Previous studies have found that use of hexaminolevulinate (HAL) and blue light cystoscopy (BLC) during treatment of bladder cancer had a positive impact on overall survival after later cystectomy, indicating a potential treatment effect beyond improved diagnostic accuracy. The aim of our study was to determine whether HAL and BL mimicking clinically relevant doses in an orthotopic rat model could have therapeutic effect by inducing modulation of a tumor-specific immune response. We also assessed whether administration with a checkpoint inhibitor could potentiate any effects observed. Rats were subjected to HAL BL alone and in combination with anti-PD-L1 and assessed for anti-tumor effects and effects on immune markers. Positive anti-tumor effect was observed in 63% and 31% of rats after, respectively, 12 and 30 days after the procedure, together with a localization effect of CD3+ and CD8+ cells after 30 days. Anti-tumor effect at 30 days increases from 31% up to 38% when combined with intravesical anti-PD-L1. In conclusion, our study demonstrated treatment effects with indications of systemic immune activation at diagnostic doses of HAL and blue light. The observed treatment effect seemed to be enhanced when used in combination with intravesically administrated immune checkpoint inhibitor.

The etiology of Alzheimer's disease (AD) is poorly understood. A growing body of literature suggests that amyloid beta oligomers (AβO) as the root cause of this disease. Here, we describe new translational in vitro and in vivo models of AD, induced by minute amount of in-house preparation of human AβO.AβO were reproducibly prepared from human Aβ 1-42 monomers. Rodent primary neurons were used to assess the neurotoxic activities of AβO in vitro and elderly wild-type mice administered by a single intracerebral injection AβO in vivo.The oligomeric preparations were characterized by SDS-page, electron microscopy and dot-plot tests. In vitro, AβO induced a dose-dependent neurodegeneration on rodent primary neurons based on various read-outs. Interestingly, neurotoxicity was greater with AβO than fibrillar Aβ, while Aβ monomers did not induce any neuronal damage. AβO-induced neurotoxicity was significantly attenuated by brain-derived neurotrophic factor (BDNF). In vivo, a single intracerebral microinjection of AβO in 18-month-old wild-type mice, led to a significant memory impairment, synaptic loss in hippocampus and increased secretion of brain proinflammatory cytokines 15 days post administration. Moreover, the memory deficits were significantly reversed with Donepezil used as reference drug.In conclusion, we characterized new tools for drug screening based on the soluble AβO hypothesis of AD. In vivo, this new non-inherited Alzheimer model can be used both to evaluate disease modifying and symptomatic drugs. Finally, these models are also valuable tools to understand the mechanisms underlying AD.

Oral probiotics potential for the management of dermatological diseases is vast. However, results of available studies in skin diseases, such as atopic dermatitis (AD), are inconsistent, partly because probiotic effects are strain specific. Careful selection of probiotic strains is therefore indispensable to ensure efficacy of treatment. In this study, Lactobacillus salivarius LA307, Lactobacillus rhamnosus LA305 and Bifidobacterium bifidum PI22, three strains that were previously identified for their interesting immunomodulatory properties in allergy and/or colitis models, were assessed in the prevention of chronic skin inflammation induced by repeated applications of 12- O -tetradecanoylphorbol-13-acetate in hairless SKH-1 mice. Macroscopic and microscopic evaluation of skin lesions was performed together with measurements of serum levels of interleukin (IL)-1β, IL-6, tumour necrosis factor alpha (TNF-α), IL-17, IL-22, IL-10 and IL-4. Daily oral treatment with the three strains at the dose of 1×10 9 cfu/day for 3 weeks limited the development of chronic skin inflammation, the effects being strain dependent. Indeed the two Lactobacillus strains significantly limited the intensity of skin inflammation both at the macroscopic and microscopic levels. Macroscopic observations were correlated to the histological observations and the resulting microscopic score. This limitation of the development of AD-like skin lesions involved the modulation of cytokine production. Treatment with the two Lactobacillus strains induced a decrease in the serum levels of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, IL-17, IL-22 and at the opposite an increase in the production of the anti-inflammatory cytokine IL-10 and also of IL-4. Globally, B. bifidum PI22 had lower benefits. These results obtained in mice suggest that L. salivarius LA307 and L. rhamnosus LA305 could be good candidates for preserving skin integrity and homeostasis via the modulation of the gut microbiota and that their use could be beneficial in dermatological conditions such as AD.

