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Background: The use of herbal extracts could represent an advantageous approach for treating sleeping disorders, especially in mild-to-moderate conditions, before the onset of a specific therapy with first-line drugs. Specifically, the focus was posed about the use of extracts from Valeriana officinalis, Ziziphus jujuba, and Humulus lupulus. Multiple studies demonstrated the efficacy of these medicinal plants to positively manage insomnia symptoms. Additionally, their efficacy in the treatment of sleeping disorders could also be improved by their pharmacological association. In the present study, extracts from Valeriana officinalis, Ziziphus jujuba, Humulus lupulus, melatonin, and their pharmacological association, Vagonotte® MEL, were studied for potential application in the treatment of insomnia. Methods: The extracts and melatonin were tested on hypothalamic neurons and tissue for evaluating biocompatibility and protective and neuromodulatory effects. The neuromodulatory effects were evaluated as orexin A gene expression and serotonin steady state level, in the hypothalamus. Results: The extracts and melatonin, although with evident differences, were effective as antioxidant and anti-inflammatory agents; additionally, they were also able to reduce the hypothalamic gene expression of orexin A and the steady state level of serotonin, playing master roles in wakefulness. It is noteworthy that the formulation displayed all the effects of the single ingredients, without any sign of toxicity and pharmacological interference in the hypothalamus. Conclusions: Concluding, the present study explored the biological effects of melatonin and herbal extracts with phytotherapy interest in V. officinalis, Z. jujuba, and H. lupulus. The study demonstrated their intrinsic scavenging/reducing activity, together with protective and neuromodulatory effects in the hypothalamus, with a significant reduction of both orexin A gene expression and serotonin steady state level. Additionally, the study also considered their pharmacological association, which displayed an overall pharmacological spectrum mirroring, including all the effects of the single ingredients, without showing any sign of toxicity in the brain and interference between the extracts and melatonin.

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Cannabis sativa L. belongs to the Cannabaceae family and includes annual, robust, fast-growing and generally dioecious plants. Industrial hemp, and particularly the inflorescences, has been recently recognized as important source of bioactive extracts with antioxidant and antimicrobial effects. The goal of the present study was to explore botanical, phytochemical, and biological properties of water and hydroalcoholic hemp pollen extracts from male inflorescences. The extracts from hemp pollen were found rich in phenolic compounds, such as hydroxytyrosol, coumaric acid, and hesperitin. The phenolic profile was also consistent with the observed scavenging/reducing, enzyme inhibitory, and antimicrobial properties of the extracts. Regarding the antimicrobial effects, Escherichia coli, Trycophyton rubrum, and T. tonsurans were the most sensitive to growth inhibitory effects (MIC values: 9.92-79.37 µg/mL) of the extracts. Whereas, null effects on prostate PC3 and myocyte C2C12 cell viability, in the range 1-1000 µg/mL, are consistent with MIC values and suggest extracts' biocompatibility. The experimental data obtained, which are not reflected in the literature as the topic of hemp pollen is almost completely unexplored, confirm the innovativeness of a product obtained directly from bees, which in the face of greater variability and complexity can reserve promising applications in food, pharmaceutical, and cosmetic sectors.

Grape (Vitis vinifera L.) pomace is a residue derived from the winemaking process, which contains bioactive compounds displaying noteworthy health-promoting properties. The aim of the present study was to investigate the phenolic composition and protective effects of a water extract of grape pomace (WEGP) in colorectal cancer cell line SW480 and in isolated mouse colon exposed to Escherichia coli lipopolysaccharide (LPS). The extract decreased SW-480 cell viability, as well as vascular endothelial factor A (VEGFA), hypoxia-induced factor 1α (HIF1α), and transient receptor potential M8 (TRPM8) LPS-induced gene expression. Moreover, the extract inhibited mRNA levels of nuclear factor kB (NFkB), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)α, interleukin (IL)-6, IL-1β, IL-10, inducible nitric oxide synthase (iNOS), and interferon (IFN)γ, in isolated colon. Conversely, WEGP increased the gene expression of antioxidant catalase (CAT) and superoxide dismutase (SOD), in the same model. The modulatory effects exerted by WEGP could be related, at least in part, to the phenolic composition, with particular regards to the catechin level. Docking calculations also predicted the interactions of catechin toward TRPM8 receptor, deeply involved in colon cancer; thus further suggesting the grape pomace as a valuable source of bioactive extracts and phytochemicals with protective effects in the colon.

