Sepsis is a life-threatening condition that requires urgent care. Thus, the identification of specific and sensitive biomarkers for its early diagnosis and management are of clinical importance. The alarmin prothymosin alpha (proTα) and its decapeptide proTα(100-109) are immunostimulatory peptides related to cell death. In this study, we generated bacterial models of sepsis in mice using two Klebsiella pneumoniae strains (L-78 and ATCC 43816) and monitored sepsis progression using proTα(100-109) as a biomarker. Serum concentration of proTα(100-109) gradually increased as sepsis progressed in mice infected with L-78, a strain which, unlike ATCC 43816, was phagocytosed by monocytes/macrophages. Analysis of splenocytes from L-78-infected animals revealed that post-infection spleen monocytes/macrophages were gradually driven to caspase-3-mediated apoptosis. These results were verified in vitro in L-78-infected human monocytes/macrophages. Efficient phagocytosis of L-78 by monocytes stimulated their apoptosis and the concentration of proTα(100-109) in culture supernatants increased. Human macrophages strongly phagocytosed L-78, but resisted cell death. This is the first report suggesting that high levels of proTα(100-109) correlate, both in vitro and in vivo, with increased percentages of cell apoptosis. Moreover, we showed that low levels of proTα(100-109) early post-infection likely correlate with sepsis resolution and thus, the decapeptide could eventually serve as an early surrogate biomarker for predicting bacteria-induced sepsis outcome.

Prothymosin α (proTα) and its C-terminal decapeptide proTα(100-109) were shown to pleiotropically enhance innate and adaptive immune responses. Their activities have been broadly studied in vitro, focusing primarily on the restoration of the deficient immunoreactivity of cancer patients' leukocytes. Previously, we showed that proTα and proTα(100-109) act as danger-associated molecular patterns (DAMPs), ligate Toll-like receptor-4, signal through TRIF- and MyD88-dependent pathways, promote the maturation of dendritic cells and elicit T-helper type 1 (Th1) immune responses in vitro, leading to the optimal priming of tumor antigen-reactive T-cell functions. Herein, we assessed their activity in a preclinical melanoma model. Immunocompetent mice bearing B16.F1 tumors were treated with two cycles of proTα or proTα(100-109) together with a B16.F1-derived peptide vaccine. Coadministration of proTα or proTα(100-109) and the peptide vaccine suppressed melanoma-cell proliferation, as evidenced by reduced tumor-growth rates. Higher melanoma infiltration by CD3+ T cells was observed, whereas ex vivo analysis of mouse total spleen cells verified the in vivo induction of melanoma-reactive cytotoxic responses. Additionally, increased levels of proinflammatory and Th1-type cytokines were detected in mouse serum. We propose that, in the presence of tumor antigens, DAMPs proTα and proTα(100-109) induce Th1-biased immune responses in vivo. Their adjuvant ability to orchestrate antitumor immunoreactivities can eventually be exploited therapeutically in humans.

Skin health is heavily affected by ultraviolet irradiation from the sun. In addition, senile skin is characterized by major changes in the collagen, elastin and in the hyaluronan content. Natural products (NPs) have been shown to delay cellular senescence or in vivo aging by regulating age-related signaling pathways. Moreover, NPs are a preferable source of photoprotective agents and have been proven to be useful against the undesirable skin hyperpigmentation. Greek flora harvests great plant diversity with approximately 6000 plant species, as it has a wealth of NPs. Here, we report an extensive screening among hundreds of plant species. More than 440 plant species and subspecies were selected and evaluated. The extracts were screened for their antioxidant and anti-melanogenic properties, while the most promising were further subjected to various in vitro and cell-based assays related to skin aging. In parallel, their chemical profile was analyzed with High-Performance Thin-Layer Chromatography (HPTLC) and/or Ultra-Performance Liquid Chromatography High-Resolution Mass Spectrometry (UPLC-HRMS). A variety of extracts were identified that can be of great value for the cosmetic industry, since they combine antioxidant, photoprotective, anti-melanogenic and anti-aging properties. In particular, the methanolic extracts of Sideritis scardica and Rosa damascena could be worthy of further attention, since they showed interesting chemical profiles and promising properties against specific targets involved in skin aging.