BACKGROUND There is a growing interest in the effects of probiotics for the prevention and treatment of skin diseases due to their immunomodulatory and antiinflammatory properties. OBJECTIVE To assess a mixture of five bacterial strains in the prevention of chronic skin inflammation in mice. METHODS Hairless SKH-1 mice received daily oral treatment with the probiotic mixture at the dose of 1x109 Colony-Forming Unit (CFU)/day (or vehicle) for three weeks. Chronic skin inflammation was induced by repeated applications of 12-O-tetradecanoylphorbol-13- acetate (TPA; control mice received acetone). Macroscopic and microscopic evaluations of skin lesions were performed and serum levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, IL-17, IL-22, IL-10 and IL-4 measured at the end of the study. RESULTS Treatment with the probiotic mixture significantly limited the induced chronic skin inflammation at both the macroscopic and microscopic levels. This limitation was consistent with downregulated levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IL- 17 and IL-22) and up-regulated levels of the anti-inflammatory cytokines, IL-10 and IL-4. CONCLUSION The results suggest that the probiotic mixture tested could help in preserving skin integrity and homeostasis and that its use could be beneficial in dermatological conditions such as atopic dermatitis and psoriasis.

OBJECTIVES:The green seaweed Ulva sp. contains a large amount of ulvans, a family of sulphated polysaccharides. The present study was designed to investigate in rats the antidepressant- and anxiolytic-like effects of a hydrophilic extract of Ulva sp. (MSP) containing about 45% of ulvans. METHODS:After a 14-day administration of MSP at doses of 10, 20 and 40 mg/kg/day, 48 and 60 male adult Wistar rats were respectively tested in the elevated plus-maze (EPM) and the forced swimming test (FST). In the FST, MSP effects were compared to the reference antidepressant drug imipramine (IMI) (10 mg/kg/day). Acute and sub-chronic toxicities of the extract were also assessed in male and female rats following OECD guidelines. RESULTS:MSP treatment did not modify anxiety-related behaviour in the EPM. In contrast, MSP induced a dose-dependent reduction of immobility behaviour in the FST. At the highest tested dose of 40 mg/kg, MSP displayed a significant antidepressant-like effect similar to IMI. MSP did not modify the exploratory behaviour of rats in the open field test and did not produce any toxic effect. DISCUSSION:MSP may potentially represent a good adjunct or alternative to existing antidepressant therapeutics. Further studies are necessary to confirm the mechanism of action of MSP and its modulation of brain functioning.

Probiotics have been shown to have preventive and therapeutic effects on diarrhea. Because effects tend to be strain specific, benefit of a strain or mixture has to be substantiated by experimental evidence. The aim of this study was to investigate the antidiarrheal and antinociceptive effects of a probiotic mixture (Lactibiane Imedia®, PiLeJe). Castor oil-induced diarrhea test was performed in Wistar rats following oral administration of probiotics (20 × 109, 30 × 109 or 40 × 109 CFU/kg), loperamide (5 mg/kg) or vehicle (water; 10 mL/kg). Time to initial evacuation, number of feces and diarrheal feces, fresh weight and water content of the feces and body weight loss were monitored. Behavioral parameters (eye closing, abnormal posture, activity, fur aspect) were used as pain indices. Probable mechanisms of action were evaluated by using the castor oil-induced enteropooling and charcoal meal transit tests. Probiotics significantly and dose-dependently delayed onset time to first feces and had a beneficial effect on all other parameters (p 90%) at the two highest doses tested (30 × 109 or 40 × 109 CFU/kg) without completely blocking defecation (65.7% at 30 × 109 CFU/kg). Behavioral parameters were improved with probiotics compared to vehicle, improvement that was not observed with loperamide. Probiotics significantly and dose-dependently decreased the volume of intestinal fluid (p<0.05 versus vehicle) in the enteropooling test and transit time of charcoal meal. These results indicate that the probiotic mixture tested is strongly antidiarrheic through the combination of antimotility and antisecretory properties. Observations are also in favor of an antinociceptive effect. Agents that can decrease both intestinal hypersecretion and motility are very useful in the management of diarrhea therefore, our probiotic mixture could be an effective alternative to standard drugs.

