Silvia Graziano

Chronic wasting disease and atypical forms of bovine spongiform encephalopathy and scrapie are not transmissible to mice expressing wild-type levels of human prion protein

The association between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) has demonstrated that cattle transmissible spongiform encephalopathies (TSEs) can pose a risk to human health and raises the possibility that other ruminant TSEs may be transmissible to humans. In recent years, several novel TSEs in sheep, cattle and deer have been described and the risk posed to humans by these agents is currently unknown. In this study, we inoculated two forms of atypical BSE (BASE and H-type BSE), a chronic wasting disease (CWD) isolate and seven isolates of atypical scrapie into gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP). Upon challenge with these ruminant TSEs, gene-targeted Tg mice expressing human PrP did not show any signs of disease pathology. These data strongly suggest the presence of a substantial transmission barrier between these recently identified ruminant TSEs and humans.

Successful Nicotine Intake in Medical Assisted Use of E-Cigarettes: A Pilot Study

The electronic cigarette (e-cig) has gained popularity as an aid in smoking cessation programs mainly because it maintains the gestures and rituals of tobacco smoking. However, it has been shown in inexperienced e-cig users that ineffective nicotine delivery can cause tobacco craving that could be responsible for unsuccessful smoking reduction/cessation. Moreover, the incorrect use of an e-cig could also led to potential nicotine overdosage and intoxication. Medically assisted training on the proper use of an e-cig plus behavioral support for tobacco dependence could be a pivotal step in avoiding both issues. We performed an eight-month pilot study of adult smokers who started e-cig use after receiving a multi-component medically assisted training program with monitoring of nicotine intake as a biomarker of correct e-cig use. Participants were tested during follow-up for breath carbon monoxide (CO), plasma cotinine and trans-3’-hydroxycotinine, and number of tobacco cigarettes smoked. At the end of the first, fourth, and eighth month of follow-up, 91.1, 73.5, and 76.5% of participants respectively were e-cig users (‘only e-cig’ and ‘dual users’). They showed no significant variation in plasma cotinine and trans-3’-hydroxycotinine with respect to the start of the study when they smoked only tobacco cigarettes, but a significant reduction in breath CO. The proposed medically assisted training program of e-cig use led to a successful nicotine intake, lack of typical cigarette craving and overdosage symptoms and a significant decrease in the biomarker of cigarette combustion products.

Creutzfeldt‐Jakob disease: hopes for therapy

Despite an incredible clinical and research interest for the animal and human transmissible spongiform encephalopathies (TSE) or prion diseases (a constantly increase of scientific publications during the last 30 years [1]), the scientific community is still debating on the nature of the infectious agent, there are no preclinical diagnostic tests to screen infected but otherwise healthy individuals, and there is no therapy that is able to even slightly prolong the course of disease. Of course, these difficulties are shared with most of neurodegenerative disorders but in Creutzfeldt-Jakob disease (CJD) scientists had access to excellent animal models for more than 40 years. In these experimental models, attempts to modify the progression of disease started in the late 60s and since then a large set of compounds have been tested [2,3]. Amongst them, several are efficacious in delaying the onset of disease but only if given very close to the time of experimental infection, an event which is unrealistic in human diseases. Pentosan polysulphate (PPS) was one of them [4], but when Doh-ura et al. [5] reported that the intraventricular infusion of the drug in mice at a late stage of TSE infection was also effective and that this treatment was safe in dogs, a new hope for the treatment of CJD was perceived by neurologists and some well-informed relatives of CJD patients. In this issue of EJN, Prof. Bone et al. [6] report on the British experience of PPS infusion in different forms of human TSE diseases. There are two important conclusions from this study. The first is that intraventricular infusion of PPS is welltolerated despite some complications from the surgical procedure and the second is that three of four treated patients with variant CJD were still alive (August 2007) with a clinical duration clearly longer than those of historical series of untreated patients. Another variant CJD patient in the UK, not included in the series reported by Bone et al. [6], had also a prolonged clinical duration [7]. Taken together, these studies indicate that the intraventricular infusion of PPS might have some effect in prolonging survival in variant CJD. Whether PPS has an effect in sporadic, iatrogenic or genetic forms of human TSE diseases is still unclear [6,8]. Despite this good news, however, there is still a long way to go before claiming for an efficacious treatment in CJD. The major goal for a treatment is the cure of disease or a prolongation of clinical course of disease, hopefully with a benefit in the patients quality of life. This is difficult to assess in patients with severe cognitive impairment where judgments usually rely on relatives perspective whose only hope is often to keep alive their dearest. Difficulties in assessing various stages of disease is an impediment in making an objective evaluation of the efficacy of any therapy in CJD, which forces neurologists to determine any beneficial effect of therapy almost exclusively in terms of the prolongation of clinical duration of disease. This is not easy to ascertain because onset is often difficult to establish unanimously and because survival varies substantially in different forms of human TSE diseases and depends on various other factors, including age at onset, gender, polymorphism at codon 129 of the prion protein gene, and the type of the pathological prion protein accumulating in the brain [9]. As a result of the relative rarity of CJD the only possible way to enroll a significant number of patients is through international collaboration, such as envisaged in the Theraprion project supported by the EU network of excellence on prion diseases (Neuroprion). As for other fatal and rare diseases, all patients (or relatives of patients) should be given the possibility to receive a potentially useful treatment leaving clinicians with the difficulty in conducting a randomized controlled study. One way to solve this issue is to use historical data, but this should be conducted properly [10]. Each treated patient should be matched with untreated patients not only by sex and age but also by the same molecular sub-type cohort of disease. In other words, any effect on survival should be assessed at the end of the study when all known determinants of survival have been collected, including neuropathology and the biochemical characteristic of the pathological prion protein deposition in the brain. Even taking into account all these parameters, survival is a poorly predictive event in CJD and other factors, yet unknown, must clearly play an important role, including the quality of everyday care given to patients. There are many areas of future investments in the development of new therapeutic strategies. The intense research activity in experimental models has clearly shown that any treatments should start as early as possible to have a chance of success. This implies that searching for early or, even better, pre-clinical markers in CJD is mandatory and that preventive treatment might be more successful than attempting therapy in clinically affected individuals. Carriers of the mutated prion protein gene might be excellent targets but, although some have been treated, we are not aware of any trials in these subjects. The reason is that there are many ethical and practical aspects to be unravelled

