Metabotropic glutamate receptors and brain ischemia: differential effects on phospoinositide turnover in young and aged rats.

Neuronal damage and phosphoinositide hydrolysis stimulated by neurotransmitter receptor agonists in cerebral cortex of 3- and 24-month Fischer 344 rats, following an episode of brain ischemia, were compared. Transient global ischemia was induced by occlusion of common carotid arteries for 15 minutes in conditions of moderate hypoxia. Seven days after, histological examination of cerebral cortex showed cell loss, neurons with nuclear pyknosis, cytoplasmatic degeneration, and glial proliferation. Ischemic lesions appeared moderate to severe in young rats and intermediate in all the aged animals. In young rats inositol phosphates accumulation stimulated by excitatory amino acids (ACPD, ibotenate and quisqualate), but not by norepinephrine or carbachol, was enhanced significantly with respect to sham-operated animals. No potentiation at all was observed in aged rats. This finding suggests that the events leading to the increased metabotropic response in the post-ischemia period is impaired by the ageing process.

Detection of bovine mitochondrial DNA in ruminant feeds: a molecular approach to test for the presence of bovine-derived materials.

A ban on ruminant-derived proteins in ruminant feeds has been introduced as a preventive measure to avoid the spread of bovine spongiform encephalopathy (BSE), as well as to minimize any potential risk of BSE transmission from bovines to humans. In the absence of commercially available efficient methods for identification of bovine-derived proteins in animal feeds, we developed a rapid and sensitive polymerase chain reaction (PCR)-based assay which allows detection and identification of a bovine-specific mitochondrial DNA sequence from feedstuffs. The amplified product encodes for the whole ATPase subunit 8 and the amino-terminal portion of the ATPase subunit 6 proteins, which are known to exhibit a relatively low degree of conservation among vertebrates. The specific amplification of such a bovine mitochondrial sequence from reference feedstuff samples was demonstrated by means of both direct sequencing and single-strand conformational analysis of the PCR product. Specificity was also confirmed by the absence of detectable homologous PCR product when using reference feedstuff samples lacking bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method allows detection of the presence of bovine mitochondrial DNA in feedstuffs containing less than 0.125% of bovine-derived meat and bonemeals. Furthermore, it does not appear to be considerably affected by prolonged heat treatment. DpnII and SspI restriction endonuclease digestions of the unpurified PCR product may be used routinely to confirm the bovine origin of the amplified sequence. Since this method is specific, rapid, and sensitive, it could be successfully utilized as a routine control assay to evaluate the presence of bovine-derived meat and bonemeals in ruminant feeds.

Transient global brain hypoxia-ischemia in adult rats: neuronal damage, glial proliferation, and alterations in inositol phospholipid hydrolysis.

A model of ischemic-hypoxic brain injury which combines bilateral occlusion of common carotid arteries for 10 min and mild hypoxia (15% O2 for 10 min before and during occlusion) was developed. Global ischemia was assessed by a simplified EEG recording indicating isoelectric line, i.e. full arrest of cortical electrical activity. Histological examination of brain 7 days after ischemic insult showed from moderate to severe damage, mainly in the cerebral cortex (layers III, V and VI) and hippocampus (mainly CA1 subfield). The injury consisted of neuronal degeneration and necrosis with nuclear pyknosis and karyorrhexis. Immunohistochemical staining for gliofibrillar acidic protein showed a marked glial proliferation in the cerebral cortex and hippocampus. In the cortical slices, inositol phosphates accumulation stimulated by excitatory amino acid agonists (ACPD, ibotenate and quisqualate), as well as by norepinephrine and carbachol, was enhanced significantly (p < 0.01) with respect to sham-operated rats 7 days, but not 24 h, after the ischemic insult. The overall data show that the relatively simple transient brain hypoxia/ischemia rat model produces full arrest of cortical EEG, histopathological alterations and those relative to post-receptor neurochemical mechanisms characteristic of four-vessel occlusion model.

Lymphocyte proliferative response to rabies virus antigen.