Chlorella sp. is a green microalgae containing nutrients, vitamins, minerals, and chlorophyll. In some communities, Chlorella sp. is a traditional medicinal plant used for the management of inflammation-related diseases. In a rat model, ROQUETTE Chlorella sp. (RCs) benefits were investigated on visceral pain and associated inflammatory parameters related to cystitis both induced by cyclophosphamide (CYP). RCs was orally administered every day from day 1-16 (250 and 500 mg/kg body weight). Six hours after an intraperitoneal injection of 200 mg/kg body weight of CYP, body temperature, general behavior, food intake, and body weight were recorded. Twenty-four hours after CYP injection, rats were tested in two behavioral tests, an open field and the aversive light stimulus avoidance conditioning test, to evaluate the influence of pain on general activity and learning ability of rats. After euthanasia, bladders were weighed, their thickness was scored, and the urinary hemoglobin was measured. RCs orally administered at the two dosages significantly reduced visceral pain and associated inflammatory parameters related to cystitis both induced by CYP injection, and improved rat behavior. To conclude, RCs demonstrated beneficial effects against visceral pain and cystitis.

The antihypertensive potential of a land snail by-product hydrolysate (SBH), obtained after an industrial treatment of the raw material, was studied in vitro and in vivo. The ACE inhibitory activity of SBH was characterised by an IC50 value of 23 µg⋅mL−1, which was not affected by in vitro digestion. SBH enhanced the Caco-2 intestinal cell metabolic activity and did not induce any toxicity in Wistar rats. The partial purification of SBH led to the obtainment of an active fraction characterised by an IC50 of 0.007 µg⋅mL−1. The sequences of the 17 most abundant peptides of the fraction were identified by LC/MS/MS analysis. Seven of them (YG, YA, VY, SF, FG, GF and VW) are known ACE inhibitory peptides. Finally, in vivo study on SHR rats showed that SBH significantly reduced systolic blood pressure. SBH represents therefore a new candidate as an ingredient for the design of functional foods against hypertension.

Objective: The purpose of our study was to investigate the effect of three probiotic strains on skin homeostasis in a model of inflammation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Methodology: Four groups of 6 mice received one of the probiotic strains (Lactobacillus salivarius LA307, Lactobacillus rhamnosus LA305 or Bifidobacterium bifidum S12; 3.109 CFU/day) or the vehicle by orogastric gavage daily for three consecutive weeks starting two weeks before the induction of skin inflammation. Macroscopic and microscopic evaluation of skin inflammation were performed as well as measurement of cytokine serum levels. Results: Treatment with LA305 or LA307 strains reduced the severity and extension of TPA-induced skin lesions with significantly lowered macroscopic and microscopic scores. It also led to a significant decrease in serum levels of pro-inflammatory cytokines and a significant increase of anti-inflammatory cytokines. The observed modifications were more pronounced with LA307 than LA305 and S12. Discussion: The probiotic strains LA305 and LA307 can prevent the development of skin inflammation and could be good candidates for preserving skin integrity and homeostasis. Cytokine analysis indicated that these effects are at least in part due to anti-inflammatory actions. Further studies will be needed to elucidate the mechanisms of action involved.

Fluorene is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) in the environment by reason of its high volatility. Demonstrated to be a neurotoxicant through inhalation, it was also identified as a contributive PAH to food contamination. Since no data are available on its oral neurotoxicity, the purpose of the present study was to assess the behavioral and physiological toxicity of repeated oral administration of fluorene to adult Wistar male rats. Animals were daily treated with fluorene at 1, 10 or 100mg/kg/day for 28 consecutive days. Administration was intraperitoneal (i.p.) or oral (p.o.) to evaluate the influence of the route of exposure on fluorene toxicity. Following this period of treatment, animals in both groups were subjected to similar cognitive evaluations, namely anxiety (elevated-plus maze), locomotor activity (open-field) and learning and memory abilities (eight-arm maze and avoidance test of an aversive light stimulus), as well as physiological measurements. The behavioral testing occurred from the 28th to the 60th day of the experiment during which fluorene treatment continued uninterrupted. At the end of this period, the concentration levels of fluorene and of three of its monohydroxylated metabolites in blood and brain were determined using a GC-MS/MS method. The results demonstrated a reduction in rat anxiety level at the lowest doses administered (1 and 10mg/kg/day) regardless of the treatment route, whereas locomotor activity and learning abilities remained unchanged. Moreover, a less significant weight gain was noticed in animals i.p.- and p.o.-treated with 100mg/kg/day during the 28-day period of treatment, which, upon comparison with the three other groups, induced a body weight gap that was maintained throughout the experiment. Significant increases in relative liver weight were also observed in a dose-dependent manner in orally treated rats and only in animal treated i.p. with 100mg/kg/day. According to the dose, higher concentration levels of fluorene and its monohydroxylated metabolites were measured in blood and brain compartments of i.p.-treated rats compared to p.o.-treated animals. In conclusion, fluorene reduced the anxiety level of rats related to dose, treatment route, duration of exposure and blood concentration levels of metabolites.

