surrounded hair follicle from the epidermis to hypodermis, highlighting differences between normal and AA affected the scalp. Our results highlight, for the first time, the presence of a microbial shift on the scalp of patients suffering from AA and gives the basis for a larger and more complete study of microbial population involvement in hair disorders.

Randomized controlled trial on a PRP-like cosmetic, biomimetic peptides based, for the treatment of alopecia areata.

Background: Alopecia areata (AA) is a non-scarring auto-immune hair disorder. Recent researches explained the role of growth factors (GFs) in hair follicle cycling. The main reservoir of GFs are alpha-granules of platelets and novel procedures have been implemented aimed at collecting platelet-rich plasma (PRP). PRP has been safely implemented in many medical applications and has also been successfully used as alternative cell-based therapy for the treatment of hair growth disorders, among which also AA. Objectives: By means of a randomized double-blinded, placebo and active-controlled, parallel group study we have studied the efficacy of a cosmetic product (named TR-M-PRP plus) comprising biomimetic peptides specific for hair growth, mimicking PRP composition for the treatment of AA. Subjects were treated for three months and evaluated, at the end of the study and after one month of follow-up, as regards hair growth using SALT score. Results: TR-M-PRP plus-like topic produced a statistically significant (p < .001) clinical improvement in SALT score after 3 months of therapy, compared to baseline. Hair growth results further improved after 1 month of follow-up. Conclusions: This clinical investigation suggests that the biotechnological designed PRP-like cosmetic could represent a valid and safer alternative to autologous PRP for the treatment of AA.

A Single-Arm, Open-Label, Phase IV Study to Evaluate the Efficacy of a Topical Formulation for Hyperkeratotic Actinic Keratosis Lesions.

INTRODUCTION: Actinic keratosis (AKs) are epidermal lesions that commonly occur in skin exposed to chronic cumulative UV irradiation. Untreated AK lesions can advance to squamous cell carcinoma. Current treatments of AK have many shortcomings; for instance, not all treatments can be used for the hyperkeratotic form of AK. The aim of this study was to test the efficacy and tolerability of a topical product containing 2,4,6-octatrienoic acid and urea for the treatment of hyperkeratotic AK lesions. METHODS: Forty male and female subjects with at least two hyperkeratotic AK lesions were enrolled in this single-arm, open-label phase IV study. The product was applied twice daily for two consecutive months. The efficacy endpoints were the reductions in the mean number of AK lesions per subject from baseline (T0) to the end of the trial (T1) and to three months after the end of the treatment period (T2). RESULTS: At T0, the mean (SD) number of lesions per subject was 3.65 (1.25). At the end of the treatment period (T1), this number had dropped (significantly, p < 0.0001) by 83.56%. The mean number of lesions per subject then decreased by 41.47% (p < 0.0001) between T1 and the three-month follow-up visit (T2). Complete elimination of lesions had occurred in 57.5% of the subjects at T1, and 82.5% (55% who had remained completely clear of lesions since T1, and 27.5% who had fully eliminated their lesions during the period from T1 to T2) at T2. No side effects were reported. CONCLUSION: The application of a topical combination of 2,4,6-octatrienoic acid and urea twice daily for 60 consecutive days is a safe and effective treatment for hyperkeratotic AK lesions. FUNDING: Giuliani SpA.

New multi-targeting strategy in hair growth promotion: in vitro and in vivo studies.

BACKGROUND: Considering the importance of hair in our modern society and the impact of hair loss, the efforts of researchers are addressed to better understand the mechanisms behind the hair cycle regulation and dysregulation. Because hair loss is multifactorial, differenced and new approaches are required. In particular we addressed our attention to two recently identified targets in hair cycling and growth control: olfactory receptor and autophagy. The aim of the study was to evaluate: the possible pro-autophagic effect of N1-methylspermidine (a spermidine analogue) in vitro and, in a double blind clinical trial, the safety and efficacy of topical daily application of a lotion containing N1-methylspermidine and Sandalore®. METHODS: Autophagic modulation by N1-methylspermidine was monitored in vitro by LC3 and p62 fluorescent signal cell line. Topical daily application of the lotion was tested in 60 male and female subjects with chronic telogen effluvium by means of non-invasive objective evaluation. RESULTS: The results obtained by in vitro tests showed the capacity of N1-methylspermidine to increase autophagic process while the clinical trials performed confirmed the safety and anti hair loss efficacy of the lotion reporting a reduction of hair loss (modified wash test) and hair growth stimulation as evaluated by hair density, hair shaft diameter, % of anagen hair and Hair Mass Index increase after 3 months of treatment. The lotion efficacy remained statistically significant for the above-mentioned parameters, with the exception of hair lost during wash, also 3 months after the end of treatment. CONCLUSIONS: Based on the obtained results, the daily use of the N1-methylspermidine and Sandalore®-based lotion is efficient to counteract hair loss and increase hair growth by a multifunctional targeting approach.

