Immunotherapeutic effect of anti-PrP monoclonal antibodies in transmissible spongiform encephalopathy mouse models: pharmacokinetic and pharmacodynamic analysis.

Prion diseases are transmissible neurodegenerative disorders for which no therapeutic or prophylactic regimens exist. Passive immunization with appropriate antibodies directed against the cellular form of the prion protein (PrPC) can delay the onset of prion disease after peripheral infection, but mechanisms and parameters determining their in vivo efficacy remain unknown. In the present study, we characterized the main pharmacokinetic properties of anti-PrP antibodies in different mouse models expressing various levels of PrPC (Prnp(0/0), C57BL/6 and tga20 mice) in correlation with therapeutic effect. Plasma levels of free antibodies, total endogenous PrPC and PrPC-antibody complexes were monitored after a single intraperitoneal monoclonal antibody (mAb) injection. Efficacy in delaying PrPSc peripheral accumulation seemed to be associated with mAb capacity to form long-lasting complexes with endogenous PrPC in the plasma. In agreement with previous observations on cellular models of transmissible spongiform encephalopathy infection, we observed that injection of anti-PrP antibodies induced a large (up to 100-fold) increase in circulating PrPC. Finally, the most efficient antibody extended the lifespan of infected animals greatly. These results allowed us to define critical characteristics of anti-PrP mAbs associated with therapeutic efficacy and could constitute a useful reference for designing optimized passive immunotherapies for prion diseases.

Prions in milk from ewes incubating natural scrapie.

Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrP(Sc) accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 microg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.

Dose-response study of effect of oleuropein, an olive oil polyphenol, in an ovariectomy/inflammation experimental model of bone loss in the rat.

BACKGROUND & AIMS: This study was carried out to assess the dose-dependent bone-sparing effect of oleuropein, an olive oil phenolic compound with anti-inflammatory and anti-oxidative properties, on bone loss induced by talc granulomatosis in oestrogen-deficient rat. METHODS: Among 98 rats, 20 were sham-operated (SH) while the others (78) were ovariectomised (OVX). The SH and 26 OVX rats (controls) were given a standard diet for 100 days. The 52 remaining OVX rats were allocated to 4 groups that received oleuropein at 2.5, 5, 10 or 15 mg/kg body weight per day for 100 days. Three weeks before necropsy, an inflammation was induced by subcutaneous injections of talc in half of the SH and OVX rats and in all oleuropein-treated animals. RESULTS: Castration was associated with a decreased bone mineral density (BMD). In OVX rats, inflammation, characterised by an increase of the spleen weight and plasma fibrinogen levels, exacerbated this bone loss, as shown by values of BMD of the total femur metaphyseal and diaphyseal subregions. The 4 doses of oleuropein reduced bone loss and improved inflammatory biomarkers excepted for 5mg/kg BW. CONCLUSIONS: Every dose of oleuropein elicited protective effects on bone mass in this model of ovariectomy associated with inflammation, probably by modulating inflammatory parameters.

A simple, versatile and sensitive cell-based assay for prions from various species.

Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies.

Classic scrapie in sheep with the ARR/ARR prion genotype in Germany and France.

In the past, natural scrapie and bovine spongiform encephalopathy (BSE) infections have essentially not been diagnosed in sheep homozygous for the A136R154R171 haplotype of the prion protein. This genotype was therefore assumed to confer resistance to BSE and classic scrapie under natural exposure conditions. Hence, to exclude prions from the human food chain, massive breeding efforts have been undertaken in the European Union to amplify this gene. We report the identification of 2 natural scrapie cases in ARR/ARR sheep that have biochemical and transmission characteristics similar to cases of classic scrapie, although the abnormally folded prion protein (PrP(Sc)) was associated with a lower proteinase-K resistance. PrP(Sc) was clearly distinct from BSE prions passaged in sheep and from atypical scrapie prions. These findings strongly support the idea that scrapie prions are a mosaic of agents, which harbor different biologic properties, rather than a unique entity.

Bovine spongiform encephalopathy agent in spleen from an ARR/ARR orally exposed sheep.

Oral contamination with bovine spongiform encephalopathy (BSE) agent in susceptible PRNP genotype sheep results in widespread distribution of prion in the host. Because ARR homozygous sheep are considered to be resistant to transmissible spongiform encephalopathies, they have been selected to eradicate scrapie from sheep flocks and to protect the human food chain from small ruminant BSE risk. However, results presented here show that several months after an oral challenge with BSE agent, healthy ARR/ARR sheep can accumulate significant amounts of PrP(Sc) in the spleen.

Isoflavones and functional foods alter the dominant intestinal microbiota in postmenopausal women.

Dietary phytoestrogens, such as isoflavones, are used as food additives to prevent menopause-related disorders. In addition to other factors, their bioavailability strongly depends on the activity of intestinal bacteria but the underlying interactions remain poorly understood. A randomized, double-blind, placebo-controlled study was undertaken with 39 postmenopausal women to characterize changes in the dominant microbial communities of the intestinal tract after 2 mo of isoflavone supplementation with and without pro- or prebiotic. The diversity and composition of the dominant microbiota were analyzed by temporal temperature-gradient gel electrophoresis (TTGE) and fluorescent in situ hybridization. Isoflavones alone stimulated dominant microorganisms of the Clostridium coccoides-Eubacterium rectale cluster, Lactobacillus-Enterococcus group, Faecalibacterium prausnitzii subgroup, and Bifidobacterium genus. The stimulation of the Clostridium coccoides-Eubacterium rectale cluster depended on the women's equol excretion and was transient, with the exception of a prolonged bifidogenic effect. Lasting changes in the diversity of the dominant species were also observed. The probiotic strain supplied could be detected by TTGE during its passage through the intestinal tract, and ingestion of fructooligosaccharides triggered a marked and specific bifidogenic effect. In conclusion, this is the first human study that shows changes in the diversity and composition of dominant bacterial communities in response to dietary supplementation with hormone-related compounds combined with functional foods.

Isoflavone consumption does not increase the bone mass in osteopenic obese female zucker rats.

Some controversy exists in the literature concerning the effects of leptin on bone metabolism. Thus we have compared femoral bone density and biochemical markers of bone metabolism in male and female fatty (leptin-resistant) Zucker rats and their lean homozygous controls at 3 and 6 months of age. At 3 months, no differences concerning total, diaphyseal (cortical bone), and distal metaphyseal (trabecular bone) femoral bone densities, plasma osteocalcin concentrations, and urinary deoxypyridinoline excretion were observed between fatty and lean rats. On the opposite, at 6 months of age, in both males and females, total, diaphyseal, and distal metaphyseal femoral bone densities and plasma osteocalcin concentrations were lower in Zucker than in lean rats. Soybean isoflavone consumption (40 microg/g body weight/day for 90 days, a dose which prevents osteopenia following ovariectomy both in lean Zucker homozygous controls and in Wistar rats) by obese female Zucker rats had no significant effect upon their bone mass.