First Exploration of the Altered Microbial Gut-Lung Axis in the Pathogenesis of Human Refractory Chronic Cough.

PURPOSE: Cough represents a natural mechanism that plays an important defensive role in the respiratory tract, but in some conditions, it may become persistent, nonproductive, and harmful. In general, refractory chronic cough (RCC) occurs in about 20% of individuals; hence, we aimed to assess the presence of altered gut-lung communication in RCC patients through a compositional and functional characterization of both gut (GM) and oral microbiota (OM). METHODS: 16S rRNA sequencing was used to characterize both GM and OM composition of RCC patients and healthy controls (HC). PICRUST2 assessed functional changes in microbial communities while gas chromatography was used to evaluate fecal short-chain fatty acid levels and serum-free fatty acid (FFA) abundances. RESULTS: In comparison with HC, RCC patients reported increased saliva alpha-diversity and statistically significant beta-diversity in both GM and OM. Also, a, respectively, significant increased or reduced Firmicutes/Bacteroidota ratio in stool and saliva samples of RCC patients has been shown, in addition to a modification of the abundances of several taxa in both GM and OM. Moreover, a potential fecal over-expression of lipopolysaccharide biosynthesis and lipoic acid metabolism pathways and several differences in serum FFA levels have been reported in RCC patients than in HC. CONCLUSION: Since differences in both GM and OM of RCC patients have been documented, these findings could provide new information about RCC pathogenesis and also pave the way for the development of novel nutritional or pharmacological interventions for the management of RCC through the restoration of eubiotic gut-lung communication.

Tisochrysis lutea F&M-M36 Mitigates Risk Factors of Metabolic Syndrome and Promotes Visceral Fat Browning through ß3-Adrenergic Receptor/UCP1 Signaling.

Pre-metabolic syndrome (pre-MetS) may represent the best transition phase to start treatments aimed at reducing cardiometabolic risk factors of MetS. In this study, we investigated the effects of the marine microalga Tisochrysis lutea F&M-M36 (T. lutea) on cardiometabolic components of pre-MetS and its underlying mechanisms. Rats were fed a standard (5% fat) or a high-fat diet (20% fat) supplemented or not with 5% of T. lutea or fenofibrate (100 mg/Kg) for 3 months. Like fenofibrate, T. lutea decreased blood triglycerides (p < 0.01) and glucose levels (p < 0.01), increased fecal lipid excretion (p < 0.05) and adiponectin (p < 0.001) without affecting weight gain. Unlike fenofibrate, T. lutea did not increase liver weight and steatosis, reduced renal fat (p < 0.05), diastolic (p < 0.05) and mean arterial pressure (p < 0.05). In visceral adipose tissue (VAT), T. lutea, but not fenofibrate, increased the ß3-adrenergic receptor (ß3ADR) (p < 0.05) and Uncoupling protein 1 (UCP-1) (p < 0.001) while both induced glucagon-like peptide-1 receptor (GLP1R) protein expression (p < 0.001) and decreased interleukin (IL)-6 and IL-1ß gene expression (p < 0.05). Pathway analysis on VAT whole-gene expression profiles showed that T. lutea up-regulated energy-metabolism-related genes and down-regulated inflammatory and autophagy pathways. The multitarget activity of T. lutea suggests that this microalga could be useful in mitigating risk factors of MetS.

Colon fibroblasts from Pirc rats (F344/NTac-Apcam1137 ) exhibit a proliferative and inflammatory phenotype that could support early stages of colon carcinogenesis.