IL-17 inhibition: is it the long-awaited savior for alopecia areata?

Interleukin-17 (IL-17) has been implicated in the pathogenesis of a large number of inflammatory and autoimmune conditions, including skin disorders such as psoriasis. Recently, much data have accumulated on the possible role of IL-17 in the pathogenesis of alopecia areata (AA). In this review, the available information on the connection between AA and IL-17 is described. While IL-17 levels are consistently reported to be elevated in the serum and lesional skin of AA patients, there is no clear connection between IL-17 levels and disease severity or duration. Some evidence has suggested an association between IL-17 and an early-onset disease, although this awaits further confirmation. While there is enough information to support clinical trials with IL-17-targeted treatments, it is possible that they will be effective only in a subset of AA patients. Further studies are warranted to better delineate the exact role of IL-17 in AA pathogenesis.

Drug Treatment for Androgenetic Alopecia: First Italian Questionnaire Survey on What Dermatologists Think about Finasteride.

INTRODUCTION: Treatment with finasteride 1 mg/day represents the therapy of choice for androgenetic alopecia (AGA). We investigated how Italian dermatologists approach use of finasteride for treatment of AGA and common side effects reported by patients. METHODS: A tablet-based survey was conducted from February 2017 to January 2018 in Italy to investigating use of 1 mg/day finasteride in the treatment of AGA. Approximately 1153 Italian dermatologists were surveyed about prescription frequency, therapy duration, treatment practices, and side effects eventually reported. RESULTS: Dermatologists considered treatment with 1 mg/day finasteride to be the most efficacious treatment for AGA, as reflecting by its long-term (5 years) prescription. Data on sexual side effects from our survey are in line with previous scientific evidence, especially regarding loss of libido, erectile dysfunction, and problems with ejaculation, but also in the psychological sphere and regarding physical impairments such as myalgia and loss of muscle tone. CONCLUSIONS: This is the first preliminary observational study on how Italian dermatologists approach use of finasteride to treat AGA. Although side effects have been reported, especially in the sexual sphere, lack of alternative treatments with the same efficacy leads dermatologists to prescribe 1 mg/day finasteride with a tendency to prolong therapy in the long term. FUNDING: Giuliani S.p.A.

A spermidine-based nutritional supplement prolongs the anagen phase of hair follicles in humans: a randomized, placebo-controlled, double-blind study.

BACKGROUND: Spermidine has been shown both in vitro and in mice models to have an anagen-prolonging effect on hair follicles (HFs). OBJECTIVES: To evaluate the effects of a spermidine-based nutritional supplement on the anagen phase of HFs in healthy human subjects in a randomized, double-blind, placebo-controlled trial. METHODS: One hundred healthy males and females were randomized to receive a tablet containing a spermidine-based nutritional supplement or a placebo once daily for 90 days. At the beginning and the end of the treatment period, 100 HFs were plucked and subjected to microscopic evaluation to determine the number of anagen V-VI HFs, and immunohistochemical examination was performed to quantify the Ki-67 and c-Kit levels in the hair bulbs. Pull test was performed after three and six months. RESULTS: The spermidine-based nutritional supplement increased the number of anagen V-VI HFs after three months of treatment, accompanied by increased Ki-67, a marker for cellular proliferation, and decreased c-Kit, a marker for apoptosis, levels. All results were also significantly better when compared to the placebo group. The pull test remained negative after six months in all patients receiving the spermidine supplement, while 68% of the subjects in the placebo group had a positive pull test. CONCLUSIONS: This preliminary study shows that a spermidine-based nutritional supplement can prolong the anagen phase in humans, and therefore might be beneficial for hair loss conditions. Further studies are needed to evaluate its effects in specific different clinical settings.

Synthesis and Biological Evaluation of New Natural Phenolic (2E,4E,6E)-Octa-2,4,6-trienoic Esters.