In the last few decades, there has been a growing worldwide interest in the use of plant extracts for the prevention of oral diseases. The main focus of this interest lies in the identification and isolation of substances that limit the formation of microbial biofilm which plays a major role in the development of caries, periodontitis, and peri-implantitis. In this clinical ex vivo study, we investigated the antimicrobial effects of Rosmarinus officinalis extract against oral microorganisms within in situ initial oral biofilms.

Oxaliplatin is a widely used chemotherapeutic agent. Despite its many beneficial aspects in fighting many malignancies, it shares an aversive effect of neuropathy. Many substances have been used to limit this oxaliplatin-driven neuropathy in patients. This study evaluates the neuroprotective role of a grape pomace extract (GPE) into an oxaliplatin induced neuropathy in rats. For this reason, following the delivery of the substance into the animals prior to or simultaneously with oxaliplatin, their performance was evaluated by behavioral tests. Blood tests were also performed for the antioxidant activity of the extract, along with a histological and pathological evaluation of dorsal root ganglion (DRG) cells as the major components of the neuropathy. All behavioral tests were corrected following the use of the grape pomace. Oxidative stressors were also limited with the use of the extract. Additionally, the morphometrical analysis of the DRG cells and their immunohistochemical phenotype revealed the fidelity of the animal model and the changes into the parvalbumin and GFAP concentration indicative of the neuroprotective role of the pomace. In conclusion, the grape pomace extract with its antioxidant properties alleviates the harmful effects of the oxaliplatin induced chronic neuropathy in rats.

In light of the growing antibiotic resistance, the usage of plant-derived antimicrobial agents could serve as an effective alternative treatment against oral infections. The aim of this study was to investigate the antimicrobial and antibiofilm activity of Mediterranean herb extracts against representative oral microorganisms. The extraction procedures and the analysis of the obtained extracts were performed under established experimental conditions. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of the methanol extracts of Cistus creticus ssp. creticus, Cistus monspeliensis, Origanum vulgare, Rosmarinus officinalis, Salvia sclarea and Thymus longicaulis against eight typical oral bacteria and the fungus Candida albicans were determined. The antibiofilm activity against Streptococcus mutans was also quantified using the microtiter plate test. Overall, all tested extracts inhibited effectively the screened obligate anaerobic microorganisms and in concentrations ≥0.3 mg ml-1 had moderate to high antibiofilm activity comparable to that of chlorhexidine (CHX) against S. mutans. In particular, R. officinalis (MIC: 0.08-5.00 mg ml-1) and S. sclarea (MIC: 0.08-2.50 mg ml-1) showed the highest antibacterial activity, while Cistus spp., R. officinalis and S. sclarea significantly inhibited S. mutans biofilm formation at 0.60, 1.25 and 2.50 mg ml-1, respectively. Porphyromonas gingivalis and Parvimonas micra were high susceptible to O. vulgare (MIC = 0.30 mg ml-1), whereas T. longicaulis eradicated all oral bacteria (MBC: 0.15-2.50 mg ml-1). Nevertheless, C. albicans showed no sensitivity to the tested extracts. In conclusion, the tested plant extracts could serve as alternative natural antibacterial and antibiofilm components against oral infections.