The role of fibroblast APC mutation in carcinogenesis is not clear. Apc+/- colon fibroblasts have been previously characterized: however, little is known about their behavior at very early-stage of colon carcinogenesis. We cultured colon mucosa fibroblasts (PCF, Apc+/- ) of Pirc rats (F344/NTac-Apcam1137 ) at an early stage of tumorigenesis, in absence of preneoplastic lesions, and of age-matched wt (WCF): DNA damage levels, inflammatory phenotype and the expression of known markers of CAFs were analyzed. The latter were also assessed by microarray analysis on colon normal mucosa of Pirc and wt animals. PCF exhibited higher proliferative rates (P < .001) and delayed replicative senescence onset (P < .05) compared to WCF, along with a lower level of oxidative DNA damage (P < .05). Furthermore, a constitutively higher expression of COX-2 and sensitivity to inflammatory stimuli was found in PCF compared to WCF (P < .05), accompanied by higher invasive capability (P < .05) and presence of cytoplasmic chromatin foci (cytoplasmic chromatin foci, P < .05). However, they neither expressed CAFs markers (α-SMA, IL-6) nor responded to CAFs activating stimuli (TGF-ß). Accordingly, CAFs markers and activating stimuli resulted down-regulated in Pirc normal mucosa compared to wt, whereas DNA damage response and tolerance pathways were overexpressed. These data show for the first time that a proliferative and inflammatory phenotype characterizes Apc+/- colon fibroblasts since very early stages of colon tumorigenesis, and indicate a role of Apc mutation in driving fibroblast phenotypic alterations that could support the establishment of a protumorigenic environment. Early pharmacological targeting of these dysfunctions might impact on tumor prevention in FAP patients.

Extracellular vesicles miR-210 as a potential biomarker for diagnosis and survival prediction of oral squamous cell carcinoma patients.

BACKGROUND: The identification of early diagnostic and prognostic biomarkers in oral squamous cell carcinoma (OSCC) is an unmet clinical need. We hypothesized that extracellular vesicles miR-210 expression (EV-miR-210) could be a potential biomarker for OSCC diagnosis and follow-up. METHODS: The expression of EV-miR-210 was measured in the plasma of OSCC patients (n = 30) and compared to that of controls (n = 14). RESULTS: The median EV-miR-210 expression was significantly higher in OSCC patients compared to controls who had often, undetectable levels (p < 0.0001). We performed receiver operating characteristic (ROC) analysis for discriminating OSCC cases from controls. EV-miR-210 yielded an area under the curve (AUC) of 0.9513 with sensitivity 92.3% and specificity 86.6%. Kaplan-Meier curves indicated that high EV-miR-210 expression was associated with worse 3 years' survival (p < 0.05). Cox regression hazard model indicated that high EV-miR-210, G2, and G3 grading and pathological nodal status (pN)>1 were independent predictors of worse survival in OSCC patients. CONCLUSION: These preliminary data suggest that EV-mir-210 may be a novel diagnostic and prognostic biomarker in OSCC.

Is the age at surgery in Crohn's disease clinically relevant? Differences and peculiarities: a wide single centre experience after long-term follow-up.

PURPOSE: The Montreal classification for Crohn's disease includes "age at diagnosis" as a parameter but few is reported about the age at surgery. The aim of this study is to evaluate the short- and long-term differences in the postoperative surgical outcome and disease behaviour, according to the age at the first surgery. METHODS: Patients consecutively operated for abdominal Crohn's disease during the period 1986-2012 at our centre were systematically analysed according to their age at first surgery. In our retrospective cohort, the age at first surgery ranged from 13 to 83 years, and patients were arbitrarily divided into four groups: ≤ 19 (G1), 20-39 (G2), 40-59 (G3) and ≥ 60 (G4) years old. RESULTS: In total, 1051 patients were included with a median follow-up time of 232 months. The four groups exhibited statistically significant differences in age at diagnosis, smoke habit, time between diagnosis and surgery, disease location and behaviour, history of perianal fistula or abscess, severe malnutrition requiring total parental nutrition before surgery, type of surgery, total length of resected bowel, median duration of hospitalization, incidence of abdominal recurrences and number of surgical recurrences. G1 displays an inverse linear trend with time in the severity of clinical characteristics when compared to G4 groups. On the contrary, the incidence of short-term complications, types of abdominal recurrence and presence of concomitant perianal disease did not vary among groups. In addition, at multivariate analysis, the age at surgery and the disease location were the only independent risk factors for abdominal surgical recurrence. CONCLUSION: Despite first surgery is extremely more frequent between 20 and 59 years, patients from G1 and G4 groups showed clinical differences and peculiarities when compared to the other age groups. The most indolent CD behaviour and occurrence of surgical recurrence was observed in patients having their first abdominal surgery in the elderly, while patients operated before the age of 19 experienced a more aggressive disease course.

