Giorgio Poli

Tetracyclines affect prion infectivity

Prion diseases are transmissible neurodegenerative disorders of humans and animals for which no effective treatment is available. Conformationally altered, protease-resistant forms of the prion protein (PrP) termed PrPSc are critical for disease transmissibility and pathogenesis, thus representing a primary target for therapeutic strategies. Based on previous findings that tetracyclines revert abnormal physicochemical properties and abolish neurotoxicity of PrP peptides in vitro, we tested the ability of these compounds to interact with PrPSc from patients with the new variant of Creutzfeldt–Jakob disease (vCJD) and cattle with bovine spongiform encephalopathy (BSE). The incubation with tetracycline hydrochloride or doxycycline hyclate at concentrations ranging from 10 μM to 1 mM resulted in a dose-dependent decrease in protease resistance of PrPSc. This finding prompted us to investigate whether tetracyclines affect prion infectivity by using an animal model of disease. Syrian hamsters were injected intracerebrally with 263K scrapie-infected brain homogenate that was coincubated with 1 mM tetracycline hydrochloride, 1 mM doxycycline hyclate, or vehicle solution before inoculation. Hamsters injected with tetracycline-treated inoculum showed a significant delay in the onset of clinical signs of disease and prolonged survival time. These effects were paralleled by a delay in the appearance of magnetic-resonance abnormalities in the thalamus, neuropathological changes, and PrPSc accumulation. When tetracycline was preincubated with highly diluted scrapie-infected inoculum, one third of hamsters did not develop disease. Our data suggest that these well characterized antibiotics reduce prion infectivity through a direct interaction with PrPSc and are potentially useful for inactivation of BSE- or vCJD-contaminated products and prevention strategies.

Shifts in circulating lymphocyte subsets in cats with feline infectious peritonitis (FIP): pathogenic role and diagnostic relevance

Abstract Cats with feline infectious peritonitis (FIP) are usually lymphopenic and have lymphoid depletion evident in spleen and lymph nodes. In particular, the number of CD4+ lymphocytes in tissues decreases during the evolution of FIP lesions. This decrease is most likely due to increased lymphocyte apoptotic rate. In contrast, cats infected with the Feline Coronavirus (FCoV) develop a follicular hyperplasia in the peripheral lymph nodes. The current study was devised to evaluate the possible pathogenic role of shifts in circulating lymphocyte subsets in FIP. Peripheral blood from cats with FIP was evaluated and compared with peripheral blood from clinically healthy cats living in both FCoV-free and FCoV-endemic catteries. Blood from cats with diseases other than FIP was also examined in order to define the diagnostic relevance of the changes. Lymphocyte subsets were analysed by flow cytometry, using a whole blood indirect immunofluorescence technique and mAbs specific for feline CD5, CD4, CD8, CD21. The results of the current study suggest that cats recently infected with FCoV that do not develop the disease have a transient increase in T cells; cats from groups with high prevalence of FIP have a moderate but persistent decrease in T cell subsets; cats with FIP have a very severe decrease in all the subsets of lymphocytes. Moreover, during FIP many lymphocytes do not express any membrane antigen, most likely due to early apoptosis. Cats with diseases other than FIP also had decreased number of lymphocytes: as a consequence, the diagnostic relevance of these findings is very low. Nevertheless, the lack of flow cytometric changes had a high negative predictive value (NPV), thus allowing to exclude FIP from the list of possible diagnoses in cats with normal cytograms.

Dermatophytes in clinically healthy laboratory animals.

Mice, rats, guineapigs and rabbits that were apparently healthy and did not present skin lesions were examined for the presence of dermatophytes. A small proportion of them were found to be carriers of Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis (8/140 = 5·7% of mice; 2/185 = 1·1% of rats; 1/70 = 1·4% of guineapigs; 1/215 = 0·5% of rabbits).

Effects of clioquinol on memory impairment and the neurochemical modifications induced by scrapie infection in golden hamsters

Prion protein (PrP) is a glycoprotein expressed on the surface of neurons and glial cells. Its pathological isoform (PrP(res)) is protease resistant, and involved in the pathogenesis of a number of transmissible encephalopathies (TSEs). One common feature of neurodegenerative diseases, including TSEs, is oxidative stress, which may be responsible not only for the dysfunction or death of neuronal cells, but also cognitive deficits. Clioquinol (5-chloro-7-iodo-8-quinolinol) chelates zinc and copper, which are involved in the deposition of amyloid plaques and acts as an antioxidant; increased lipid peroxidation has also been demonstrated in the early phases of PrP propagation. The aim of this study was to investigate the effects of clioquinol on the changes in motor and cognitive behaviours induced by scrapie infection, as well as its effects on oxidative stress and the neurotransmitters known to be involved in motor and cognitive functions. The results show that clioquinol counteracts the massive memory deficit induced by scrapie infection. This effect is not paralleled by neurochemical changes because the levels of all of the biogenic amines and their metabolites were reduced despite clioquinol treatment. The main biochemical change induced by clioquinol was a marked reduction in lipid peroxidation at all time points. The antioxidant effect of clioquinol can reduce functional impairment and thus improve memory, but clioquinol does not reduce PrP deposition or synapse loss, as indicated by the unchanged Western blot, histopathological and histochemical findings.

