Angelina Valanzano

Preparation of soluble infectious samples from scrapie-infected brain: a new tool to study the clearance of transmissible spongiform encephalopathy agents during plasma fractionation

BACKGROUND:  Concern about the safety of blood, blood components, and plasma‐derived products with respect to prions has increased since the report of two blood‐related infections of variant Creutzfeldt‐Jakob disease in the United Kingdom. Efforts were directed toward the development of procedures able to remove or inactivate prions from blood components or plasma‐derived products with brain fractions of transmissible spongiform encephalopathy (TSE)‐infected rodents as spiking materials. These spiking materials, however, are loaded with pathological prion protein (PrPTSE) aggregates that are likely not associated to blood infectivity. The presence of these aggregates may invalidate these studies.

Progressive behavioural changes in the spatial open-field in the quinolinic acid rat model of Huntington's disease.

Huntingtons disease (HD) is a progressive neurodegenerative disorder, characterized by severe degeneration of basal ganglia, motor abnormalities, impaired cognitive functions and emotional disturbances. Intrastriatal injection of the excitotoxin quinolinic acid (QA), an N-methyl-D-aspartate receptor agonist, appears to reproduce in rats some of the clinical features of human HD, included motor and behavioural deficits. Aim of this study was to assess whether the behavioural alterations described in the QA rat model of HD progressed over time. We analysed the effects of bilateral striatal injection of QA (300 nmol/1 microl) to adult rats in the spatial open-field test, a nonaversive task in which exploratory activity and responses to both spatial rearrangement of familiar objects and object novelty are measured. Rats were tested 2 weeks, 2 and 6 months after the QA lesion. Lesioned rats showed progressive alterations in performance in this task. Whereas sham and QA rats did not markedly differ 2 weeks post-lesion, lesioned rats were significantly more active than controls 2 and 6 months after surgery. Specifically, frequency and duration of rearing and wall rearing increased progressively over time, while grooming was enhanced at 2 months post-lesion only. Spatial and object novelty discrimination was not affected. These results show that a single injection of QA excitotoxin can induce behavioural changes that progress over time. The main implication of these findings is that, besides genetic mice models of HD, QA-lesioned rats may represent a suitable mean to test the ability of new drugs to slow down disease progression.

Adenosine A2A receptor blockade before striatal excitotoxic lesions prevents long term behavioural disturbances in the quinolinic rat model of Huntington's disease.

Huntingtons disease (HD) is a progressive neurodegenerative disorder, characterised by severe degeneration of basal ganglia, motor abnormalities, impaired cognitive function and emotional disturbances. Many of the distinct neuropathological features of HD are reproduced in rats by intrastriatal injections of the excitotoxin quinolinic acid (QA), and QA-induced excitotoxicity is partially prevented by administration of the A(2A) receptor antagonist prior to the QA injection. In this study, we assessed the neuroprotective effects of the adenosine A(2A) receptor antagonist SCH 58261 on the progressive behavioural alterations reported in the QA rat model of Huntingtons disease. Male rats received i.p. SCH 58261 (0.01mg/kg) or vehicle 20min before a bilateral injection of quinolinic acid (QA, 300nmol/1mul) or its vehicle in the dorsal striatum. Motor activity and anxiety levels were analyzed in an open-field arena and in an elevated plus-maze at 2 weeks, 2 months and 6 months post-lesion. In QA-lesioned rats SCH 58261 prevented alterations of wall rearing behaviour starting from 2 weeks post-lesion while emotional changes (reduced anxiety) were back to control levels by 6 months post-lesion. These findings extend to the behavioural parameters the protective effects of SCH 58261 in the QA model of Huntingtons disease.

Systemic administration of anti-NGF antibodies to neonatal mice impairs 24-h retention of an inhibitory avoidance task while increasing ChAT immunoreactivity in the medial septum