Mentha spicata is one of the most popular species in the genus, and it is of great interest as a gastrointestinal and sedative agent in the folk medicine system. In this study, different M. spicata extracts, obtained by the use of four solvents (hexane, chloroform, acetone and acetone/water) were chemically characterized using HPLC-ESI-MS n, which allowed for identification of 27 phenolic compounds. The extracts’ antioxidant and enzyme inhibitory properties were investigated. In addition, neuroprotective effects were evaluated in hypothalamic HypoE22 cells, and the ability of the extracts to prevent the hydrogen peroxide-induced degradation of dopamine and serotonin was observed. The best antioxidant effect was achieved for all the extraction methods using acetone/water as a solvent. These extracts were the richest in acacetin, eriodictyol, hesperidin, sagerinic acid, naringenin, luteolin, chlorogenic acid, chrysoeriol and apigenin. The intrinsic antioxidant and enzyme inhibition properties of the acetone/water extract could also explain, albeit partially, its efficacy in preventing prostaglandin E2 overproduction and dopamine depletion (82.9% turnover reduction) in HypoE22 cells exposed to hydrogen peroxide. Thus, our observations can provide a scientific confirmation of the neuromodulatory and neuroprotective effects of M. spicata.

Outer Membrane Vesicles (OMV) constitute a promising platform for the development of efficient vaccines. OMV can be decorated with heterologous antigens (proteins or polysaccharides), becoming attractive novel carriers for the development of multicomponent vaccines. Chemical conjugation represents a tool for linking antigens, also from phylogenetically distant pathogens, to OMV. Here we develop two simple and widely applicable conjugation chemistries targeting proteins or lipopolysaccharides on the surface of Generalized Modules for Membrane Antigens (GMMA), OMV spontaneously released from Gram-negative bacteria mutated to increase vesicle yield and reduce potential reactogenicity. A Design of Experiment approach was used to identify optimal conditions for GMMA activation before conjugation, resulting in consistent processes and ensuring conjugation efficiency. Conjugates produced by both chemistries induced strong humoral response against the heterologous antigen and GMMA. Additionally, the use of the two orthogonal chemistries allowed to control the linkage of two different antigens on the same GMMA particle. This work supports the further advancement of this novel platform with great potential for the design of effective vaccines.

Industrial hemp is characterized by a huge amount of by-products, such as inflorescences, that may represent high-quality sources of biomolecules with pharmaceutical interest. In the present study, we have evaluated the phytochemical profile, including terpene and terpenophenolic compounds, of the essential oils (EOs) of Futura 75, Carmagnola selezionata and Eletta campana hemp varieties. The EOs were also tested for antifungal properties toward Trichophyton mentagrophytes, Trichophyton rubrum, Arthroderma crocatum, Arthroderma quadrifidum, Arthroderma gypseum, Arthroderma curreyi, and Arthroderma insingulare. In parallel, we investigated the inhibitory effects of the EOs against tyrosinase, and the production of prostaglandin E2 in isolated mouse skin exposed to hydrogen peroxide. In human H1299 lung adenocarcinoma cells, we also evaluated the influence of the EOs on the gene expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2), which are involved in SARS-CoV-2 entry in human host. E-caryophyllene and α-pinene were the prominent terpenes in the EOs, whereas the cannabidiolic acid was the terpenophenol present at higher concentration. The EOs inhibited the growth of all tested dermatophytes species. In isolated skin specimens, EOs prevented the hydrogen-peroxide-induced synthesis of prostaglandin E2, consistent with the intrinsic antityrosinase activity. Finally, in H1299 cells, all tested EOs reduced the gene expression of ACE-2 and TMPRSS2, as well. Therefore, the present findings highlight the rationale for the use of the present EOs against infectious diseases.