In the present study the esterification of the OH groups of resveratrol, caffeic acid, ferulic acid, and ß-sitosterol with an antioxidant polyconjugated fatty acid, (2E,4E,6E)-octa-2,4,6-trienoic acid, was achieved. As the selective esterification of OH groups of natural compounds can affect their biological activity, a selective esterification of resveratrol and caffeic acid was performed by an enzymatic approach. The new resulting compounds were characterized spectroscopically (FT-IR, NMR mono, and bidimensional techniques); when necessary the experimental data were integrated by quantum chemical calculations. The antioxidant, anti-inflammatory and proliferative activity was evaluated. The good results encourage the use of these molecules as antioxidant and/or anti-inflammatory agents in dermocosmetic application.

Exploitation of grape marc as functional substrate for lactic acid bacteria and bifidobacteria growth and enhanced antioxidant activity.

This study aimed at using grape marc for the growth of lactic acid bacteria and bifidobacteria with the perspective of producing a functional ingredient having antioxidant activity. Lactobacillus plantarum 12A and PU1, Lactobacillus paracasei 14A, and Bifidobacterium breve 15A showed the ability to grow on grape marc (GM) based media. The highest bacterial cell density (>9.0 CFU/g) was found in GM added of 1% of glucose (GMG). Compared to un-inoculated and incubated control fermented GMG showed a decrease of carbohydrates and citric acid together with an increase of lactic acid. The content of several free amino acids and phenol compounds differed between samples. Based on the survival under simulated gastro-intestinal conditions, GMG was a suitable carrier of lactic acid bacteria and bifidobacteria strains. Compared to the control, cell-free supernatant (CFS) of fermented GMG exhibited a marked antioxidant activity in vitro. The increased antioxidant activity was confirmed using Caco-2 cell line after inducing oxidative stress, and determining cell viability and radical scavenging activity through MTT and DCFH-DA assays, respectively. Supporting these founding, the SOD-2 gene expression of Caco-2 cells also showed a lowest pro-oxidant effect induced by the four CFS of GMG fermented by lactic acid bacteria and bifidobacteria.

Improving the antioxidant properties of quinoa flour through fermentation with selected autochthonous lactic acid bacteria.

Lactic acid bacteria strains, previously isolated from the same matrix, were used to ferment quinoa flour aiming at exploiting the antioxidant potential. As in vitro determined on DPPH and ABTS radicals, the scavenging activity of water/salt-soluble extracts (WSE) from fermented doughs was significantly (P<0.05) higher than that of non-inoculated doughs. The highest inhibition of linoleic acid autoxidation was found for the quinoa dough fermented with Lactobacillus plantarum T0A10. The corresponding WSE was subjected to Reverse Phase Fast Protein Liquid Chromatography, and 32 fractions were collected and subjected to in vitro assays. The most active fraction was resistant to further hydrolysis by digestive enzymes. Five peptides, having sizes from 5 to 9 amino acid residues, were identified by nano-Liquid Chromatography-Electrospray Ionisation-Mass Spectra/Mass Spectra. The sequences shared compositional features which are typical of antioxidant peptides. As shown by determining cell viability and radical scavenging activity (MTT and DCFH-DA assays, respectively), the purified fraction showed antioxidant activity on human keratinocytes NCTC 2544 artificially subjected to oxidative stress. This study demonstrated the capacity of autochthonous lactic acid bacteria to release peptides with antioxidant activity through proteolysis of native quinoa proteins. Fermentation of the quinoa flour with a selected starter might be considered suitable for novel applications as functional food ingredient, dietary supplement or pharmaceutical preparations.

Italian legumes: effect of sourdough fermentation on lunasin-like polypeptides.