Long-Term Follow-Up, Association between CARD15/NOD2 Polymorphisms, and Clinical Disease Behavior in Crohn's Disease Surgical Patients.

BACKGROUND: CARD15/NOD2 is the most significant genetic susceptibility in Crohn's disease (CD) even though a relationship between the different polymorphisms and clinical phenotype has not been described yet. The study is aimed at analyzing, in a group of CD patients undergoing surgery, the relationship between CARD15/NOD2 polymorphisms and the clinical CD behavior after a long-term follow-up, in order to identify potential clinical biomarkers of prognosis. METHODS: 191 surgical CD patients were prospectively characterized both for the main single nucleotide polymorphisms of CARD15/NOD2 and for many other environmental risk factors connected with the severe disease form. After a mean follow-up of 7.3 years, the correlations between clinical features and CD natural history were analyzed. RESULTS: CARD15/NOD2 polymorphisms were significantly associated with younger age at diagnosis compared to wild type cases (p < 0.05). Moreover, patients carrying a 3020insC polymorphism presented a larger Δ between diagnosis and surgery (p = 0.0344). Patients carrying an hz881 and a 3020insC exhibited, respectively, a lower rate of responsiveness to azathioprine (p = 0.012), but no difference was found in biologic therapy. Finally, the risk of surgical recurrence was significantly associated, respectively, to age at diagnosis, to familial CD history, to diagnostic delay, to arthritis, and to the presence of perioperative complications. CONCLUSIONS: 3020insC CARD15 polymorphism is associated with an earlier CD onset, and age at CD diagnosis < 27 years was confirmed to have a detrimental effect on its clinical course. In addition, the familiarity seems to be connected with a more aggressive postoperative course. Finally, for the first time, we have observed a lower rate of responsiveness to azathioprine in patients carrying an hz881 and a 3020insC.

A Comparative In Vitro Evaluation of the Anti-Inflammatory Effects of a Tisochrysis lutea Extract and Fucoxanthin.

In this study, we compared the effects of a Tisochrysis lutea (T. lutea) F&M-M36 methanolic extract with those of fucoxanthin (FX) at equivalent concentration, on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The T. lutea F&M-M36 methanolic extract contained 4.7 mg of FX and 6.22 mg of gallic acid equivalents of phenols per gram. HPLC analysis revealed the presence of simple phenolic acid derivatives. The T. lutea F&M-M36 extract exhibited a potent and concentration-dependent inhibitory activity against COX-2 dependent PGE2 production compared to FX alone. Compared to LPS, T. lutea F&M-M36 extract and FX reduced the expression of IL-6 and of Arg1 and enhanced that of IL-10 and of HO-1; T. lutea F&M-M36 extract also significantly abated the expression of NLRP3, enhanced mir-223 expression and reduced that of mir-146b, compared to LPS (p < 0.05). These findings indicate that T. lutea F&M-M36 methanolic extract has a peculiar anti-inflammatory activity against COX-2/PGE2 and NLRP3/mir-223 that might be attributable to the known anti-inflammatory effects of simple phenolic compounds found in the extract that may synergize with FX. Our data suggest that T. lutea F&M-M36 may serve as a source of anti-inflammatory compounds to be further evaluated in in vivo models of inflammation.

Aromatic Sulfonamides including a Sulfonic Acid Tail: New Membrane Impermeant Carbonic Anhydrase Inhibitors for Targeting Selectively the Cancer-Associated Isoforms.

