Toxicity and behavioural effects of ocfentanil and 2-furanylfentanyl in zebrafish larvae and mice.

The introduction of the so-called New Psychoactive Substances represents a problem of global concern due to several factors, including multiplicity of structures, poorly known activity, short half-life in the market, lack of pure standards etc. Among these problems, of the highest relevance is also the lack of information about metabolism and adverse effects, which must be faced using simple and low-cost animal models. On these grounds, the present work has been carried out on 5 days post fertilization zebrafish (Danio rerio) larvae in comparison with adult mice (Mus musculus). Ocfentanil and 2-furanylfentanyl were administered at different concentrations to zebrafish larvae (1, 10 µM) and mice (0.1, 1, 6, 15 mg/kg). The behavioural assay showed a decrease in basal locomotor activity in zebrafish, whereas in mice this effect was evident only after the mechanical stimulus. Larva extracts and mice urine were analysed by using liquid chromatography coupled to high resolution mass spectrometry to identify the metabolic pathways of the fentanyl analogs. For 2-furanylfentanyl, the most common biotransformations observed were hydroxylation, hydration and oxidation in zebrafish larvae, whereas mice produced mainly the dihydrodiol metabolite. Hydroxylation was the major route of metabolism for ocfentanil in zebrafish larvae, while in mice the O-demethylated derivative was the main metabolite. In addition, a study was conducted to evaluate morphological effects of the two drugs on zebrafish larvae. Malformations were noticeable only at the highest concentration of 2-furanylfentanyl, whereas no significant damage was observed with ocfentanil. In conclusion, the two animal models show similarities in behavioral response and in metabolism, considering the different biological investigated.

Zebrafish larvae: A new model to study behavioural effects and metabolism of fentanyl, in comparison to a traditional mice model.

In an effort to find alternatives to study in vivo the so-called New Psychoactive Substances (NPS), the present work was undertaken to investigate the use of zebrafish larvae as animal model in pharmaco-toxicology, providing behavioural and metabolism information. For this purpose, fentanyl, the progenitor of an extremely dangerous group of NPS, was administered at different doses to zebrafish larvae (1, 10, 50, 100 µM) in comparison to mice (0.1, 1, 6, 15 mg/kg), as a well-established animal model. A behavioural assay was performed at the time of the peak effect of fentanyl, showing that the results in larvae are consistent with those observed in mice. On the other hand, several morphological abnormalities (namely yolk sac edema, abnormal pericardial edema, jaw defect and spinal curvature) were found in larvae mostly at high fentanyl doses (50, 100 µM). Larva extract and mice urine were analyzed by using liquid chromatography coupled to high resolution mass spectrometry to identify the metabolic pathways of fentanyl. The main metabolites detected were norfentanyl and hydroxyfentanyl in both the tested models. In conclusion, the present study provides evidence that fentanyl effects on zebrafish larvae and metabolism are similar to rodents and consequently support the hypothesis of using zebrafish larvae as a suitable rapid screening tool to investigate new drugs, and particularly NPS.

Recombinant HIV-1 vaccine candidates based on replication-defective flavivirus vector.

Multiple approaches utilizing viral and DNA vectors have shown promise in the development of an effective vaccine against HIV. In this study, an alternative replication-defective flavivirus vector, RepliVax (RV), was evaluated for the delivery of HIV-1 immunogens. Recombinant RV-HIV viruses were engineered to stably express clade C virus Gag and Env (gp120TM) proteins and propagated in Vero helper cells. RV-based vectors enabled efficient expression and correct maturation of Gag and gp120TM proteins, were apathogenic in a sensitive suckling mouse neurovirulence test, and were similar in immunogenicity to recombinant poxvirus NYVAC-HIV vectors in homologous or heterologous prime-boost combinations in mice. In a pilot NHP study, immunogenicity of RV-HIV viruses used as a prime or boost for DNA or NYVAC candidates was compared to a DNA prime/NYVAC boost benchmark scheme when administered together with adjuvanted gp120 protein. Similar neutralizing antibody titers, binding IgG titers measured against a broad panel of Env and Gag antigens, and ADCC responses were observed in the groups throughout the course of the study, and T cell responses were elicited. The entire data demonstrate that RV vectors have the potential as novel HIV-1 vaccine components for use in combination with other promising candidates to develop new effective vaccination strategies.