Background: Shigellosis accounts for substantial morbidity and mortality worldwide and is the second most common cause of moderate and severe diarrhoea in children, after rotavirus. Methods: This phase 2b study (NCT03527173) evaluated vaccine efficacy (VE) against shigellosis, safety, and immunogenicity of a Shigella sonnei GMMA candidate vaccine (1790GAHB) in adults, using a S. sonnei 53G controlled human infection model (CHIM). Participants (randomized 1:1) received two doses of 1790GAHB or placebo (GAHB-Placebo), at day (D) 1 and D29, and an oral challenge of S. sonnei 53G at D57. VE was evaluated using several clinical endpoints, reflecting different case definitions of shigellosis. The success criterion was defined as a lower limit of the 90% confidence interval >0 for the primary endpoint. Findings: Thirty-six and 35 participants received 1790GAHB or placebo, respectively; 33 and 29 received the bacterial challenge, 15 and 12 participants developed shigellosis. Efficacy was not demonstrated for any endpoint. Solicited/unsolicited adverse events were more frequent in 1790GAHB versus placebo recipients post-vaccination. Anti-S. sonnei lipopolysaccharide (LPS) immunoglobulin G (IgG) responses increased at D29 and remained stable through D57 in the 1790GAHB group; no increase was shown in the placebo group. Interpretation: 1790GAHB had an acceptable safety profile and induced anti-LPS IgG responses but did not demonstrate clinical efficacy against shigellosis. Baseline and pre-challenge antibody levels were higher in participants who did not develop shigellosis post-challenge, thus suggesting a role of anti-LPS IgG antibodies in clinical protection. Although a precise threshold for clinical protection remains unknown, antibody levels derived from convalescent sera have demonstrated protection from shigellosis in field studies; however, this concept did not translate to the current CHIM. For further development of the vaccine, an increased content of S. sonnei O-antigen is likely needed to enhance anti-LPS immune responses. Trial Registration: NCT03527173 Funding Statement: GlaxoSmithKline Biologicals SA, the Bill and Melinda Gates Foundation. Declaration of Interests: AP, LBM, RR, GLC, PF, IDR, FM, FN, EM, JA, AA, OR and VC are employees of the GSK group of companies. AP, LBM, EM, and JA hold shares in the GSK group of companies. JA, LBM, AS, GLC, AGWN, PF, and IDR report grants from the Bill and Melinda Gates Foundation and the Wellcome Trust during the conduct of the study. LBM reports grants from the Bill and Melinda Gates foundation and the Wellcome Trust, outside the submitted work. AS, AGWN, and JPG were employees of the GSK group of companies at the time of study conduct. AA report personal fees from GSK Vaccines Institute for Global Health during the conduct of the study. AS hold shares in the GSK group of companies. LBM and AS are inventors of patents owned by the GSK group of companies and relevant to Shigella vaccine. AES, KAC, LC, MMcN, and RWK report grants from the GSK group of companies during the conduct of the trial. MMcN reports grants from NIH, outside the submitted work. MD, SP, and RWF have nothing to disclose. Ethics Approval Statement: The study was conducted in accordance with all applicable regulatory requirements, International Conference on Harmonisation - Good Clinical Practice guidelines, and the Declaration of Helsinki. The protocol and study-related documents were reviewed and approved by the institutional review board at the site. All participants provided electronic consent through REDcap system (www.projectredcap.org), except for one who provided written consent because the REDcap system was not working.

Industrial hemp is a multiuse crop whose phytocomplex includes terpenophenolics and flavonoids. In the present study, the phenolic and terpenophenolic compounds were assayed in the water extract of the hemp variety Futura 75. Protective effects were also investigated in human fibroblast and keratinocytes and isolate mouse skin specimens, which were exposed to hydrogen peroxide and/or to the extract (1&ndash, 500 &micro, g/mL). The results of phytochemical analysis suggested the cannabidiol, cannabidiolic acid and rutin as the prominent phytocompounds. In the in vitro system represented by human keratinocytes and fibroblasts, the hemp extract was found to be able to protect cells from cytotoxicity and apoptosis induced by oxidative stress. Moreover, modulatory effects on IL-6, a key mediator in skin proliferation, were found. In isolated rat skin, the extract reduced hydrogen peroxide-induced l-dopa turnover, prostaglandin-E2 production and the ratio kynurenine/tryptpophan, thus corroborating anti-inflammatory/antioxidant effects. The in silico docking studies also highlighted the putative interactions between cannabidiol, cannabidiolic acid and rutin with tyrosinase and indoleamine-2,3-dioxygenase, involved in l-dopa turnover and tryptophan conversion in kynurenine, respectively. In conclusion, the present findings showed the efficacy of hemp water extract as a skin protective agent. This could be partly related to the extract content in cannabidiol, cannabidiolic acid and rutin.

