Development of a semi-nested PCR using degenerate primers for the generic detection of small ruminant lentivirus proviral DNA.

A PCR assay was developed for the reliable detection of small ruminant lentivirus (SRLV) proviral DNA. The method involved the use of degenerate deoxyinosine-substituted primers and a second semi-nested PCR step that increased the polyvalency and sensitivity of the detection, respectively. Primers were designed from the pol gene conserved motifs of 85 SRLV isolates and were evaluated using different SRLV isolates together with Maedi-Visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) reference strains. The method successfully detected SRLV proviral DNA in total DNA extracts originating from whole blood samples, separated peripheral blood mononuclear cells (PBMCs) and tissue cultures. The semi-nested PCR was compared with the agar gel immunodiffusion test and proved to be highly sensitive, specific and capable of detecting many SRLV variants in infected or suspect animals. Therefore, it would be useful in the diagnosis of natural SRLV infections, in eradication programs and epidemiological studies. Whole blood samples can be used directly, thus alleviating the need for PBMC separation, and thereby enables a simple, fast and cost-effective analysis of a large number of samples.

Antigenic profile of human recombinant PrP: generation and characterization of a versatile polyclonal antiserum.

We describe the quality of a rabbit polyclonal antiserum (Sal1) that was raised against mature human recombinant prion protein (rhuPrP). Epitope mapping demonstrated that the Sal1 antiserum recognized six to eight linear antigenic sites, depending on the animal species. The versatility of the antiserum was evident from the range of animal species and immunochemical techniques where it could be applied successfully. Antigen absorption studies revealed differences in the location and number of epitopes remaining after incubation with soluble or aggregated antigen.Our knowledge concerning immunoprophylaxis against prion diseases and the important role played by conformational changes of PrP is increasing rapidly. The findings reported here should add to this body of knowledge.

Increased incidence of sporadic Creutzfeldt-Jakob disease on the island of Crete associated with a high rate of PRNP 129-methionine homozygosity in the local population.

Since the spring of 1997, when the Neurology Department of the University Hospital of Crete admitted its first patient, nine cases (eight neuropathologically confirmed and one probable) of sporadic Creutzfeldt-Jakob disease (sCJD) have been recorded. This represents an annual incidence five-fold higher than expected based on the island's population (0.54 million). Molecular analysis of the prion-protein gene (PRNP) showed no mutations in any of the seven CJD cases studied. Five patients (ages 64-88 years) were homozygous for methionine-129 of PRNP and showed the classic sCJD triad (subacute dementia, myoclonus, periodic electroencephalogram). Brains contained type 1 (unglycosylated 21.5 kDa band) protease-resistant prion protein (PrPres). Two patients (ages 56 and 57 years), both homozygous for valine-129, showed cerebellar ataxia and later dementia not associated with periodic electroencephalogram; brain PrPres was type 2. Genotyping of 205 Cretan controls showed that methionine-129 homozygosity, a susceptibility factor for sCJD, was significantly higher in this population than in other Caucasian populations (57.0% n = 205 vs. 41.5% n = 859, p < 0.0001). These data are the first to relate a high regional incidence rate for sCJD to the distribution of PRNP 129 genotypes in the local population; however, additional factors may be operational.

Marked increase of neuronal prion protein immunoreactivity in Alzheimer's disease and human prion diseases.

In neurodegenerative disorders including Alzheimer's disease (AD), free radical damage to lipids, carbohydrates, proteins and DNA has been demonstrated to play a key pathogenetic role. In vitro studies have suggested a function of the cellular prion protein (PrPc) in the defense against oxidative stress. Therefore, we investigated the distribution of PrPc immunoreactivity in hippocampus (sectors CA4-CA1), subiculum (Sub), entorhinal (EC), and temporal cortex (TC) in sections from AD, human transmissible spongiform encephalopathy (TSE) and control brains. Compared to control cases, AD brains revealed an increase in the proportion of PrPc-immunoreactive neurons, which was statistically significant in CA2, Sub, and TC. In TSEs, a statistically significant increase of PrPc-immunoreactive neurons was observed in CA2, CA1, Sub, EC, and TC. In conclusion, our data show a striking up-regulation of PrPc in neurodegeneration and provide additional support for the concept that PrPc may be involved in the defense against oxidative stress.