Due to increasing antibiotic resistance, the application of antimicrobial photodynamic therapy (aPDT) is gaining increasing popularity in dentistry. The aim of this study was to investigate the antimicrobial effects of aPDT using visible light (VIS) and water-filtered infrared-A (wIRA) in combination with a Hypericum perforatum extract on in situ oral biofilms. The chemical composition of H. perforatum extract was analyzed using ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HRMS). To obtain initial and mature oral biofilms in situ, intraoral devices with fixed bovine enamel slabs (BES) were carried by six healthy volunteers for two hours and three days, respectively. The ex situ exposure of biofilms to VIS + wIRA (200 mWcm-2) and H. perforatum (32 mg ml-1, non-rinsed or rinsed prior to aPDT after 2-min preincubation) lasted for five minutes. Biofilm treatment with 0.2% chlorhexidine gluconate solution (CHX) served as a positive control, while untreated biofilms served as a negative control. The colony-forming units (CFU) of the aPDT-treated biofilms were quantified, and the surviving microorganisms were identified using MALDI-TOF biochemical tests as well as 16 S rDNA-sequencing. We could show that the H. perforatum extract had significant photoactivation potential at a concentration of 32 mg ml-1. When aPDT was carried out in the presence of H. perforatum, all biofilms (100%) were completely eradicated (p = 0.0001). When H. perforatum was rinsed off prior to aPDT, more than 92% of the initial viable bacterial count and 13% of the mature oral biofilm were killed. Overall, the microbial composition in initial and mature biofilms was substantially altered after aPDT, inducing a shift in the synthesis of the microbial community. In conclusion, H. perforatum-mediated aPDT using VIS + wIRA interferes with oral biofilms, resulting in their elimination or the substantial alteration of microbial diversity and richness. The present results support the evaluation of H. perforatum-mediated aPDT for the adjunctive treatment of biofilm-associated oral diseases.

Hot flashes are considered the most bothersome complaint during menopause. Although hormone therapy is an effective option to relieve hot flashes, it has been associated with significant side effects. The aim of our study is to suggest a novel combination of different plant extracts with distinct mechanisms of action against hot flashes. We selected the rhizome of Glycyrrhiza glabra L. (Fabaceae), the rhizome of Actaea racemosa L. (Ranunculaceae), the aerial parts of Hypericum perforatum L. (Hypericaceae) to produce extracts rich in bioactive phytochemicals and the seed oil of Oenothera biennis L. (Onagraceae). We investigated their estrogenic and antioxidant potential and their inhibitory effect against prostaglandin D2 receptor 1 (DP1) as a novel mechanistic pathway for vasodilation in hot flashes, alone or in combination. The phytochemical footprint of the extracts was analyzed using HPLC-PDA and UPLC-HRMS. We observed that the tested extracts possess different mechanisms of action. A. racemosa exerts a beneficial activation of the estrogen receptor, H. perforatum possesses the highest antioxidant capacity and the seed oil of O. biennis inhibits the DP1 receptor. The triple combination in the optimal doses pertains to efficacy against all three mechanisms of action, serves as a multitarget plant-based therapy and could serve as a novel strategy for the alleviation of hot flashes in postmenopausal women.

Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine, which possesses a contributing role in cancer progression and metastasis and, thus, is now considered a promising anticancer drug target. Many MIF-inactivating strategies have proven successful in delaying cancer growth. Here, we report on the synthesis of ISO-66, a novel, highly stable, small-molecule MIF inhibitor, an analog of ISO-1 with improved characteristics. The MIF:ISO-66 co-crystal structure demonstrated that ISO-66 ligates the tautomerase active site of MIF, which has previously been shown to play an important role in its biological functions. In vitro, ISO-66 enhanced specific and non-specific anticancer immune responses, whereas prolonged administration of ISO-66 in mice with established syngeneic melanoma or colon cancer was non-toxic and resulted in a significant decrease in tumor burden. Subsequent ex vivo analysis of mouse splenocytes revealed that the observed decrease in tumor growth rates was likely mediated by the selective in vivo expansion of antitumor-reactive effector cells induced by ISO-66. Compared to other MIF-inactivating strategies employed in vivo, the anticancer activity of ISO-66 is demonstrated to be of equal or better efficacy. Our findings suggest that targeting MIF, via highly specific and stable compounds, such as ISO-66, may be effective for cancer treatment and stimulation of anticancer immune responses.

Background: Ocimum sanctum L. (holy basil; Tulsi in Hindi) is an important medicinal plant, traditionally used in India. Methods: The phytochemical study of the nonpolar (dichloromethane 100%) and polar (methanol:water; 7:3) extracts yielded fourteen compounds. Compounds 6, 7, 9, 11, 12, and 13, along with the methanol:water extract were evaluated for their cytotoxicity against the human cancer cell lines MCF-7, SKBR3, and HCT-116, and normal peripheral blood mononuclear cells (PBMCs). Results: Five terpenoids, namely, ursolic acid (1), oleanolic acid (2), betulinic acid (3), stigmasterol (4), and β-caryophyllene oxide (5); two lignans, i.e., (-)-rabdosiin (6) and shimobashiric acid C (7); three flavonoids, luteolin (8), its 7-O-β-D-glucuronide (9), apigenin 7-O-β-D-glucuronide (10); and four phenolics, (E)-p-coumaroyl 4-O-β-D-glucoside (11), 3-(3,4-dihydroxyphenyl) lactic acid (12), protocatechuic acid (13), and vanillic acid (14) were isolated. Compound 6 was the most cytotoxic against the human cancer lines assessed and showed very low cytotoxicity against PBMCs. Conclusions: Based on these results, the structure of compound 6 shows some promise as a selective anticancer drug scaffold.