Tanacetum parthenium (feverfew) has traditionally been employed as a phytotherapeutic remedy in the treatment of migraine. In this study, a commercial T. parthenium water extract was investigated to explore its anti-inflammatory and neuromodulatory effects. Isolated mouse cortexes were exposed to a K+ 60 mM Krebs-Ringer buffer and treated with T. parthenium water extract. The prostaglandin E2 (PGE2) level, brain-derived neurotrophic factor (BDNF), interleukin-10 (IL-10), and IL-1&beta, gene expression were evaluated in the cortex. The effects on dopamine (DA) release and dopamine transporter (DAT) gene expression were assayed in hypothalamic HypoE22 cells. A bioinformatics analysis was conducted to further investigate the mechanism of action. The extract was effective in reducing cortex PGE2 release and IL-1&beta, gene expression. In the same experimental system, IL-10 and BDNF gene expressions increased, and in HypoE22 cells, the extract decreased the extracellular dopamine level and increased the DAT gene expression due to the direct interaction of parthenolide with the DAT. Overall, the present findings highlight the efficacy of T. parthenium water extract in controlling the inflammatory pathways that occur during cortical-spreading depression. Additionally, the inhibition of the hypothalamic DA release observed in this study further supports the role of dopaminergic pathways as key targets for novel pharmacological approaches in the management of migraine attacks.

Generalized modules for membrane antigens (GMMA) represent a technology particularly attractive for designing affordable vaccines against Gram-negative bacteria. We explored such technology for the development of O-antigen-based vaccines against Shigella and nontyphoidal Salmonella. Adsorption of GMMA on Alhydrogel was required for abrogation of pyrogenicity in rabbits, and Shigella sonnei GMMA on Alhydrogel was well tolerated and immunogenic in humans. Quantification of key antigens in formulated vaccines was fundamental for release and to check stability overtime. Traditionally, the direct quantification of antigens adsorbed on aluminum salts has been challenging, and the quantification of each active ingredient in multicomponent formulated vaccines has been even more complicated. To directly quantify each active ingredient and unbound drug substances in formulated vaccines, we developed the Formulated Alhydrogel competitive ELISA (FAcE) and the competitive ELISA method, respectively. The methods were both fully characterized, assessing specificity, repeatability, intermediate precision, and accuracy, for S. sonnei OAg quantification, both in a single component or multicomponent GMMA formulation also containing S. flexneri GMMA. The developed immunological methods allowed us to fully characterize Shigella GMMA drug products, supporting their preclinical and clinical development. The same methods, already extended to GMMA from nontyphoidal Salmonella and Neisseria meningitidis, could be potentially extended to any antigen formulated on Alhydrogel.

The mixture of water extracts from Tanacetum parthenium and Salix alba was studied in an ex vivo assessment of neurotoxicity constituted by isolated mouse cortex specimens challenged with K+ 60 mM Krebs–Ringer buffer (neurotoxicity stimulus). The effects of the mixture on lactate dehydrogenase (LDH), nitrite and serotonin levels were investigated. The phytochemical profile of the mixture was also evaluated. A docking approach was conducted to predict, albeit partially, the putative mechanism underlying the observed effects. The extracts displayed a good profile of polyphenolic compounds (22 chromatographic peaks detected), with caftaric acid and epicatechin being the prominent phenols. In isolated cortex, the association of T. parthenium and S. alba extracts was effective in reducing the K+ 60 mM-induced levels of LDH and nitrites, whereas the neurotoxicity stimulus-induced serotonin depletion was prevented by the treatment. Regarding the inhibition of serotonin catabolism, epicatechin (44.65 µg/mg) and caftaric acid (10.51 µg/mg) were putatively the main compounds involved in the inhibition of monoamineoxidase-A, which is known to play a master role in serotonin turnover. Collectively, the results of the present study point to the efficacy of the present extract mixture as an innovative pharmacological tool to prevent the onset of migraine.