Acquired cancer resistance to combination immunotherapy from transcriptional loss of class I HLA.

Understanding mechanisms of late/acquired cancer immunotherapy resistance is critical to improve outcomes; cellular immunotherapy trials offer a means to probe complex tumor-immune interfaces through defined T cell/antigen interactions. We treated two patients with metastatic Merkel cell carcinoma with autologous Merkel cell polyomavirus specific CD8+ T cells and immune-checkpoint inhibitors. In both cases, dramatic remissions were associated with dense infiltration of activated CD8+s into the regressing tumors. However, late relapses developed at 22 and 18 months, respectively. Here we report single cell RNA sequencing identified dynamic transcriptional suppression of the specific HLA genes presenting the targeted viral epitope in the resistant tumor as a consequence of intense CD8-mediated immunologic pressure; this is distinguished from genetic HLA-loss by its reversibility with drugs. Transcriptional suppression of Class I loci may underlie resistance to other immunotherapies, including checkpoint inhibitors, and have implications for the design of improved immunotherapy treatments.

A phase 2, randomized, double-blind, placebo- controlled study of chemo-immunotherapy combination using motolimod with pegylated liposomal doxorubicin in recurrent or persistent ovarian cancer: a Gynecologic Oncology Group partners study.

BACKGROUND: A phase 2, randomized, placebo-controlled trial was conducted in women with recurrent epithelial ovarian carcinoma to evaluate the efficacy and safety of motolimod-a Toll-like receptor 8 (TLR8) agonist that stimulates robust innate immune responses-combined with pegylated liposomal doxorubicin (PLD), a chemotherapeutic that induces immunogenic cell death. PATIENTS AND METHODS: Women with ovarian, fallopian tube, or primary peritoneal carcinoma were randomized 1 : 1 to receive PLD in combination with blinded motolimod or placebo. Randomization was stratified by platinum-free interval (≤6 versus >6-12 months) and Gynecologic Oncology Group (GOG) performance status (0 versus 1). Treatment cycles were repeated every 28 days until disease progression. RESULTS: The addition of motolimod to PLD did not significantly improve overall survival (OS; log rank one-sided P = 0.923, HR = 1.22) or progression-free survival (PFS; log rank one-sided P = 0.943, HR = 1.21). The combination was well tolerated, with no synergistic or unexpected serious toxicity. Most patients experienced adverse events of fatigue, anemia, nausea, decreased white blood cells, and constipation. In pre-specified subgroup analyses, motolimod-treated patients who experienced injection site reactions (ISR) had a lower risk of death compared with those who did not experience ISR. Additionally, pre-treatment in vitro responses of immune biomarkers to TLR8 stimulation predicted OS outcomes in patients receiving motolimod on study. Immune score (tumor infiltrating lymphocytes; TIL), TLR8 single-nucleotide polymorphisms, mutational status in BRCA and other DNA repair genes, and autoantibody biomarkers did not correlate with OS or PFS. CONCLUSIONS: The addition of motolimod to PLD did not improve clinical outcomes compared with placebo. However, subset analyses identified statistically significant differences in the OS of motolimod-treated patients on the basis of ISR and in vitro immune responses. Collectively, these data may provide important clues for identifying patients for treatment with immunomodulatory agents in novel combinations and/or delivery approaches. TRIAL REGISTRATION: Clinicaltrials.gov, NCT 01666444.

Relationship between pharmacokinetics and pharmacodynamics of clopidogrel in patients undergoing percutaneous coronary intervention: comparison between vasodilator-stimulated phosphoprotein phosphorylation assay and multiple electrode aggregometry.