We report here a new drug design strategy for producing membrane-impermeant carbonic anhydrase (CA; EC 4.2.1.1) inhibitors selectively targeting the tumor-associated, membrane-bound human CAs IX and XII over off-target cytosolic isoforms. To date, this approach has only been pursued by including permanent positively charged pyridinium type or highly hydrophilic glycosidic moieties into the structure of aromatic sulfonamide CA inhibitors (CAIs). Aliphatic (propyl and butyl) sulfonic acid tails, deprotonated at physiological pH, were thus incorporated onto a benzenesulfonamide scaffold by a common 1,2,3-triazole linker and different types of spacers. Twenty such derivatives were synthesized and tested for their inhibition of target (hCAs IV, IX, and XII) and off-target CAs (hCAs I and II). Most sulfonate CAIs induced a potent inhibition of hCAs II, IX, and XII up to a low nanomolar KI range (0.9-459.4 nM) with a limited target/off-target CA selectivity of action. According to the drug design schedule, a subset of representative derivatives was assessed for their cell membrane permeability using Caco-2 cells and a developed FIA-MS/MS method. The complete membrane impermeability of the sulfonate tailed CAIs (≥98%) validated these negatively charged moieties as being suitable for achieving, in vivo, the selective targeting of the tumor-associated CAs over off-target ones.

Nanostructured Lipid Carriers Can Enhance Oral Absorption of Khellin, a Natural Pleiotropic Molecule.

A novel formulation based on nanostructured lipid carriers (NLCs) was developed to increase solubility and intestinal absorption of khellin. K-NLCs were prepared with stearic acid, hempseed oil, Brij S20, and Labrafil M 1944 CS, using the emulsification-ultrasonication method. Developed nanoparticles were chemically and physically characterized by liquid chromatography, light scattering techniques, and electron microscopy. The size, about 200 nm, was optimal for oral delivery, and the polydispersity index (around 0.26), indicated high sample homogeneity. Additionally, K-NLCs showed a spherical morphology without aggregation by microscopic analysis. The encapsulation efficiency of khellin was about 55%. In vitro release studies were carried out in media with different pH to mimic physiological conditions. K-NLCs were found to be physically stable in the simulated gastric and intestinal fluids, and they preserved about 70% of khellin after 6 h incubation. K-NLCs were also successfully lyophilized testing different lyoprotectants, and obtained freeze-dried K-NLCs demonstrated good shelf life over a month. Lastly, permeability studies on Caco-2 cells were performed to predict khellin passive diffusion across the intestinal epithelium, demonstrating that nanoparticles increased khellin permeability by more than two orders of magnitude. Accordingly, developed NLCs loaded with khellin represent a versatile formulation with good biopharmaceutical properties for oral administration, possibly enhancing khellin's bioavailability and therapeutic effects.

A flavonoid-rich extract from bergamot juice prevents carcinogenesis in a genetic model of colorectal cancer, the Pirc rat (F344/NTac-Apcam1137).

PURPOSE: To determine the potential of a flavonoid-rich extract from bergamot juice (BJe) to prevent colorectal carcinogenesis (CRC) in vivo. MAIN METHODS: Pirc rats (F344/NTac-Apcam1137), mutated in Apc, the key gene in CRC, were treated with two different doses of BJe (35 mg/kg or 70 mg/kg body weight, respectively) mixed in the diet for 12 weeks. Then, the entire intestine was surgically removed and dissected for histological, immunohistochemical and molecular analyses. RESULTS: Rats treated with BJe showed a significant dose-related reduction in the colon preneoplastic lesions mucin-depleted foci (MDF). Colon and small intestinal tumours were also significantly reduced in rats supplemented with 70 mg/kg of BJe. To elucidate the involved mechanisms, markers of inflammation and apoptosis were determined. Compared to controls, colon tumours from BJe 70 mg/kg-supplemented rats showed a significant down-regulation of inflammation-related genes (COX-2, iNOS, IL-1ß, IL-6 and IL-10 and Arginase 1). Moreover, in colon tumours from rats fed with 70 mg/kg BJe, apoptosis was significantly higher than in controls. Up-regulation of p53 and down-regulation of survivin and p21 genes was also observed. CONCLUSIONS: These data indicate a strong chemopreventive activity of BJe that, at least in part, is due to its pro-apoptotic and anti-inflammatory actions. This effect could be exploited as a strategy to prevent CRC in high-risk patients.