BACKGROUND: There is an increasing interest toward the use of legumes in food industry, mainly due to the quality of their protein fraction. Many legumes are cultivated and consumed around the world, but few data is available regarding the chemical or technological characteristics, and especially on their suitability to be fermented. Nevertheless, sourdough fermentation with selected lactic acid bacteria has been recognized as the most efficient tool to improve some nutritional and functional properties. This study investigated the presence of lunasin-like polypeptides in nineteen traditional Italian legumes, exploiting the potential of the fermentation with selected lactic acid bacteria to increase the native concentration. An integrated approach based on chemical, immunological and ex vivo (human adenocarcinoma Caco-2 cell cultures) analyses was used to show the physiological potential of the lunasin-like polypeptides. RESULTS: Italian legume varieties, belonging to Phaseulus vulgaris, Cicer arietinum, Lathyrus sativus, Lens culinaris and Pisum sativum species, were milled and flours were chemically characterized and subjected to sourdough fermentation with selected Lactobacillus plantarum C48 and Lactobacillus brevis AM7, expressing different peptidase activities. Extracts from legume doughs (unfermented) and sourdoughs were subjected to western blot analysis, using an anti-lunasin primary antibody. Despite the absence of lunasin, different immunoreactive polypeptide bands were found. The number and the intensity of lunasin-like polypeptides increased during sourdough fermentation, as the consequence of the proteolysis of the native proteins carried out by the selected lactic acid bacteria. A marked inhibitory effect on the proliferation of human adenocarcinoma Caco-2 cells was observed using extracts from legume sourdoughs. In particular, sourdoughs from Fagiolo di Lamon, Cece dell'Alta Valle di Misa, and Pisello riccio di Sannicola flours were the most active, showing a decrease of Caco-2 cells viability up to 70 %. The over-expression of Caco-2 filaggrin and involucrin genes was also induced. Nine lunasin-like polypeptides, having similarity to lunasin, were identified. CONCLUSIONS: The features of the sourdough fermented legume flours suggested the use for the manufacture of novel functional foods and/or pharmaceuticals preparations.

N(1)-methylspermidine, a stable spermidine analog, prolongs anagen and regulates epithelial stem cell functions in human hair follicles.

Spermidine (Spd), the prototypic polyamine, has been shown to be essential for hair follicle (HF) growth. However, Spd can be readily converted into other polyamines, and is physiologically unstable. Therefore, to assess its individual functions on HFs, we used the metabolically stable Spd analog N(1)-methylspermidine (N(1)-MeSpd). N(1)-MeSpd was confirmed to be a metabolically stable compound, with a half life of 90 h. 0.5 µM N(1)-MeSpd strongly prolonged anagen and decreased cell apoptosis in HFs in culture after 6 days, accompanied by specific stimulation of the expression of the epithelial stem cell-associated keratin, K15. N(1)-MeSpd also reduced lactate dehydrogenase activity in the culture supernatant, a parameter of cell death and cell lysis. N(1)-MeSpd diminished intracellular reactive oxygen species production in cultured keratinocytes, and reduced tumor necrosis factor-α, interleukin (IL)-1ß and IL-6 gene and protein expression after lipopolysaccharide stimulation. This suggests that some effects of N(1)-MeSpd may be mediated by anti-oxidative and anti-inflammatory effects. These additional properties of N(1)-MeSpd could be clinically important for the treatment of inflammatory alopecias and inflammatory scalp diseases.

Lactic acid fermentation as a tool to enhance the antioxidant properties of Myrtus communis berries.

BACKGROUND: Myrtle (Myrtus communis L.) is a medicinal and aromatic plant belonging to Myrtaceae family, which is largely diffused in the Mediterranean areas and mainly cultivated in Tunisia and Italy. To the best of our knowledge, no studies have already considered the use of the lactic acid fermentation to enhance the functional features of M. communis. This study aimed at using a selected lactic acid bacterium for increasing the antioxidant features of myrtle berries, with the perspective of producing a functional ingredient, dietary supplement or pharmaceutical preparation. The antioxidant activity was preliminarily evaluated through in vitro assays, further confirmed through ex vivo analysis on murine fibroblasts, and the profile of phenol compounds was characterized. RESULTS: Myrtle berries homogenate, containing yeast extract (0.4%, wt/vol), was fermented with Lactobacillus plantarum C2, previously selected from plant matrix. Chemically acidified homogenate, without bacterial inoculum and incubated under the same conditions, was used as the control. Compared to the control, fermented myrtle homogenate exhibited a marked antioxidant activity in vitro. The radical scavenging activity towards DPPH increased by 30%, and the inhibition of linoleic acid peroxidation was twice. The increased antioxidant activity was confirmed using Balb 3 T3 mouse fibroblasts, after inducing oxidative stress, and determining cell viability and radical scavenging activity through MTT and DCFH-DA assays, respectively. The lactic acid fermentation allowed increased concentrations of total phenols, flavonoids and anthocyanins, which were 5-10 times higher than those found for the non-fermented and chemically acidified control. As shown by HPLC analysis, the main increases were found for gallic and ellagic acids, and flavonols (myricetin and quercetin). The release of these antioxidant compounds would be strictly related to the esterase activities of L. plantarum. CONCLUSIONS: The lactic acid fermentation of myrtle berries is a suitable tool for novel applications as functional food dietary supplements or pharmaceutical preparations.