UNLABELLED: ESSENTIALS: The reliability of platelet tests as markers of the variable bioavailability of clopidogrel is not yet defined. Kinetics of clopidogrel active metabolite (CAM) and platelet response were studied in ischemic heart disease. CAM plasma maximum concentration (Cmax ) predicted vasodilator-stimulated phosphoprotein (VASP-P). Timely performed VASP-P, not an aggregation-based test, may be a surrogate for clopidogrel bioavailability. BACKGROUND: The high inter-individual variability in the inhibition of platelet function by clopidogrel is mostly explained by high variability in its transformation to an active metabolite (CAM). Objective We investigated the relations between pharmacokinetics and pharmacodynamics of CAM by comparing two methods of platelet function. METHODS: We enrolled 14 patients undergoing percutaneous coronary interventions for non-ST-segment elevation acute coronary syndrome or inducible myocardial ischemia. Plasma concentrations of clopidogrel and CAM, phosphorylation of vasodilator-stimulated phosphoprotein (VASP-P), expressed as a platelet reactivity index (PRI) and whole-blood platelet aggregation (multiple electrode aggregometer, MEA) were measured before and after a 600-mg clopidogrel loading dose (nine time-points) and before and after 75-mg maintenance doses on days 2, 7 and 30. RESULTS: Plasma concentrations of clopidogrel and CAM were highly variable. CAM reached maximal concentration (Cmax ) (median, 110.8 nm; range, 41.9-484.8) 0.5-2 h after the loading dose. A sigmoid dose-response curve defined the relations between CAMCmax and PRI after 3 to 24 h (IC50 , 459.6 nm; 95% confidence interval, 453.4-465.7; R(2) = 0.82). PRI was unchanged from baseline in patients with the lowest CAMCmax (< 83 nm, n = 7), indicating low sensitivity of VASP-P. PRI values were also predicted by CAMCmax at days 2, 7 and 30. Platelet aggregation measured by MEA did not show significant relations with either PRI or with CAM pharmacokinetics at any time-point. CONCLUSIONS: After 600 mg clopidogrel, VASP-P, but not whole-blood platelet aggregation measured by MEA, is almost entirely predicted by CAMCmax . VASP-P could be useful in studies aimed at investigating relations between CAM bioavailability and clinical events.

Use of elephant grass for energy production in Campos dos Goytacazes-RJ, Brazil.

Elephant grass is a tropical forage plant widely distributed throughout Brazil. It was first exclusively used in the livestock sector as cattle feed. The grass is characterized by its high productivity and photosynthetic capacity and is considered as an alternative source of renewable energy. Here, we estimated the general combining ability of the parents and specific combining ability of the hybrids based on morpho-agronomic biomass-quality traits. The experiment was conducted in a randomized block design with 3 replicates. The diallel was composed of 16 hybrids and 2 groups of genitors. In the diallel analysis of variance, we observed a significant difference among treatments. A significant difference was observed among genitors for dry matter production (DMP). For the general combining ability of group 1, the traits leaf blade width, DMP, height, percentage of neutral detergent fiber, percentage of hemicellulose, percentage of lignin, percentage of acid detergent fiber, and percentage of cellulose were significant. For the estimates of general combining ability of DMP, parents Porto Rico 534-B, Vruckwona, Taiwan A-146, and Mercker S. E. A. were 0.4748, 3.2819, 1.1659, and 0.4317. The parents of Mercker S. E. A. and Porto Rico 534-B produced the highest percentage of detergent fiber and percentage of lignin with values of 0.1482 and 0.0856. Thus, parents Vruckwona, Porto Rico 534-B, and Taiwan A-146 are promising for integration into breeding programs. The best hybrid combinations for DMP were 1 x 5, 1 x 8, 2 x 6, 3 x 7, and 4 x 5.

Identification and analysis of murine pancreatic islet enhancers.