Formulation of a Phenol-Rich Extract from Unripe Olives (Olea europaea L.) in Microemulsion to Improve Its Solubility and Intestinal Permeability.

The beneficial properties of phenolic compounds from Olea europaea L. are well-known. An olive extract (OE) was prepared from unripe olives (Moraiolo cultivar). The study aimed to formulate OE into a microemulsion (ME) in oral dosage form. OE was extracted from olives with EtOH:H2O (80:20) and characterized by HPLC-DAD. ME composition was stated by a solubility and pseudo-ternary diagram. The ME was chemically and physically characterized, and its stability at 4 °C was analyzed for three months. The ability of the formulation to ameliorate the solubility and the intestinal permeability of OE was evaluated by a Parallel Artificial Membrane Permeability Assay (PAMPA) assay and Caco-2 cells. The total phenolic content of the extract was 39% w/w. The main constituent was oleuropein (31.0%), together with ligstroside (3.1%) and verbascoside (2.4%). The ME was prepared using Capryol 90 as the oily phase, and Cremophor EL and Transcutol (2:1) as surfactant and co-surfactant, respectively. ME droplet size was 14.03 ± 1.36 nm, PdI 0.20 ± 0.08, ζ-potential -1.16 ± 0.48. Stability of ME was confirmed for at least three months. The formulation was loaded with 35 mg/mL of OE, increasing the solubility of the extract by about four times. The enhanced permeability of OE was evaluated by PAMPA, as demonstrated by the Pe value (1.44 ± 0.83 × 10-6 cm/s for OE hydroalcoholic solution, 3.74 ± 0.34 × 10-6 cm/s for OE-ME). Caco-2 cell transport studies confirmed the same results: Papp was 16.14 ± 0.05 × 10-6 cm/s for OE solution and 26.99 ± 0.45 × 10-6 cm/s for OE-ME. ME proved to be a suitable formulation for oral delivery.

Formulation of Nanomicelles to Improve the Solubility and the Oral Absorption of Silymarin.

Two novel nanomicellar formulations were developed to improve the poor aqueous solubility and the oral absorption of silymarin. Polymeric nanomicelles made of Soluplus and mixed nanomicelles combining Soluplus with d-α-tocopherol polyethylene glycol 1000 succinate (vitamin E TPGS) were prepared using the thin film method. Physicochemical parameters were investigated, in particular the average diameter, the homogeneity (expressed as polydispersity index), the zeta potential, the morphology, the encapsulation efficiency, the drug loading, the critical micellar concentration and the cloud point. The sizes of ~60 nm, the narrow size distribution (polydispersity index ≤0.1) and the encapsulation efficiency >92% indicated the high affinity between silymarin and the core of the nanomicelles. Solubility studies demonstrated that the solubility of silymarin increased by ~6-fold when loaded into nanomicelles. Furthermore, the physical and chemical parameters of SLM-loaded formulations stored at room temperature and in refrigerated conditions (4 °C) were monitored over three months. In vitro stability and release studies in media miming the physiological conditions were also performed. In addition, both formulations did not alter the antioxidant properties of silymarin as evidenced by the 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) assay. The potential of the nanomicelles to increase the intestinal absorption of silymarin was firstly investigated by the parallel artificial membrane permeability assay. Subsequently, transport studies employing Caco-2 cell line demonstrated that mixed nanomicelles statistically enhanced the permeability of silymarin compared to polymeric nanomicelles and unformulated extract. Finally, the uptake studies indicated that both nanomicellar formulations entered into Caco-2 cells via energy-dependent mechanisms.

New NO- and H2S-releasing doxorubicins as targeted therapy against chemoresistance in castration-resistant prostate cancer: in vitro and in vivo evaluations.