The aim of this study was to determine the cognitive and behavioral effects of extra virgin olive oil total phenolic content (TPC) and Sideritis (SID) extracts in female mice, and identify the associated neurochemical changes in the hippocampus and the prefrontal cortex. All animals received intraperitoneal low or high doses of TPC, SID or vehicle treatment for 7 days and were subjected to the Open Field (OF), Novel Object Recognition (NOR) and Tail Suspension Test (TST). The prefrontal cortex and hippocampus were dissected for analysis of neurotransmitters and aminoacids with high performance liquid chromatography with electrochemical detection (HPLC-ED). Both TPC doses enhanced vertical activity and center entries in the OF, which could indicate an anxiolytic-like effect. In addition, TPC enhanced non-spatial working memory and, in high doses, exerted antidepressant effects. On the other hand, high SID doses remarkably decreased the animals' overall activity. Locomotor and exploratory activities were closely associated with cortical increases in serotonin turnover induced by both treatments. Cognitive performance was linked to glutamate level changes. Furthermore, TPC reduced cortical taurine levels, while SID reduced cortical aspartate levels. TPC seems to have promising cognitive, anxiolytic and antidepressant effects, whereas SID has sedative effects in high doses. Both extracts act in the brain, but their specific actions and properties merit further exploration.

Given the undesirable side effects of commercially used mouth rinses that include chemically synthesized antimicrobial compounds such as chlorhexidine, it is essential to discover novel antimicrobial substances based on plant extracts. The aim of this study was to examine the antimicrobial effect of Inula viscosa extract on the initial microbial adhesion in the oral cavity. Individual test splints were manufactured for the participants, on which disinfected bovine enamel samples were attached. After the initial microbial adhesion, the biofilm-covered oral samples were removed and treated with different concentrations (10, 20, and 30 mg/mL) of an I. viscosa extract for 10 min. Positive and negative controls were also sampled. Regarding the microbiological parameters, the colony-forming units (CFU) and vitality testing (live/dead staining) were examined in combination with fluorescence microscopy. An I. viscosa extract with a concentration of 30 mg/mL killed the bacteria of the initial adhesion at a rate of 99.99% (log10 CFU value of 1.837 ± 1.54). Compared to the negative control, no killing effects were determined after treatment with I. viscosa extract at concentrations of 10 mg/mL (log10 CFU value 3.776 ± 0.831; median 3.776) and 20 mg/mL (log10 CFU value 3.725 ± 0.300; median 3.711). The live/dead staining revealed a significant reduction (p < 0.0001) of vital adherent bacteria after treatment with 10 mg/mL of I. viscosa extract. After treatment with an I. viscosa extract with a concentration of 30 mg/mL, no vital bacteria could be detected. For the first time, significant antimicrobial effects on the initial microbial adhesion in in situ oral biofilms were reported for an I. viscosa extract.