Lactic acid fermentation as a tool to enhance the functional features of Echinacea spp.

BACKGROUND: Extracts and products (roots and/or aerial parts) from Echinacea ssp. represent a profitable market sector for herbal medicines thanks to different functional features. Alkamides and polyacetylenes, phenols like caffeic acid and its derivatives, polysaccharides and glycoproteins are the main bioactive compounds of Echinacea spp. This study aimed at investigating the capacity of selected lactic acid bacteria to enhance the antimicrobial, antioxidant and immune-modulatory features of E. purpurea with the prospect of its application as functional food, dietary supplement or pharmaceutical preparation. RESULTS: Echinacea purpurea suspension (5%, wt/vol) in distilled water, containing 0.4% (wt/vol) yeast extract, was fermented with Lactobacillus plantarum POM1, 1MR20 or C2, previously selected from plant materials. Chemically acidified suspension, without bacterial inoculum, was used as the control to investigate functional features. Echinacea suspension fermented with Lb. plantarum C2 exhibited a marked antimicrobial activity towards Gram-positive and -negative bacteria. Compared to control, the water-soluble extract from Echinacea suspension fermented with Lactobacillus plantarum 1MR20 showed twice time higher radical scavenging activity on DPPH. Almost the same was found for the inhibition of oleic acid peroxidation. The methanol extract from Echinacea suspension had inherent antioxidant features but the activity of extract from the sample fermented with strain 1MR20 was the highest. The antioxidant activities were confirmed on Balb 3T3 mouse fibroblasts. Lactobacillus plantarum C2 and 1MR20 were used in association to ferment Echinacea suspension, and the water-soluble extract was subjected to ultra-filtration and purification through RP-FPLC. The antioxidant activity was distributed in a large number of fractions and proportional to the peptide concentration. The antimicrobial activity was detected only in one fraction, further subjected to nano-LC-ESI-MS/MS. A mixture of eight peptides was identified, corresponding to fragments of plantaricins PlnH or PlnG. Treatments with fermented Echinacea suspension exerted immune-modulatory effects on Caco-2 cells. The fermentation with Lb. plantarum 1MR20 or with the association between strains C2 and 1MR20 had the highest effect on the expression of TNF-α gene. CONCLUSIONS: E. purpurea subjected to lactic acid fermentation could be suitable for novel applications as functional food dietary supplements or pharmaceutical preparations.

A specific combination of zeaxanthin, spermidine and rutin prevents apoptosis in human dermal papilla cells.

Hair follicle (HF) regression is characterized by the activation of apoptosis in HF cells. Dermal papilla cells play a leading role in the regulation of HF development and cycling. Human follicular dermal papilla cells (HFDPC) were used to investigate the protective activities of rutin, sperimidine and zeaxanthine. HFDP cell incubation with staurosporine caused apoptosis, which was completely inhibited by exposure to rutin (2.2 µM), spermidine (1 µM) and zeaxanthin (80 µM). These agents were much less effective when applied as single compounds. Moreover, treatment preserved the expression of anti-apoptotic molecules such as Bcl-2, MAP-kinases and their phosphorylated forms. In conclusion, the investigated agents may represent an effective treatment for the prevention of apoptosis, one of the leading events involved in hair bulb regression.

The antimicrobial peptide pheromone Plantaricin A increases antioxidant defenses of human keratinocytes and modulates the expression of filaggrin, involucrin, ß-defensin 2 and tumor necrosis factor-α genes.