Chemotherapy for castration-resistant prostate cancer (CRPC) is only temporarily effective due to the onset of chemoresistance. We investigated the efficacy of NO- and H2S-releasing doxorubicins (NitDox and H2SDox) in overcoming drug resistance and evaluated their safety. New and innovative NO- and H2S-releasing doxorubicins (NitDox and H2SDox) showed a good intracellular accumulation and high cytotoxic activity in vitro in an androgen-independent and doxorubicin-resistant DU-145 prostate cancer cell line. Nude mice were subcutaneously injected with 4*106 DU-145 cells and treated once a week for 3 weeks with 5 mg/kg doxorubicin, NitDox, H2SDox or vehicle, i.p. Animal weight, tumor volume, intra-tumoral drug accumulation, apoptosis and the presence of nitrotyrosine and sulfhydryl (SH) groups within the tumor, were evaluated. Cardiotoxicity was assessed by measuring troponin plasma levels and the left ventricular wall thickness. In vivo, NitDox and H2SDox accumulated inside the tumors, significantly reduced tumor volumes by 60%, increased the percentage of apoptotic cells in both the inner and the outer parts of the tumors and the presence of nitrotyrosine and SH groups. Doxorubicin treatment was associated with reduced body weight and cardiotoxicity. On the contrary, NitDox and H2SDox were well tolerated and had a better safety profile. Combining efficacy with reduced cardiovascular side effects, NitDox and H2SDox are promising novel therapeutic agents for reversing chemoresistance in CRCP.

Enhanced Solubility and Permeability of Salicis cortex Extract by Formulating as a Microemulsion.

A microemulsion system was developed and investigated as a novel oral formulation to increase the solubility and absorption of Salicis cortex extract. This extract possesses many pharmacological activities, in particular, it is beneficial for back pain and osteoarthritic and rheumatic complaints. In this work, after qualitative and quantitative characterization of the extract and the validation of an HPLC/diode array detector analytical method, solubility studies were performed to choose the best components for microemulsion formulation. The optimized microemulsion consisted of 2.5 g of triacetin, as the oil phase, 2.5 g of Tween 20 as the surfactant, 2.5 g of labrasol as the cosurfactant, and 5 g of water. The microemulsion was visually checked, characterized by light scattering techniques and morphological observations. The developed formulation appeared transparent, the droplet size was around 40 nm, and the ζ-potential result was negative. The maximum loading content of Salicis cortex extract resulted in 40 mg/mL. Furthermore, storage stability studies and an in vitro digestion assay were performed. The advantages offered by microemulsion were evaluated in vitro using artificial membranes and cells, i.e., parallel artificial membrane permeability assay and a Caco-2 model. Both studies proved that the microemulsion was successful in enhancing the permeation of extract compounds, so it could be useful to ameliorate the bioefficacy of Salicis cortex.

A nutrigenomics approach for the study of anti-aging interventions: olive oil phenols and the modulation of gene and microRNA expression profiles in mouse brain.

PURPOSE: Middle-aged C57Bl/6J mice fed for 6 months with extra-virgin olive oil rich in phenols (H-EVOO, phenol dose/day: 6 mg/kg) showed cognitive and motor improvement compared to controls fed the same olive oil deprived of phenolics (L-EVOO). The aim of the present study was to evaluate whether these behavioral modifications were associated with changes in gene and miRNA expression in the brain. METHODS: Two brain areas involved in cognitive and motor processes were chosen: cortex and cerebellum. Gene and miRNA profiling were analyzed by microarray and correlated with performance in behavioral tests. RESULTS: After 6 months, most of the gene expression changes were restricted to the cerebral cortex. The genes modulated by aging were mainly down-regulated, and the treatment with H-EVOO was associated with a significant up-regulation of genes compared to L-EVOO. Among those, we found genes previously associated with synaptic plasticity and with motor and cognitive behavior, such as Notch1, BMPs, NGFR, GLP1R and CRTC3. The agrin pathway was also significantly modulated. miRNAs were mostly up-regulated in old L-EVOO animals compared to young. However, H-EVOO-fed mice cortex displayed miRNA expression profiles similar to those observed in young mice. Sixty-three miRNAs, out of 1203 analyzed, were significantly down-regulated compared to the L-EVOO group; among them, we found miRNAs whose predicted target genes were up-regulated by the treatment, such as mir-484, mir-27, mir-137, mir-30, mir-34 and mir-124. CONCLUSIONS: We are among the first to report that a dietary intervention starting from middle age with food rich in phenols can modulate at the central level the expression of genes and miRNAs involved in neuronal function and synaptic plasticity, along with cognitive, motor and emotional behavior.