Comparison of nanofiltration efficacy in reducing infectivity of centrifuged versus ultracentrifuged 263K scrapie‐infected brain homogenates in “spiked” albumin solutions

BACKGROUND: The safety of plasma‐derived products is of concern for possible transmission of variant Creutzfeldt‐Jakob disease. The absence of validated screening tests requires the use of procedures to remove or inactivate prions during the manufacture of plasma‐derived products to minimize the risk of transmission. These procedures need proper validation studies based on spiking human plasma or intermediate fractions of plasma fractionation with prions in a form as close as possible to that present in blood.

Role of proteomics in understanding prion infection

Transmissible spongiform encephalopathies or prion diseases are fatal neurodegenerative pathologies characterized by the autocatalytic misfolding and polymerization of a cellular glycoprotein (cellular prion protein [PrPC]) that accumulates in the CNS and leads to neurodegeneration. The detailed mechanics of PrPC conversion to its pathological isoform (PrPTSE) are unclear but one or more exogenous factors are likely involved in the process of PrP misfolding. In the last 20 years, proteomic investigations have identified several endogenous proteins that interact with PrPC, PrPTSE or both, which are possibly involved in the prion pathogenetic process. However, current approaches have not yet produced convincing conclusions on the biological value of such PrP interactors. Future advancements in the comprehension of the molecular pathogenesis of prion diseases, in experimental techniques and in data analysis procedures, together with a boost in more productive international collaborations, are therefore needed to improve the understanding on the role of PrP interactors. Finally, the advancement of ‘omics’ techniques in prion diseases will contribute to the development of novel diagnostic tests and effective drugs.

Genomic and post-genomic analyses of human prion diseases

Prion diseases share common features of neurodegenerative disorders, infectious diseases and pathologies linked to misfolded proteins. Whether these aspects are independently and fortuitously present in prion diseases or are somewhat linked together remains unsettled, but the contribution of genomic, proteomic, metabolomic and spectroscopic techniques might give insights into this puzzle, and likely give hope for therapy to patients. Although the prion protein gene (PRNP) governs most of the clinical and pathological features of prion diseases and plays a pivotal role in determining host susceptibility, there are still many uncertainties and unknown risk factors that need to be clarified and identified. Several genes, other than PRNP, have recently been found to be associated with a risk of developing sporadic or variant Creutzfeldt-Jakob disease, but these novel data have been produced in a relatively small number of patients and controls and, therefore, need further confirmation. The same criticism applies to the identification of the over 20 new cerebrospinal fluid or plasma markers of disease. Some of these markers seem related to the massive brain damage that occurs, rather than being specific to prion infection. Nevertheless, genomic and post-genomic approaches have shown that these techniques are very powerful, and the best way to overcome the scantiness of samples would be to encourage strong collaboration between different centers of excellence in prion diseases. In this review, we describe the most recent and outstanding advances offered by genomics and post-genomics analyses in the field of human prion diseases.

Monitoring nicotine intake from e-cigarettes: measurement of parent drug and metabolites in oral fluid and plasma.