Plantaricin A (PlnA) is a peptide with antimicrobial and pheromone activities. PlnA was synthesized chemically and used as a pure peptide or synthesized biologically using Lactobacillus plantarum DC400 co-cultured with Lactobacillus sanfranciscensis DPPMA174. Cell-free supernatant (CFS) was used as a crude PlnA preparation. As estimated using the 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide and the 2',7'-dichlorofluorescein diacetate assays, both PlnA preparations increased the antioxidant defenses of human NCTC 2544 keratinocytes. PlnA (10 µg/ml) had a higher activity than hyaluronic acid or 125 µg/ml α-tocopherol. Effects on the transcriptional regulation of filaggrin (FLG), involucrin (IVL), hyaluronan synthase (HAS2), human ß-defensin-2 (HBD-2) and tumor necrosis factor-alpha (TNF-α) genes were assayed. Compared with the control, expression of the FLG gene in NCTC 2544 cells increased in cells treated with hyaluronic acid, 1 or 10 µg/ml PlnA. Compared with the control, the level of IVL gene expression increased in NCTC 2544 cells treated with 10 µg/ml PlnA. No significant difference was found between the level of the HAS2 gene expressed by control cells and cells treated with PlnA. Compared with chemically synthesized PlnA, the up-regulation of the HBD-2 gene by CFS was higher. Compared with the control, expression of TNF-α decreased in NCTC 2544 cells after treatment with 1 or 10 µg/ml of chemically synthesized PlnA. In contrast, the level of TNF-α was highest in the presence of 10 µg/ml CFS-PlnA. These findings suggest that the PlnA was positively sensed by human keratinocytes, promoting antioxidant defenses, barrier functions and antimicrobial activity of the skin.

Plantaricin A synthesized by Lactobacillus plantarum induces in vitro proliferation and migration of human keratinocytes and increases the expression of TGF-ß1, FGF7, VEGF-A and IL-8 genes.

This work showed the effect of pheromone plantaricin A (PlnA) on the proliferation and migration of the human keratinocytes NCTC 2544. PlnA was chemically synthesized and used as pure peptide or biologically synthesized during co-cultivation of Lactobacillus plantarum DC400 and Lactobacillus sanfranciscensis DPPMA174. The cell-free supernatant (CFS) was used as the crude preparation containing PlnA. The inductive effect of PlnA on the proliferation of NCTC 2544 cells was higher than that found for hyaluronic acid, a well known skin protective compound. As shown by scratch assay and image analyses, PlnA enhanced the migration of NCTC 2544 cells. Compared to the basal serum free medium (control), the highest inductive effect was found using 10µg/ml of chemically synthesized PlnA. Similar results (P>0.05) were found for CFS. In agreement, the percentage of the starting scratch area was decreased after treatment (24h) with PlnA. The expression of transforming growth factor-ß1 (TGF-ß1), keratinocyte growth factor 7 (FGF7), vascular endothelial growth factor (VEGF-A), and interleukin-8 (IL-8) genes was affected by PlnA. Compared to control, TGF-ß1 gene was under expressed in the first 4h of treatments and up-regulated after 8-24h. On the contrary, FGF7 gene was strongly up-regulated in the first 4h of treatments. Compared to control, VEGF-A and IL-8 genes were always up-regulated during the 4-24h from scratching. Since capable of promoting the proliferation and migration of the human keratinocytes and of stimulating IL-8 cytokine, the use of PlnA for dermatological purposes should be considered.

Antioxidant supplementation had positive effects in old rat muscle, but through better oxidative status in other organs.

OBJECTIVE: Aged muscle is characterized by a defect in the ability of leucine to stimulate protein synthesis. We showed previously that antioxidant supplementation improved the anabolic response to leucine of old muscle and reduced inflammation. The aim of the present study was to determine if the positive effects observed in muscle could be related to an improvement of local muscle oxidative status. METHODS: Two groups of 20-mo-old male Wistar rats were supplemented or not with rutin, vitamin E, vitamin A, zinc, and selenium during 7 wk. We measured body weight, food intake, oxidative status in muscle and other tissues, gastrocnemius muscle proteolytic activities, and liver glutathione metabolism. RESULTS: Antioxidant supplementation had no effect on muscle antioxidant capacity, superoxide dismutase activities, and myofibrillar protein carbonyl content and induced an increase in muscle cathepsin activities. In other tissues, antioxidant supplementation increased liver glutathione (reduced plus oxidized glutathione) content, reduced oxidative damage in the liver and spleen (as measured by γ-keto-aldehyde content), and reduced heart thiobarbituric acid-reactive substances. CONCLUSION: Our results showed that the positive effects of antioxidant supplementation observed previously on the anabolic response to leucine of old muscle were not directly related to an improvement of in situ muscle oxidative status. It could result from reduced systemic inflammation/oxidative stress. The dialog between muscle and other organs should be studied more thoroughly, especially during aging.