Prediction of Permeation and Cellular Transport of Silybum marianum Extract Formulated in a Nanoemulsion by Using PAMPA and Caco-2 Cell Models.

The present study explores the potential of nanoemulsion, a lipid drug delivery system, to improve solubility and oral absorption of Silybum marianum extract. The optimized formulation contained 40 mg/mL of commercial extract (4 % w/w) and it was composed of 2.5 g labrasol (20 %) as the oil phase, 1.5 g cremophor EL as the surfactant, and 1 g labrafil as the cosurfactant (mixture surfactant/cosurfactant, 20 %).The system was characterized by dynamic light scattering, transmission electron microscopy, and HPLC-DAD analyses in order to evaluate size, homogeneity, morphology, and encapsulation efficiency. Physical and chemical stabilities were assessed during 40 days at 4 °C and 3 months at 25 °C. Stability in simulated gastric fluid followed by simulated intestinal conditions was also considered. In vitro permeation studies were performed to determine the suitability of the prepared nanoemulsion for oral delivery. Different models such as the parallel artificial membrane permeability assay and Caco-2 cell lines were applied.The nanoemulsion showed a good solubilizing effect of the extract, with a pronounced action also on its permeability, in respect to a saturated aqueous solution. The Caco-2 test confirmed the parallel artificial membrane permeability assay results and they revealed the suitability of the prepared nanoemulsion for oral delivery.

Multiple mucin depleted foci, high proliferation and low apoptotic response in the onset of colon carcinogenesis of the PIRC rat, mutated in Apc.

PIRC rats (F344/NTac-Apc (am1137) ) mutated in the Apc gene spontaneously develop colon tumors thus mimicking familial adenomatous polyposis (FAP) and sporadic colorectal cancer (CRC) more closely than Apc-based rodent models developing tumors mostly in the small intestine. To understand whether microscopic dysplastic lesions precede the development of macroscopic tumors, PIRC rat colon was examined for the presence of mucin depleted foci (MDF), microadenomas of the rodent and human colon. Few MDF (about 4/animal) were already present in 1-month-old rats and their number rapidly increases to about 250 in 8-month-old rats. These lesions showed Wnt signaling activation (nuclear ß-catenin accumulation) and were dramatically decreased by sulindac (320 ppm), a drug with chemopreventive activity (MDF/rat at 4 months: 156 ± 8 and 38 ± 6 in controls and sulindac-treated rats, respectively, means ± SE, p < 0.001). Since altered proliferation and apoptosis could underlie the early phases of carcinogenesis, we studied these processes in the apparently normal colon mucosa (NM) of 1-month-old PIRC and wt rats. Colon proliferation (PCNA expression) was significantly higher in PIRC rats. Notably, PIRC rat NM showed resistance to apoptosis since it sustained proliferation and had lower apoptosis after a cytotoxic insult with 1,2 dimethylhydrazine. Gene expression of Myc, p21, Birc5, Ogg1, Apex1 and Sod2 were significantly up-regulated in the NM of PIRC rat. The overall results put forward PIRC rat as useful model of colon carcinogenesis, either to study the process itself or to test in vivo chemopreventive agents in both short- and long-term studies.

Sulindac, 3,3'-diindolylmethane and curcumin reduce carcinogenesis in the Pirc rat, an Apc-driven model of colon carcinogenesis.