Group B Streptococcus (GBS) is a normal inhabitant of recto-vaginal mucosae in up to 30% of healthy women. Colonization is a major risk factor for perinatal infection which can lead to severe complications such as stillbirth and neonatal invasive disease. Intra-partum antibiotic prophylaxis in colonized women is a safe and cost-effective preventive measure against early-onset disease in the first days of life, but has no effect on late-onset manifestations or on early maternal infection. Maternal immunization with capsular polysaccharide-based vaccines shows promise for the prevention of both early-onset and late-onset neonatal infections, although ability to prevent maternal colonization and ascending infection has been less studied. Here we investigated the effect of a GBS glycoconjugate vaccine since the very early stage of maternal GBS acquisition to neonatal outcome by rodent models of vaginal colonization and ascending infection. Immunization of female mice and rats with a type III glycoconjugate reduced vaginal colonization, infection of chorioamniotic/ placental membranes and bacterial transmission to fetuses and pups. Type III specific antibodies were detected in the blood and vagina of vaccinated mothers and their offspring. The obtained data support a potential preventive effect of GBS glycoconjugate vaccines during the different stages of pregnancy.

Vitis vinifera (grape) contains various compounds with acknowledged phytochemical and pharmacological properties. Among the different parts of the plant, pomace is of particular interest as a winemaking industry by-product. A characterization of the water extract from grape pomace from Montepulciano d’Abruzzo variety (Villamagna doc) was conducted, and the bioactive phenolic compounds were quantified through HPLC-DAD-MS analysis. HypoE22, a hypothalamic cell line, was challenged with an oxidative stimulus and exposed to different concentrations (1 µg/mL−1 mg/mL) of the pomace extract for 24, 48, and 72 h. In the same conditions, cells were exposed to the sole catechin, in a concentration range (5–500 ng/mL) consistent with the catechin level in the extract. Cell proliferation was investigated by MTT assay, dopamine release through HPLC-EC method, PGE2 amount by an ELISA kit, and expressions of neurotrophin brain-derived neurotrophic factor (BDNF) and of cyclooxygenase-2 (COX-2) by RT-PCR. The extract reverted the cytotoxicity exerted by the oxidative stimulus at all the experimental times in a dose-dependent manner, whereas the catechin was able to revert the oxidative stress-induced depletion of dopamine 48 h and 72 h after the stimulus. The extract and the catechin were also effective in preventing the downregulation of BDNF and the concomitant upregulation of COX-2 gene expression. In accordance, PGE2 release was augmented by the oxidative stress conditions and reverted by the administration of the water extract from grace pomace and catechin, which were equally effective. These results suggest that the neuroprotection induced by the extract could be ascribed, albeit partially, to its catechin content.

Background Shigellosis accounts for substantial morbidity and mortality worldwide and is the second most common cause of moderate and severe diarrhoea in children. Methods This phase 2b study (NCT03527173), conducted between August 2018 and November 2019, evaluated vaccine efficacy (VE), safety, and immunogenicity of a Shigella sonnei GMMA candidate vaccine (1790GAHB) in adults, using a S. sonnei 53 G controlled human infection model. Participants (randomized 1:1) received two doses of 1790GAHB or placebo (GAHB-Placebo), at day (D) 1 and D29, and an oral challenge of S. sonnei 53 G at D57. VE was evaluated using several endpoints, reflecting different case definitions of shigellosis. For the primary endpoint, the success criterion was a lower limit of the 90% confidence interval >0. Findings Thirty-six and 35 participants received 1790GAHB or placebo, respectively; 33 and 29 were challenged, 15 and 12 developed shigellosis. VE was not demonstrated for any endpoint. Adverse events were more frequent in 1790GAHB versus placebo recipients post-vaccination. Anti-S. sonnei lipopolysaccharide (LPS) IgG responses increased at D29 and remained stable through D57 in group 1790GAHB; no increase was shown in placebo recipients. Interpretation 1790GAHB had an acceptable safety profile and induced anti-LPS IgG responses but did not demonstrate clinical efficacy against shigellosis. Baseline/pre-challenge antibody levels were higher in participants who did not develop shigellosis post-challenge, suggesting a role of anti-LPS IgG antibodies in clinical protection, although not fully elucidated in this study. For further vaccine development an increased S. sonnei O-antigen content is likely needed to enhance anti-LPS immune responses. Funding GlaxoSmithKline Biologicals SA, Bill and Melinda Gates Foundation