Prothymosin alpha (ProTα) is a conserved mammalian polypeptide with intracellular functions associated with cell proliferation and apoptosis and an extracellular role associated with immunopotentiation. The N-terminal fragment [1-28], which is identical with the immunostimulating peptide thymosin α1 (Tα1), was earlier considered as the immunoactive region of the polypeptide; however, recent data suggest that ProTα may exert a discrete immunomodulating action through its central or C-terminal region, via targeting Toll-like receptor- 4 (TLR4). In this work, a derivative of the C-terminal fragment ProTα[100-109] (ProTα-D1) that can be radiolabeled with (99m)Tc was developed. The biological activity of the non-radioactive (185/187)rhenium-complex of this derivative ([(185/187)Re]ProTα-D1, structurally similar with [(99m)Tc]ProTα-D1) was verified through suitable in vitro bioassays on human neutrophils. Subsequent cell-binding studies revealed specific, time-dependent and saturable binding of [(99m)Tc]ProTα-D1 on neutrophils, which was inhibited by intact ProTα and ProTα[100-109], as well as by a "prototype" TLR4-ligand (lipopolysaccharide from Escherichia coli). Overall, our results support the existence of ProTα-binding sites on human neutrophils, recognizing [(99m)Tc]ProTα-D1, which might involve TLR4. [(99m)Tc]ProTα-D1 may be a useful tool for conducting further in vitro and in vivo studies, aiming to elucidate the extracellular mode of action of ProTα and, eventually, develop ProTα-based immunotherapeutics.

Active cancer immunotherapies are beginning to yield clinical benefit, especially those using peptide-pulsed dendritic cells (DCs). Different adjuvants, including Toll-like receptor (TLR) agonists, commonly co-administered to cancer patients as part of a DC-based vaccine, are being widely tested in the clinical setting. However, endogenous DCs in tumor-bearing individuals are often dysfunctional, suggesting that ex vivo educated DCs might be superior inducers of anti-tumor immune responses. We have previously shown that prothymosin alpha (proTα) and its immunoreactive decapeptide proTα(100-109) induce the maturation of human DCs in vitro. The aim of this study was to investigate whether proTα- or proTα(100-109)-matured DCs are functionally competent and to provide preliminary evidence for the mode of action of these agents.

Apoptosis is characterized by a series of discrete biochemical events, among which is the truncation of the nuclear polypeptide prothymosin alpha (proTα) by activated caspase-3. This early apoptotic event results in the generation of a carboxy-terminal fragment of proTα, the immunoactive decapeptide proTα(100-109). We hypothesized that the detection of increased levels of proTα(100-109) in serum can be directly correlated with the induction of massive cell apoptosis, resulting from a severe bacterial infection. Thus, using high-affinity-purified polyclonal antibodies (Abs), raised in rabbits and a prototype antibody-capture system, we developed a highly sensitive and specific competitive ELISA for proTα(100-109). The sensitivity of the ELISA (0.1ng/mL to 10μg/mL) is acceptable for the quantification of the decapeptide in serum samples. To assess our initial hypothesis, we determined the concentration of proTα(100-109) in the sera of mice infected with the bacterium Streptococcus pyogenes over the course of the infection. We show that serum concentration of proTα(100-109) was marginal to undetectable before infection, increased over time and peaked at 72h postinfection. In silico analysis suggests that the Abs generated are unlikely to cross-react with any other unrelated mouse or bacterial protein. Further validation of our ELISA using serum samples from humans, infected with bacteria, may provide a useful tool to differentiate the causative agent of a potentially lethal septic infection.

Natural products are characterized by extreme structural diversity and thus they offer a unique source for the identification of novel anti-tumor agents. Herein, we report that the herbal substance acteoside being isolated by advanced phytochemical methods from Lippia citriodora leaves showed enhanced cytotoxicity against metastatic tumor cells; acted in synergy with various cytotoxic agents and it sensitized chemoresistant cancer cells. Acteoside was not toxic in physiological cellular contexts, while it increased oxidative load, affected the activity of proteostatic modules and suppressed matrix metalloproteinases in tumor cell lines. Intraperitoneal or oral (via drinking water) administration of acteoside in a melanoma mouse model upregulated antioxidant responses in the tumors; yet, only intraperitoneal delivery suppressed tumor growth and induced anti-tumor-reactive immune responses. Mass-spectrometry identification/quantitation analyses revealed that intraperitoneal delivery of acteoside resulted in significantly higher, vs. oral administration, concentration of the compound in the plasma and tumors of treated mice, suggesting that its in vivo anti-tumor effect depends on the route of administration and the achieved concentration in the tumor. Finally, molecular modeling studies and enzymatic activity assays showed that acteoside inhibits protein kinase C. Conclusively, acteoside holds promise as a chemical scaffold for the development of novel anti-tumor agents.