Rabies virus (RV) infection, as well as active immunisation using viral antigen, elicit both humoral and cellular reactions whose protective effects are still unclear. We evaluated both responses in order to find valuable monitoring parameters for the immunisation procedure. Three laboratory workers repetitively immunised with booster human diploid cell vaccine against rabies virus, 13 patients from the anti-rabies centre (vaccinated for the first time) and 10 healthy volunteers (not immunised nor exposed to rabies virus antigen), were monitored for: (i) in vivo RV-specific antibody production; (ii) in vitro anti-RV lymphocyte proliferative response and (iii) in vitro phenotype modulation induced by the viral antigen. In particular CD3, CD4, CD8, and surface immunoglobulins were monitored. All 3 subjects receiving the booster immunisation and, to a lesser extent, those receiving 4 doses of vaccine did recognise the antigen in vitro. The proliferation involved mainly CD4 positive cells leading to an increased number of cell bearing surface immunoglobulins, i.e. B cells. The proliferation index was in good correlation with the in vivo antibody production (p = less than 0.00009441). Nevertheless the presence of some cases without correlation between those parameters (in particular 5 out of 6 patients over 65 years of age failed to mount an adequate cellular proliferative response) reveals the need to use cellular response in parallel to the current humoral response, in order to evaluate and monitor the immunisation procedure. This conclusion is further stressed by the fact that protection against rabies infection is mainly cellular.

A rapid serum neutralization test in microplates for the detection of antibodies to hog cholera virus.

The fluorescent antibody serum neutralization (FASN) test for the detection of antibodies to hog cholera virus was developed utilizing 96-well and Terasaki microplates. This microtechnique, especially when performed in Terasaki plates, offers some advantage if compared with conventional FASN in coverslip cell cultures, being easier and more rapid, saving of reagents and allowing simple microscopic observation.

Susceptibility of a minipig kidney cell line (MPK) to hog cholera virus.

A comparitive study on the different susceptibility of MPK cells (Minipig Kidney cell line) and PK15 cells (Pig Kidney cell line) to the Hog Cholera Virus (HCV) was conducted. Higher HCV titres (3 log10) were reached on MPK cells compared with PK15 cells.

[Attenuated rabies virus, ERA strain, in cattle and dogs vaccinated with multiple doses]. / Recherche sur le virus attenue de la rage, souche ERA, chez des bovins et des chiens vaccines avec des doses multiples.

Investigation on the vaccination of 18 cattle and 5 dogs against rabies is reported. Each animal received multiple doses of ERA strain vaccine intramuscularly in the gluteal or masseter region. The saliva, the brain and salivary glands of the vaccinated animals were examined to detect the presence of ERA virus using immunofluorescent test and mouse inoculation. The virus was never found in the saliva and organs of treated animals. Circulating antibodies against ERA rabies virus were detected in all vaccinated cattle and dogs.

Antibody levels following ERA rabies vaccine in cattle.

The humoral response in cattle treated with ERA strain rabies vaccine, was studied utilizing the following criteria: antibody titres determined by RFFIT, seroimmunological monitoring of experimentally vaccinated animals, a comparison of data obtained from cattle vaccinated and maintained under field conditions in the absence of anamnestic information. The average antibody responses in the field experiment are in agreement with the laboratory animal response, both showing highest levels at day 15 after vaccination; at day 30 in both groups the immune response is sensibly lower, to remain then on a constant level after day 90 and stay about 0.5 I.U./ml up to the fifteenth month.

[Indirect immunofluorescence microtest in the determination of antirabies antibodies]. / Microtest di immunofluorescenza indiretta per il dosaggio degli anticorpi antirabici.

The A.A. describe an indirect fluorescent antibody microtest for the rapid detection and titration of antirabies antibodies. Slides containing rabies antigen were prepared by planting 50% infected cells onto multiwelled teflon-coated slides. Following fixation, slides were stored at -20 degrees C until used. The indirect fluorescent antibody microtest was compared to the RFFIT in titration of sera containing rabies antibodies. The test was found to be rapid, easy and reliable.