Characterization of the murine BSE infectious agent.

Bovine spongiform encephalopathy (BSE) is a prion-associated disease where the infectious agent is thought to be a host-encoded protein with a protease-resistant conformation (PrP(Sc)). Here, data are presented on the solubilization of purified murine BSE material, using guanidine-HCl as a denaturing agent. This treatment led to loss of infectivity, which was partially recovered on renaturation after dialysis to remove the chaotropic agent. The renatured product was then fractionated on an isopycnic sucrose-density gradient and the fractions were analysed for the presence of PrP(Sc), nucleic acids and infectivity. It was found that the major part of PrP(Sc) (>90%) and the endogenous nucleic acids did not contribute towards the formation of infectious particles on renaturation. Infectivity was distributed in the top three, low-density fractions. Among these, the presence of considerable infectivity in the fraction of lowest density, with barely detectable PrP(Sc), is of particular interest.

Glycosaminoglycan analysis in brain stems from animals infected with the bovine spongiform encephalopathy agent.

Increasing evidence suggests that the pathological alterations observed in brains affected by neurodegenerative disorders such as Creutzfeldt-Jakob disease and Alzheimer's disease also involve changes in glycosaminoglycans (GAGs). In the present study, we have isolated, purified, and characterized total GAGs from brain stems of healthy cows or those infected with the bovine spongiform encephalopathy (BSE) agent and we report on the differences between the two groups. Purification of the GAGs was achieved by gel filtration after homogenization, delipidation, and sequential treatment with pronase, DNase, and alkali borohydride. Fractionation of the total GAGs by Superose 6 gel filtration and HPLC revealed four major fractions, with average molecular masses of 360, 180, 15, and 2.3 kDa, respectively, both in controls and infected tissues. Enzymatic characterization, using GAG-degrading enzymes, showed that in both infected and normal brain stems, the 360- and 180-kDa fractions correspond to hyaluronic acid, which was also the most abundant GAG, while the 15-kDa fractions correspond to chondroitin sulfates as well as heparan sulfate and dermatan sulfate, the latter being the least prominent GAG. Electrophoresis on cellulose acetate membranes revealed that the relative ratio of GAGs was not significantly modified in infected brain stems, compared to controls. However, total GAGs in infected brain stems was significantly decreased by approximately 40%, compared to controls, and this decrease applied equally to all of the above GAG fractions. The diminution observed in total GAGs in infected brain stems is in good agreement with the recently reported neuroprotective role of certain GAG molecules and offers an additional criterion for differential diagnosis of BSE-infected animals.

Expression of the prion protein in the rat forebrain--an immunohistochemical study.

The cellular prion protein (PrP(C)) is crucial for the development of transmissible spongiform encephalopathies (TSEs), where the pathogenic scrapie isoform (PrP(Sc)) of the same protein, is considered to be the principal or sole infectious agent. Here, we report findings on PrP(C) expression in the rat forebrain, using immunohistochemical techniques on free floating sections of 60 microm thickness. Along with neurons and astrocytes in the gray matter, PrP(c) was detected for the first time in glial cells of the white matter and in cells of circumventricular organs. PrP(C) positive cellular processes were also found to be closely associated with intraparenchymal blood vessels, often in the form of end feet. Interestingly, PrP(C) expression was observed in areas where PrP(Sc) deposition in late stages of infection has been earlier reported in the rat and other species.

Preliminary findings on the expression of thymosin beta-10 in human breast cancer.

Paraffin sections from 30 human breast tissue specimens were stained with a specific antibody for thymosin beta-10, ATB10(38-43). The results showed that thymosin beta-10 was detected mainly in the malignant tissue, particularly in the cancerous cells, whereas the normal cell population around the lesions showed very weak staining. Also, the intensity of staining in the cancerous cells was proportionally increased with the increasing grade of the lesions.