Treatment of stroke, especially during the first hours or days, is still lacking. S-nitrosoglutathione (GSNO), a cerebroprotective agent with short life time, may help if administered early with a sustain delivery while avoiding intensive reduction in blood pressure. We developed in situ forming implants (biocompatible biodegradable copolymer) and microparticles (same polymer and solvent emulsified with an external oily phase) of GSNO to lengthen its effects and allow cerebroprotection after a single subcutaneous administration to Wistar rats. Arterial pressure was recorded for 3 days (telemetry, n = 14), whole-blood platelet aggregation up to 13 days (aggregometry, n = 58), and neurological score, cerebral infarct size and edema volume for 2 days after obstruction of the middle cerebral artery by autologous blood clots (n = 30). GSNO-loaded formulations (30 mg/kg) induced a slighter and longer hypotension (-10 vs. -56 ± 6 mmHg mean arterial pressure, 18 h vs. 40 min) than free GSNO at the same dose. The change in pulse pressure (-50%) lasted even up to 42 h for microparticles. GSNO-loaded formulations (30 mg/kg) prevented the transient 24 h hyper-aggregability observed with free GSNO and 7.5 mg/kg-loaded formulations. When injected 2 h after stroke, GSNO-loaded microparticles (30 mg/kg) reduced neurological score at 24 (-62%) and 48 h (-75%) vs. empty microparticles and free GSNO 7.5 mg/kg and, compared to free GSNO, divided infarct size by 10 and edema volume by 8 at 48 h. Corresponding implants reduced infarct size and edema volume by 2.5 to 3 times. The longer (at least 2 days) but slight effects on arterial pressures show sustained delivery of GSNO-loaded formulations (30 mg/kg), which prevent transient platelet hyper-responsiveness and afford cerebroprotection against the consequences of stroke. In conclusion, in situ GSNO-loaded formulations are promising candidates for the treatment of stroke.

INTRODUCTION:Clenbuterol has been used to alleviate chronic obstructive pulmonary disease and elicit an anabolic response in muscles. The aim of this study was to determine the influence of muscle mass variation on physical capacities in rats.METHODS:The left hindlimbs of Wistar rats were immobilized for 20 days in plantarflexion with a splint and then remobilized for 16 days. The effect of a non-myotoxic dose of clenbuterol during the immobilization period was evaluated. Physical capacities were coordination, free locomotion, grip strength, and bilateral deficit.RESULTS:Immobilization induced a loss of muscle mass, coordination, and strength without any effect on free locomotion. The positive anabolic effect of clenbuterol did not prevent a loss of physical capacities resulting from immobilization.CONCLUSIONS:Muscle mass correlated strongly with coordination and isometric strength in untreated rats. Anabolic effect, fiber phenotype modification, and perturbation in neuromuscular communication with clenbuterol improved muscle mass, but it altered physical capacities.

The human body is constantly exposed to the risk of traumatic lesions. ROQUETTE Schizochytrium sp. (SCs) is a marine microalgae containing large amounts of health-valuable nutrients, more particularly polyunsaturated fatty acids such as docosahexaenoic acid. SCs was investigated by oral administration (125, 250 and 500 mg/kg) and cutaneous application (2.5, 5.0 and 10.0%) to evaluate its impact in two dermatological disorder models in mice: skin inflammation and wound healing. For skin inflammation, it was administered during 14 days starting one week before the induction of chronic skin inflammation by repeated cutaneous application of 12-O-tetradecanoylphorbol 13-acetate (TPA). For wound healing the microalgae was administered after incisional wound healing of the skin until complete wound healing. Results indicated that oral and topical administrations of the two higher doses of SCs had significant effects on macroscopic score of skin inflammation. It had also efficient effect on healing process and duration of wound healing with a dose-response by oral administration and a maximal effect observed from the lowest to the highest dose by topical application. These findings suggest that administration of SCs by both oral and topical routes appeared to have beneficial effects on skin lesions.