Neonatal mice received subcutaneous injections of either antibody against murine NGF raised in goat (3 mg, injection volume 50 microliters) or preimmune serum on postnatal days 2, 4, 6, 8, 10, and 12. They were tested on postnatal days 15-16 or 20-21 for learning and 24-h retention of a passive avoidance step-through task. Immunostaining for choline acetyltransferase (ChAT) was measured in two cholinergic forebrain areas (septum and caudate-putamen) on postnatal day 16 or 21. Locomotor activity and exploratory behavior in an open-field test were also assessed on day 17 or 22, following a single administration of either scopolamine (2 mg/kg) or saline solution. While anti-NGF treatment did not affect acquisition on day 15, impairment in retention was evident on day 16. On days 20-21, no effects were found either on acquisition or on retention capabilities. Analysis of ChAT immunostaining revealed a significant increase of ChAT-immunopositive cells in the medial septal area in 16-day-old but not in 21-day-old mice. Behavior in the open-field test and age-typical response to scopolamine were not altered by anti-NGF at either of the two ages considered. These data support the view that immunological neutralization of endogenous NGF specifically affects the maturation of retention capabilities in altricial rodents, and confirm the involvement of forebrain cholinergic mechanisms in early memory processes.

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.

Effects of prenatal AZT+3TC treatment on open field behavior and responsiveness to scopolamine in adult mice

Treatment of pregnant seropositive women and their neonates with the nucleoside analogs (reverse transcriptase inhibitors) zidovudine (AZT), lamivudine (3TC) and their combination has become a standard of care in industrialized countries to prevent transmission of the HIV-1 virus. Animal studies indicated limited but significant behavioral changes in AZT or 3TC-prenatally exposed offspring, whereas data on the potential neurobehavioral outcomes of AZT+3TC combination are still lacking. The aim of the present study was to assess in mice prenatally exposed to AZT+3TC the functional state of cholinergic muscarinic neuroregulation at adulthood. Pregnant CD-1 mice received per orem twice daily AZT+3TC (160 and 500 mg/kg, respectively) or vehicle solution (NaCl 0.9%) from gestational day (GD) 10 to delivery (GD 19). Locomotor activity, exploratory behavior and responsiveness to the muscarinic cholinergic blocker scopolamine (2 mg/kg) were analyzed at adulthood (PND 70) in offspring of both sexes in an open field test. Results indicated that prenatal AZT+3TC exposure does not influence responsiveness to the muscarinic cholinergic antagonist as measured by analysis of the drugs effects on locomotor and exploratory activity and different behavioral items. However, AZT+3TC-treated mice displayed higher frequency of rearing, and lower frequency and duration of self-grooming behavior, consistent with an effect on dopaminergic neurotransmission. However, this would need confirmatory experiments.

Age-dependent effects of NGF and scopolamine on suckling behavior of neonatal mice.

Nerve growth factor (NGF) influences the neurochemical differentiation of central cholinergic neurons of developing rodents. In this study, NGF was given intracerebrally to mice on different postnatal days (days 5 and 7, or days 8 and 10). Pups were tested for suckling behavior 24 h after the second NGF injection, following systemic administration of either the muscarinic cholinergic antagonist scopolamine or saline solution. Scopolamine significantly impaired nipple attachment on day 11 but not on day 8, and decreased locomotor activity in 11-day pups. NGF given on days 5 and 7 increased paddling and treading on day 8, and this effect was more pronounced in scopolamine injected pups. Pretreatment with NGF on days 8 and 10 decreased activity levels in 11-day pups. The differences in the effects of scopolamine at successive ages suggest that distinct portions of the cholinergic system mature at different rates and that sensitivity to NGF is age dependent. NGF appears to influence functional maturation of that portion of the cholinergic system involved in the regulation of locomotor activity.

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.

PrpTSE in muscle-associated lymphatic tissue during the preclinical stage of mice infected orally with bovine spongiform encephalopathy

The involvement of muscles in the pathogenesis of transmissible spongiform encephalopathies (TSEs) is irregular and unpredictable. We show that the TSE-specific protein (PrP(TSE)) is present in muscles of mice fed with a mouse-adapted strain of bovine spongiform encephalopathy as early as 100 days post-infection, corresponding to about one-third of the incubation period. The proportion of mice with PrP(TSE)-positive muscles and the number of muscles involved increased as infection progressed, but never attained more than a limited distribution, even at the clinical stage of disease. The appearance of PrP(TSE) in muscles during the preclinical stage of disease was probably due to the haematogenous/lymphatic spread of infectivity from the gastrointestinal tract to lymphatic tissues associated with muscles, whereas in symptomatic animals, the presence of PrP(TSE) in the nervous system, in neuromuscular junctions and in muscle fibres suggests a centrifugal spread from the central nervous system, as already observed in other TSE models.