Abstract Background: Electronic cigarettes (e-cig) known as electronic nicotine devices recently gained popularity among smokers. Despite many studies investigating their safety and toxicity, few examined the delivery of e-cig-derived nicotine and its metabolites in alternative biological fluids. Methods: We performed a randomized, crossover, and controlled clinical trial in nine healthy smokers. Nicotine (NIC), cotinine (COT), and trans-3′-hydroxycotinine (3-HCOT) were measured in plasma and oral fluid by liquid chromatography-tandem mass spectrometry after consumption of two consecutive e-cig administrations or two consecutive tobacco cigarettes. Results: NIC and its metabolites were detected both in oral fluid and plasma following both administration conditions. Concentrations in oral fluid resulted various orders of magnitude higher than those observed in plasma. Oral fluid concentration of tobacco cigarette and e-cig-derived NIC peaked at 15 min after each administration and ranged between 1.0 and 1396 μg/L and from 0.3 to 860 μg/L; those of COT between 52.8 and 110 μg/L and from 33.8 to 94.7 μg/L; and those of 3-HCOT between 12.4 and 23.5 μg/L and from 8.5 to 24.4 μg/L. The oral fluid to plasma concentration ratio of both e-cig- and tobacco cigarette-derived NIC peaked at 15 min after both administrations and correlated with oral fluid NIC concentration. Conclusions: The obtained results support the measurement of NIC and metabolites in oral fluid in the assessment of intake after e-cig use and appear to be a suitable alternative to plasma when monitoring nicotine delivery from e-cig for clinical and toxicological studies.

Assessment of prion reduction filters in decreasing infectivity of ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" human blood and red blood cells.

The safety of red blood cells (RBCs) is of concern because of the occurrence of four transfusion‐transmitted variant Creutzfeldt‐Jakob disease (vCJD) cases in the United Kingdom. The absence of validated screening tests requires the use of procedures to remove prions from blood to minimize the risk of transmission. These procedures must be validated using infectious prions in a form that is as close as possible to one in blood.

Prion Strain Characterization of a Novel Subtype of Creutzfeldt-Jakob Disease

ABSTRACT In 2007, we reported a patient with an atypical form of Creutzfeldt-Jakob disease (CJD) heterozygous for methionine-valine (MV) at codon 129 who showed a novel pathological prion protein (PrPTSE) conformation with an atypical glycoform (AG) profile and intraneuronal PrP deposition. In the present study, we further characterize the conformational properties of this pathological prion protein (PrPTSE MVAG), showing that PrPTSE MVAG is composed of multiple conformers with biochemical properties distinct from those of PrPTSE type 1 and type 2 of MV sporadic CJD (sCJD). Experimental transmission of CJD-MVAG to bank voles and gene-targeted transgenic mice carrying the human prion protein gene (TgHu mice) showed unique transmission rates, survival times, neuropathological changes, PrPTSE deposition patterns, and PrPTSE glycotypes that are distinct from those of sCJD-MV1 and sCJD-MV2. These biochemical and experimental data suggest the presence of a novel prion strain in CJD-MVAG. IMPORTANCE Sporadic Creutzfeldt-Jakob disease is caused by the misfolding of the cellular prion protein, which assumes two different major conformations (type 1 and type 2) and, together with the methionine/valine polymorphic codon 129 of the prion protein gene, contribute to the occurrence of distinct clinical-pathological phenotypes. Inoculation in laboratory rodents of brain tissues from the six possible combinations of pathological prion protein types with codon 129 genotypes results in the identification of 3 or 4 strains of prions. We report on the identification of a novel strain of Creutzfeldt-Jakob disease isolated from a patient who carried an abnormally glycosylated pathological prion protein. This novel strain has unique biochemical characteristics, does not transmit to humanized transgenic mice, and shows exclusive transmission properties in bank voles. The identification of a novel human prion strain improves our understanding of the pathogenesis of the disease and of possible mechanisms of prion transmission.

Herbal Highs: Review on Psychoactive Effects and Neuropharmacology

Background: A new trend among users of new psychoactive substances’ the consumption of “herbal highs”: plant parts containing psychoactive substances. Most of the substances extracted from herbs, in old centuries were at the centre of religious ceremonies of ancient civilizations. Currently, these herbal products are mainly sold by internet web sites and easily obtained since some of them have no legal restriction. Objective: We reviewed psychoactive effects and neuropharmacology of the most used “herbal highs” with characterized active principles, with studies reporting mechanisms of action, pharmacological and subjective effects, eventual secondary effects including intoxications and/or fatalities Method: The PubMed database was searched using the following key.words: herbal highs, Argyreia nervosa, Ipomoea violacea and Rivea corymbosa; Catha edulis; Datura stramonium; Piper methysticum; Mitragyna speciosa. Results: Psychoactive plants here reviewed have been known and used from ancient times, even if for some of them limited information still exist regarding subjective and neuropharmacological effects and consequent eventual toxicity when plants are used alone or in combination with “classical” drugs of abuse. Conclusion: Some “herbal highs” should be classified as harmful drugs since chronic administration has been linked with addiction and cognitive impairment; for some others taking into consideration only the recent trends of abuse, studies investigating these aspects are lacking.