AIMS/HYPOTHESIS: The paucity of information on the epigenetic barriers that are blocking reprogramming protocols, and on what makes a beta cell unique, has hampered efforts to develop novel beta cell sources. Here, we aimed to identify enhancers in pancreatic islets, to understand their developmental ontologies, and to identify enhancers unique to islets to increase our understanding of islet-specific gene expression. METHODS: We combined H3K4me1-based nucleosome predictions with pancreatic and duodenal homeobox 1 (PDX1), neurogenic differentiation 1 (NEUROD1), v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MAFA) and forkhead box A2 (FOXA2) occupancy data to identify enhancers in mouse islets. RESULTS: We identified 22,223 putative enhancer loci in in vivo mouse islets. Our validation experiments suggest that nearly half of these loci are active in regulating islet gene expression, with the remaining regions probably poised for activity. We showed that these loci have at least nine developmental ontologies, and that islet enhancers predominately acquire H3K4me1 during differentiation. We next discriminated 1,799 enhancers unique to islets and showed that these islet-specific enhancers have reduced association with annotated genes, and identified a subset that are instead associated with novel islet-specific long non-coding RNAs (lncRNAs). CONCLUSIONS/INTERPRETATIONS: Our results indicate that genes with islet-specific expression and function tend to have enhancers devoid of histone methylation marks or, less often, that are bivalent or repressed, in embryonic stem cells and liver. Further, we identify a subset of enhancers unique to islets that are associated with novel islet-specific genes and lncRNAs. We anticipate that these data will facilitate the development of novel sources of functional beta cell mass.

Toxicokinetics of cocaine and metabolites: the forensic toxicological approach.

Cocaine is one of the most used psychomotor stimulants all over the world. On this basis, the interest for the pharmacological activity and the pharmacodynamic and pharmacokinetic aspects of this drug is very prominent in both clinical and forensic toxicological environments. The review presents and discusses 65 scientific publications covering all the aspects of cocaine toxicokinetic, including absorption, distribution, metabolism and elimination of the drug. Particular attention has been dedicated to the studies on the disposition of the drug in alternative biological matrices, such as oral fluid, hair, fetus fluids and tissues, and sweat. In fact, in the last years the use of these matrices has been proposed in clinical and forensic drug analysis in order to obtain additional information to that which can be obtained by analyzing the traditional biological matrices, such as blood and urine.

Determination of CDT, a marker of chronic alcohol abuse, for driving license issuing: immunoassay versus capillary electrophoresis.

The present work is aimed at a validation of the carbohydrate-deficient transferrin (CDT) determination in real cases by comparison between the a commercial immunometric method and a method based on capillary electrophoresis. Overall, 650 serum samples from subjects applying to re-obtain their driving license, previously withdrawn for "drunk driving", were investigated. A highly significant correlation (P < 0.001) was found between the results from immunoassay and capillary electrophoresis. However, particularly in the samples with CDT values around the cut-off or moderately elevated, a wide dispersion of the correlation data was found. This finding stresses the need to confirm by alternative techniques all the results from CDT immunoassays. For this purpose, capillary electrophoresis, because of its inherent characteristics of high selectivity, easy operation, high productivity and low operative costs looks well-suited for becoming the method of choice.

[A preliminary study on the tolerability and efficacy of nabumetone in the treatment of acute or chronic and very painful rheumatic diseases]. / Studio preliminare di tollerabilità ed efficacia nel trattamento di patologie acute o croniche di pertinenza reumatologica con alta risposta algica con nabumetone.

Twenty patients (2 males, 18 females, age range 33-69, average 55.85 years) with diagnoses of variously localized osteoarthritis (18 cases) or acute joint pain (2 cases) were studied. All patients were given one 1-g tablet of nabumetone every evening before going to bed, for 8 days. In three cases treatment was withdrawn immediately after the first dose due to the onset of allergic (2 cases) or dyspeptic (1 case) symptoms. Initially, all patients complained of considerable pain, both at rest and during active and passive movements with correlated functional limitation of the joints involved. Treatment resulted in considerable subsidence of symptoms from the very first days of therapy. Statistical calculation of the score showed significant improvement. Parameters of tolerability did not show significant changes from baseline. Local tolerability was generally good. The fact of the single daily dose has certainly contributed to the patients' good compliance.