Wilma Ponti

Immune function alterations in mice tolerant to Δ9-tetrahydrocannabinol: functional and biochemical parameters

We studied the effect of acute (1 h) or chronic exposure (7 and 14 days) to delta9-tetrahydrocannabinol (delta9-THC) on immune parameters in male Swiss mice. One hour after a dose of 10 mg/kg s.c., the splenocyte proliferative response to ConA and NK activity were not inhibited, but there was a significant decrease in the production of IL-2. After 7 days of treatment, when mice were tolerant to delta9-THC-induced analgesia, these functional parameters were strongly inhibited and there was a persistent reduction in IL-2 and IFNgamma. With 14 days exposure to the drug, splenocyte proliferation was significantly reduced only with 5 microg/ml ConA, and NK activity was still significantly depressed (about 37%). IL-2 had returned to the control value, whereas IFNgamma was still 40% down. Flow cytometry analysis of spleen cell composition indicated no changes after the acute and 7 day treatments, but at 14 days there was a 20% decrease in the number of T lymphocytes, mirrored by a 26% increase of B lymphocytes. In conclusion, in vivo exposure to psychoactive doses of delta9-THC has profound effects on immune function. This implies some important questions in relation to the liberalization of marijuana and its therapeutic uses.

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).

Bovine Doppel (Dpl) and Prion Protein (PrP) Expression on Lymphoid Tissue and Circulating Leukocytes

Doppel (Dpl) protein shares some structural features with prion protein (PrP), whose pathologic isoform (PrPsc) is considered to be the causative agent of transmissible spongiform encephalopathies. Dpl is mainly expressed in testes but, when ectopically expressed in the central nervous system, is neurotoxic. We have examined the expression pattern of Dpl and PrP on bovine lymphoid tissues and circulating leukocytes. A polyclonal anti-Dpl antibody along with a panel of monoclonal antibodies specific for leukocyte membrane antigens or PrP were used to examine frozen sections from spleen, lymph nodes, and bone marrow by immunohistochemistry. Blood was analyzed by flow cytometry. Double staining was used to study the possible coexpression of the two proteins and to characterize cells expressing Dpl and/or PrP. Dpl was expressed in B-cells, in dendritic cells within lymphoid follicles, bone marrow, circulating myeloid cells, and circulating B-cells. The distribution of Dpl was quite similar to that of PrP. The only differences in expression observed concerned the low number of Dpl + cells in lymph nodes and the strong Dpl positivity of circulating granulocytes. The two proteins were rarely co-expressed, suggesting an independent expression mechanism in resting cells. The role of Dpl+ leukocytes in the pathogenesis of Dpl- or PrP-induced diseases merits further investigation.

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

Seroepidemiological and clinical survey of feline immunodeficiency virus infection in Northern Italy

Abstract Four hundred and thirty-nine feline serum samples from cats with different living conditions in the north of Italy were tested for antibodies to feline immunodeficiency virus (FIV) and for antigen of Feline Leukemia Virus by enzyme-linked immunosorbent assay. A Western blot technique was also used on the positive sera in order to confirm the presence of specific antibodies to FIV. The Western blot enabled the detection of a false positive serum. The prevalence of FIV infection in this population was 12.5% and among the seropositive cats a greater proportion was male (74.5%) than female (25.5%). A correlation between the clinical status and the evolution of the pathology is described together with a score based on the severity of the stomatitis in infected cats. The Western blot patterns of positive samples were then compared with the stage of the pathology. Statistical analysis on the distribution of FIV in stray cats, cats with garden and courtyard access and strictly house-confined cats showed a highly significant risk of the infection in the first group.

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.

Pharmacokinetics and distribution of clioquinol in golden hamsters

Clioquinol (5‐chloro‐7‐iodo‐8‐quinolinol) is a zinc and copper chelator that can dissolve amyloid deposits and may be beneficial in Alzheimers disease. Prion diseases are also degenerative CNS disorders characterised by amyloid deposits. The pharmacokinetics and tissue distribution of drugs active against prions may clarify their targets of action. We describe the harmacokinetics of clioquinol in hamster plasma, spleen and brain after single and repeated oral or intraperitoneal administration (50 mg kg−1), as well as after administration with the diet. A single intraperitoneal administration led to peak plasma clioquinol concentrations after 15 min (Tmax), followed by a decay with an apparent half‐life of 2.20 ± 1.1 h. After oral administration, Tmax was reached after 30 min and was followed by a similar process of decay; the AUC0‐last was 16% that recorded after intraperitoneal administration. The Cmax and AUC values in spleen after a single administration were about 65% (i.p.) and 25% (p.o.) those observed in blood; those in liver were 35% (p.o.) those observed in blood and those in brain were 20% (i.p.) and 10% (p.o.) those observed in plasma. After repeated oral doses, the plasma, brain and spleen concentrations were similar to those observed at the same times after a single dose. One hour after intraperitoneal dosing, clioquinol was also found in the ventricular CSF. Clioquinol was also given with the diet; its morning and afternoon concentrations were similar, and matched those after oral administration. No toxicity was found after chronic administration. Our results indicate that clioquinol, after oral administration with the diet, reaches concentrations in brain and peripheral tissues (particularly spleen) that can be considered effective in preventing prion accumulation, but are at least ten times lower than those likely to cause toxicity.

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.