BACKGROUND: Recently, we showed that Sulindac (SU; 320 ppm) reduces precancerous lesions in the colon of Pirc rats, mutated in the Apc gene. Surprisingly, previous data in Apc-mutated mice showed that SU, with reported efficacy in Familial Adenomatous Polyposis (FAP), increases colon carcinogenesis. Therefore, we assessed the effect of SU 320 ppm in a long-term carcinogenesis experiment in Pirc rats. Moreover, since side effects of SU hamper its chronic use and a combination of drugs could be more effective and less toxic than single agents, we also studied whether two natural compounds, 3,3'-diindolylmethane (DIM; 250 ppm) and curcumin (CUR; 2000 ppm), with or without lower doses of SU could affect carcinogenesis METHODS: Pirc rats were fed an AIN76 diet containing SU, DIM and CUR and sacrificed at 8 months of age to measure intestinal tumours. Apoptosis and proliferation in the normal colon mucosa, as well as gene expression profile were studied RESULTS: Colon tumours were significantly reduced by SU 320 ppm (62 % reduction over Controls), by DIM and CUR without or with SU 80 and 160 ppm (50, 53 and 58 % reduction, respectively) but not by SU 80 ppm alone. Total tumours (colon and small intestine) were reduced by SU (80 and 320 ppm) and by DIM and CUR. Apoptosis in the normal mucosa was significantly increased by SU 320 ppm, and slightly increased by DIM and CUR with or without SU. A slight reduction in Survivin-Birc5 expression was observed with all the treatments compared to Controls. Proliferative activity was not varied CONCLUSIONS: The results on SU reinforce the validity of Pirc rats to identify chemopreventive products. Moreover, the efficacy of the DIM and CUR combination to lower colon tumours, suggests an alternative strategy to be exploited in patients at risk.

Exploring the effects of homeopathic Apis mellifica preparations on human gene expression profiles.

BACKGROUND: Diluted preparations obtained from Apis mellifica are reported in the homeopathic literature to have anti-inflammatory activity. The present study was designed to explore the effects on global gene expression profiles of human cells by means of microarrays, using Apis mellifica mother tincture (TM) and its 3C, 5C, 7C dynamized dilutions; the technique employed allowed us to study the changes in gene expression at concentrations much lower than those associated with pharmacological responses. METHODS: An RWPE-1 cell line (human immortalized prostate epithelial cells) was used to study the effects on global gene expression by transcriptomic analysis. RESULTS: Apis mellifica TM and its 3C, 5C, 7C dynamized dilutions modulated hundreds of genes; using cluster analysis we observed groups of genes up- or down-regulated with similar expression profiles among treatments; other genes showed opposite regulation profiles at low and high dilutions of Apis mellifica, suggesting a hormetic response. In particular, genes involved in cytokine expression, inflammatory processes, anti-oxidative responses and proteasome degradation were differentially, and sometimes divergently expressed by the TM or by Apis mellifica 3C, 5C and 7C dilutions. We confirmed these data by RT-PCR analyses on 5 selected candidate genes (IL1ß, CD46, ATF1, UBE2Q2 and MT1X). CONCLUSIONS: Apis mellifica TM modifies gene expression in human cells and has inhibitory effects on regulatory processes of inflammation; in addition, extremely diluted dynamized dilutions (3C, 5C and 7C) still exert significant effects on genes involved in inflammation and oxidative stress.

Cyclic Acetals as Novel Long-Lasting Mosquito Repellents.

The use of skin repellents against hematophagous mosquitoes is an important personal protection practice wherever these insects are abundant and where they are vectors of diseases. DEET and Icaridin are the major synthetic insect repellents in commercial formulations and are considered the most effective. Here, we tested against the mosquito Aedes albopictus several cyclic hydroxyacetals synthesized by acetalization of commercially available aliphatic carbonyl compounds (ranging from C3 to C15) with either glycerol, 1,1,1-trismethyloletane, or 1,1,1-trismethylolpropane and compared their efficacy with commercial repellents. We found that several hydroxyacetals were comparable with DEET and Icaridin both in terms of the required dose and repellence duration, while a few performed better. For those most active, toxicity was investigated, finding that a few of them were less cytotoxic than DEET and less prone to permeate through cell layers. Therefore, such results indicate that novel safe mosquito repellents could be developed among cyclic hydroxyacetals.