Rationale and Objectives This study aimed to investigate whether a simplified breast magnetic resonance imaging (MRI) protocol consisting of a localizer, one precontrast sequence, and three time-point postcontrast sequences (at 28 seconds, 84 seconds and 252 seconds after the contrast agent administration) is suitable for the characterization of breast lesions as compared to a full diagnostic protocol (FDP). This study also aimed to review the current literature concerning abbreviated breast MRI protocols and offer an alternative protocol. Materials and Methods Breast magnetic resonance (MR) examinations with detected breast lesions of 98 patients were retrospectively evaluated. Two expert radiologists in consensus reviewed the simplified breast protocol (SBP) first and only thereafter the regular FDP, recording a diagnosis for each detected lesion for both protocols. Receiver operating characteristic curve analysis was performed to determine the diagnostic performance of the SBP compared to the standard FDP. A revision of the previously reported abbreviated breast magnetic resonance protocols was also carried out. Results A total of 180 lesions were identified; of these, 110 (61%) were malignant and 70 (39%) were benign. Of the 110 malignant lesions, 86 (78%) were invasive ductal carcinoma, 18 (16%) were invasive lobular carcinoma, and 6 (6%) were ductal carcinoma in situ. Areas under the curve for the receiver operating characteristic curves for the SBP vs the FDP were equivalent (0.98 vs 0.99, respectively; P = 0.76). The SBP could be performed in approximately 6 minutes and 58 seconds, compared to 14 minutes and 48 seconds for the FDP. Conclusions An SBP protocol including a late postcontrast time point is accurate for the characterization of breast lesions and was comparable to the standard FDP protocol, allowing a potential reduction of the total acquisition and interpretation times.

This study aimed to investigate whether a simplified breast magnetic resonance imaging (MRI) protocol consisting of a localizer, one precontrast sequence, and three time-point postcontrast sequences (at 28 seconds, 84 seconds and 252 seconds after the contrast agent administration) is suitable for the characterization of breast lesions as compared to a full diagnostic protocol (FDP). This study also aimed to review the current literature concerning abbreviated breast MRI protocols and offer an alternative protocol.Breast magnetic resonance (MR) examinations with detected breast lesions of 98 patients were retrospectively evaluated. Two expert radiologists in consensus reviewed the simplified breast protocol (SBP) first and only thereafter the regular FDP, recording a diagnosis for each detected lesion for both protocols. Receiver operating characteristic curve analysis was performed to determine the diagnostic performance of the SBP compared to the standard FDP. A revision of the previously reported abbreviated breast magnetic resonance protocols was also carried out.A total of 180 lesions were identified; of these, 110 (61%) were malignant and 70 (39%) were benign. Of the 110 malignant lesions, 86 (78%) were invasive ductal carcinoma, 18 (16%) were invasive lobular carcinoma, and 6 (6%) were ductal carcinoma in situ. Areas under the curve for the receiver operating characteristic curves for the SBP vs the FDP were equivalent (0.98 vs 0.99, respectively; P = 0.76). The SBP could be performed in approximately 6 minutes and 58 seconds, compared to 14 minutes and 48 seconds for the FDP.An SBP protocol including a late postcontrast time point is accurate for the characterization of breast lesions and was comparable to the standard FDP protocol, allowing a potential reduction of the total acquisition and interpretation times.

During the last decade, it has been shown that the activation of NRF2 and the binding to electrophile-responsive element (EpREs), stimulates the expression of a great number of genes responsible for the synthesis of phase I and phase II proteins, including antioxidants enzymes and heme oxygenase-1 (HO-1). This critical cell response occurs in cardiovascular, degenerative and chronic infective diseases aggravated by a chronic oxidative stress. In our previous reports we have shown that ozonated plasma is able to up-regulate HO-1 expression in endothelial cells. In the present work we investigated a candidate mechanism involved in this process. After treatment with increasing doses of ozonated serum (20, 40 and 80 μg/mL O(3) per mL of serum), a clear dose dependent activation of NRF2 and the subsequent induction of HO-1 and NAD(P)H quinone oxidoreductase 1(NQO1) was observed. This effect was also present when cells were treated with serum and hydrogen peroxide (H(2)O(2)) or serum and 4-hydroxynonenal (4HNE). Moreover, the treatment with ozonated serum was associated with a dose-dependent activation of extracellular-signal-regulated kinases (ERK1/2) and p38 MAP kinases (p38), not directly involved in NRF2 activation. These data, provide a new insight on the mechanism responsible for the induction of HO-1 expression by ozonated serum in the endothelium, and have a practical importance as an expedient approach to the treatment of patients with both effective orthodox drugs and ozonated autohemotherapy, targeted to the restoration of redox homeostasis.