Evaluation of Anti-Prionic Activity of Clioquinol in an in vivo Model (Mesocricetus auratus)

C. Pollera1,∗, B. Lucchini1, E. Formentin1, S. Bareggi2, G. Poli1 and W. Ponti1 1Department of Veterinary Pathology, Hygiene and Public Health, Microbiology and Immunology Unit, Faculty of Veterinary Medicine, Centre of Excellence on Neurodegenerative Diseases, University of Milan, Italy; 2Department of Pharmacology, Chemoterapy and Medical Toxicology, School of Medicine, Centre of Excellence on Neurodegenerative Diseases, University of Milan, Italy ∗Correspondence: E-mail: claudia.pollera@unimi.it

Cannabinoids inhibit nitric oxide production in bone marrow derived feline macrophages

Feline immunodeficiency virus (FIV) infection causes a widespread natural immunodeficiency syndrome in cats that is considered a suitable animal model for studying human immunodeficiency virus (HIV) infection and pathogenesis. Short term cultures of bone marrow derived feline macrophages stimulated with recombinant feline interferon-gamma (r-IFN-gamma) and lipopolysaccharide (LPS) were shown to produce nitric oxide. Feline macrophages were shown to express cannabinoid receptors, and nitric oxide production decreased after in vitro exposure to synthetic cannabinoid CP-55940. Both cannabinoid receptors, CB1 and CB2, were involved in this process, since the inhibition was reversed by selective cannabinoid antagonists for both of these receptors.

Therapeutic effect of CHF5074, a new γ-secretase modulator, in a mouse model of scrapie.

In transmissible spongiform encephalopathies (TSEs) and Alzheimer disease (AD) both misfolding and aggregation of specific proteins represent key features. Recently, it was observed that PrPc is a mediator of a synaptic dysfunction induced by Aβ oligomers. We tested a novel γ-secretase modulator (CHF5074) in a murine model of prion disease. Groups of female mice were intracerebrally or intraperitoneally infected with the mouse-adapted Rocky Mountain Laboratory prions. Two weeks prior infection, the animals were provided with a CHF5074-medicated diet (375 ppm) or a standard diet (vehicle) until they showed neurological signs and eventually died. In intracerebrally infected mice, oral administration of CHF5074 did not prolong survival of the animals. In intraperitoneally-infected mice, CHF5074-treated animals showed a median survival time of 21 d longer than vehicle-treated mice (p < 0.001). In these animals, immunohistochemistry analyses showed that deposition of PrPSc in the cerebellum, hippocampus and parietal cortex in CHF5074-treated mice was significantly lower than in vehicle-treated animals. Immunostaining of glial fibrillary acidic protein (GFAP) in parietal cortex revealed a significantly higher reactive gliosis in CHF5074-treated mice compared with the control group of infected animals. Although the mechanism underlying the beneficial effects of CHF5074 in this murine model of human prion disease is unclear, it could be hypothesized that the drug counteracts PrPSc toxicity through astrocyte-mediated neuroprotection. CHF5074 shows a pharmacological potential in murine models of both AD and TSEs thus suggesting a link between these degenerative pathologies.

Expression of bovine leukemia virus ENV glycoprotein in insect cells by recombinant baculovirus

The gp51‐p30 glycoprotein constituting BLV envelope was expressed in Sf‐21 insect cells by means of recombinant baculoviruses. Post‐infection cell lysates were analyzed, in order to define the immunologic reactivity of recombinant products. Oligosaccharide chains, containing N‐acetylglucosamine, mannose, galactose and sialic acid were found on recombinant gp51‐p30. In order to investigate the timing of transcription and translation of the glycoprotein, kinetic assays were carried out on cell lysates and directly in situ on Sf‐21 cells during the course of baculovirus infection. The use of different solubilizing reagents was also evaluated in order to rescue recombinant glycoprotein from its subcellular location.

In vivo Model for the Evaluation of Molecules Active Towards Transmissible Spongiform Encephalopathies

W. Ponti1*, M. Sala2, C. Pollera1, D. Braida 2, G. Poli1 and S. Bareggi2 1Dipartimento di Patologia Animale, Igiene e Sanita Pubblica Veterinaria, Sezione di Microbiologia e Immunologia; 2Dipartimento di Farmacologia Medica, Chemioterapia e T ossicologia, Milano. Centro di Eccellenza per le Malattie Neurodegenerative *Correspondence: Dipartimento di Patologia Animale, Igiene e Sanita Pubblica Veterinaria, Sezione di Microbiologia e Immunologia. V ia Celoria 10. 20133 Milan, Italy E-mail: wilma.ponti@unimi.it

Therapeutic activity of inhibition of the soluble epoxide hydrolase in a mouse model of scrapie.

AIMS The misfolding and the aggregation of specific proteins are key features of neurodegenerative diseases, specifically Transmissible Spongiform Encephalopathies (TSEs). In TSEs, neuronal loss and inflammation are associated with the accumulation of the misfolded isoform (PrP(sc)) of the cellular prion protein (PrP(c)). Therefore we tested the hypothesis that augmenting a natural anti-inflammatory pathway mediated by epoxygenated fatty acids (EpFAs) will delay lethality. EpFAs are highly potent but enzymatically labile molecules produced by the actions of a number of cytochrome P450 enzymes. Stabilization of these bioactive lipids by inhibiting their degradation mediated by the soluble epoxide hydrolase (sEH) results in potent anti-inflammatory effects in multiple disease models. MAIN METHODS Mice were infected with the mouse-adapted RML strain of scrapie by intracerebral or intraperitoneal routes. Animals received the sEH inhibitor, by oral route, administrated in drinking water or vehicle (PEG400). Infected mice were euthanized at a standard clinical end point. Histopathological, immunohistochemical and Western blot analyses of brain tissue confirmed the presence of pathology related to prion infection. KEY FINDINGS Oral administration of the sEHI did not affect the very short survival time of the intracerebral prion infection group. However, mice infected by intraperitoneal route and treated with t-AUCB survived significantly longer than the control group mice (p<0.001). SIGNIFICANCE These findings support the idea that inhibition of sEH or augmentation of the natural EpFA signaling in the brain offers a potential and different route to understand prion diseases and may become a therapeutic strategy for diseases involving neuroinflammation.