Inflammation adversely affects the health of millions of people worldwide, and there is an unmet medical need for better anti-inflammatory drugs. We evaluated the therapeutic interest of mycolactone, a polyketide-derived macrolide produced by Mycobacterium ulcerans. Bacterial production of mycolactone in human skin causes a combination of ulcerative, analgesic, and anti-inflammatory effects. Whereas ulcer formation is mediated by the proapoptotic activity of mycolactone on skin cells via hyperactivation of Wiskott-Aldrich syndrome proteins, analgesia results from neuronal hyperpolarization via signaling through angiotensin II type 2 receptors. Mycolactone also blunts the capacity of immune cells to produce inflammatory mediators by an independent mechanism of protein synthesis blockade. In an attempt to isolate the structural determinants of mycolactone's immunosuppressive activity, we screened a library of synthetic subunits of mycolactone for inhibition of cytokine production by activated T cells. The minimal structure retaining immunosuppressive activity was a truncated version of mycolactone, missing one of the two core-branched polyketide chains. This compound inhibited the inflammatory cytokine responses of human primary cells at noncytotoxic doses and bound to angiotensin II type 2 receptors comparably to mycolactone in vitro. Notably, it was considerably less toxic than mycolactone in human primary dermal fibroblasts modeling ulcerative activity. In mouse models of human diseases, it conferred systemic protection against chronic skin inflammation and inflammatory pain, with no apparent side effects. In addition to establishing the anti-inflammatory potency of mycolactone in vivo, our study therefore highlights the translational potential of mycolactone core-derived structures as prospective immunosuppressants.

Taichi Goto, Bunkyo-ku, Tokyo, Japan , Nao Tamai, Bunkyo-ku, Tokyo, Japan , Gojiro Nakagami, Bunkyo-ku, Tokyo, Japan , Masayuki Hirokawa, Chiyoda-ku, Tokyo, Japan , Ayumi Naito, Fujisawa-city, Kanagawa, Japan , Kazuo Takahashi, Fujisawa-city, Kanagawa, Japan , Junichi Umemoto, Fujisawa-city, Kanagawa, Japan , Ayumi Amemiya, Bunkyo-ku, Tokyo, Japan , Aya Kitamura, Bunkyo-ku, Tokyo, Japan , Yuiko Koyano, Bunkyo-ku, Tokyo, Japan , Hiromi Sanada, Bunkyo-ku, Tokyo, Japan

The human body is constantly exposed to the risk of traumatic lesions. Chlorella is a green microalgae enriched with nutrients, vitamins, minerals and chlorophyll. In some communities, Chlorella is a traditional medicinal plant used for the management of inflammation-related diseases. ROQUETTE Chlorella sp. (RCs) was investigated by oral administration (125, 250 and 500 mg/kg) and cutaneous application (2.5, 5.0 and 10.0%) to evaluate its impact in two dermatological disorder models in mice: skin inflammation and wound healing. For skin inflammation, it was administered during 14 days starting one week before the induction of chronic skin inflammation by repeated cutaneous application of 12-Otetradecanoylphorbol 13-acetate (TPA). For wound healing the microalgae was administered by topical application after scarification of the skin until complete wound healing. Results indicated that oral and topical administrations of the two higher doses of RCs had significant effects on macroscopic score of skin inflammation with an efficient effect on microscopic score with cutaneous application. The microalgae had also efficient effect on healing process and duration of wound healing for both administration routes and particularly at the two highest doses of RCs. These findings suggest that administration of RCs by both oral and topical routes appeared to have beneficial effects on skin lesions.

The orange-peel derived terpene d-Limonene, probably through its metabolite, perillyl alcohol (POH), has been reported to have tissue-repair properties. Two murine models of respectively 12-O-Tetradecanoylphorbol-13-Acetate (TPA)-induced dermatitis and mechanical skin lesion were used here to assess the efficacy of d-Limonene or POH applied topically. Macroscopic and microscopic evaluation of skin lesions was performed as well as that of P-selectin expression, together with measurements of serum concentrations of IL-1β, IL-6 and TNF-α in the first model. Healing and angiogenesis around the scar were examined in the second model. Because differences in angiogenesis were noted, the effect of both d-Limonene and POH was further tested on an in vitro model of endothelial microtubules formation. Both d-Limonene and POH reduced the severity and extension of TPA-induced skin lesions with significantly lowered macroscopic and microscopic scores (p<0.04 in both cases). Moreover, the expression of P-selectin induced by TPA was abrogated by POH and significantly lower serum concentrations of IL-6 and TNF-α were observed in d-Limonene- and POH-treated mice (p<0.04 and 0.03). In the second model, tissue regeneration was improved, especially by POH, and was clearly associated with reduced neovascularization. This surprising anti-angiogenic effect was confirmed in the matrigel model of endothelial microtubules formation. These studies show that d-Limonene and POH demonstrate significant anti-inflammatory effects in murine dermal inflammation and wound-healing. The decreased systemic cytokine production as well as a consistent inhibition of endothelial P-selectin expression and neo-vascularization induced by these terpenic compounds contribute to their healing effects on the epidermal barrier.