The phosphodiesterase 4 inhibitor roflumilast prevents bleomycin- (BLM-) induced lung fibrosis in animal models. However, its mechanism of action remains unknown. We investigated whether roflumilast N-oxide (RNO), the active metabolite of roflumilast, can modulatein vitrothe oxidative effects of BLM on human lung fibroblasts (HLF). In addition, since BLM increases the production of F2-isoprostanes that haveper sefibrogenic activity, the effect of RNO on oxidative stress and fibrogenesis induced by the F2-isoprostane 8-epi-PGF2αwas investigated. HLF were preincubated either with the vehicle or with RNO and exposed to either BLM or 8-epi-PGF2α. Proliferation and collagen synthesis were assessed as [3H]-thymidine and [3H]-proline incorporation. Reactive oxygen species (ROS) and F2-isoprostanes were measured. NADPH oxidase 4 (NOX4) protein and mRNA were also evaluated. BLM increased both cell proliferation and collagen synthesis and enhanced ROS and F2-isoprostane production. These effects were significantly prevented by RNO. Also, RNO significantly reduced the increase in both NOX4 mRNA and protein, induced by BLM. Finally, 8-epi-PGF2α per sestimulated HLF proliferation, collagen synthesis, and NOX4 expression and ROS generation, and RNO prevented these effects. Thus, the antifibrotic effect of RNO observedin vivomay be related to its ability to mitigate ROS generation via downregulation of NOX4.

Oxidative stress (OS) is important in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA) and its experimental model--adjuvant arthritis (AA). Antioxidants are scarcely studied in autoimmunity, and future analyses are needed to assess its effects in ameliorating these diseases. Although there are studies about antioxidants effects on the course of RA, their role in combination therapy has not yet been studied in detail, especially on extra-articular manifestations of AA. During the 28-d administration of pinosylvin (PIN) in monotherapy and in combination with methotrexate (MTX) to AA rats, we evaluated the impact of the treatment on selected parameters. The experiment included: healthy controls, untreated AA, AA administered 50 mg/kg b.w. of PIN daily p.o., AA administered 0.4 mg/kg b.w. of MTX twice weekly p.o. and AA treated with a combination of PIN+MTX. AA was monitored using: hind paw volume, C-reactive protein, monocyte chemotactic protein-1 (MCP-1), thiobarbituric acid reactive substances (TBARS) and F2-isoprostanes in plasma, γ-glutamyltransferase activity in spleen, activity of lipoxygenase (LOX) in lung, heme oxygenase-1 (HO-1) and nuclear factor kappa B (NF-κB) in liver and lung. PIN monotherapy significantly improved the activation of NF-κB in liver and lung, HO-1 expression and activity of LOX in the lung, MCP-1 levels in plasma (on 14th d) and plasmatic levels of F2-isoprostanes. An important contribution of PIN to MTX effect was the reduction of OS (an increase of HO-1 expression in lung and reduction of plasmatic TBARS) and decrease of LOX activity in the lung.

Novel therapies for rheumatoid arthritis also include the use of naturally occurring compounds possessing antioxidant properties. In the present work, the effects of oral administration of quercetin were investigated in a rat model of adjuvant arthritis. Arthritis was induced by a single intradermal injection of heat-inactivated Mycobacterium butyricum in incomplete Freund's adjuvant. The experimental groups were treated with an oral daily dose of 150 mg/kg b.w. of quercetin for 28 days. Results indicated that quercetin was able to ameliorate all markers of inflammation and oxidative stress measured. Quercetin lowered levels of interleukin-1β, C-reactive protein, and monocyte chemotactic protein-1 and restored plasma antioxidant capacity. In addition, quercetin inhibited the enzymatic activity of pro-inflammatory 12/15-lipoxygenase in lung and liver and increased the expression of heme oxygenase-1 in joint and lung of arthritic rats. Finally, quercetin inhibited the 2-fold increase of NF-қB activity observed in lung, liver and joint after induction of arthritis.

Summary Background We present an uncommon case of Ewing’s sarcoma in a 16-year-old boy. Case Report This case can be considered unique because of the atypical presentation, normal laboratory tests and absence of the typical symptoms such as pain, masses or swelling, fatigue or weight loss, breathing problems linked to lung metastases or pathologic fractures. The only event that brought the patient to our attention was the sudden onset of left proptosis. Conclusions The final histopathology together with CT and PET-CT findings led to the diagnosis of a multi-metastatic Ewing’s sarcoma involving the orbit, skeleton, bone marrow and lymph nodes.