Human sporadic Creutzfeldt-Jakob disease (sCJD), endemic sheep scrapie, and epidemic bovine spongiform encephalopathy (BSE) are caused by a related group of infectious agents. The new U.K. BSE agent spread to many species, including humans, and clarifying the origin, specificity, virulence, and diversity of these agents is critical, particularly because infected humans do not develop disease for many years. As with viruses, transmissible spongiform encephalopathy (TSE) agents can adapt to new species and become more virulent yet maintain fundamentally unique and stable identities. To make agent differences manifest, one must keep the host genotype constant. Many TSE agents have revealed their independent identities in normal mice. We transmitted primate kuru, a TSE once epidemic in New Guinea, to mice expressing normal and Ϸ8-fold higher levels of murine prion protein (PrP). High levels of murine PrP did not prevent infection but instead shortened incubation time, as would be expected for a viral receptor. Sporadic CJD and BSE agents and representative scrapie agents were clearly different from kuru in incubation time, brain neuropathology, and lymphoreticular involvement. Many TSE agents can infect monotypic cultured GT1 cells, and unlike sporadic CJD isolates, kuru rapidly and stably infected these cells. The geographic independence of the kuru agent provides additional reasons to explore causal environmental pathogens in these infectious neurodegenerative diseases.sporadic CJD ͉ bovine spongiform encephalopathy ͉ viral adaptation ͉ prion protein receptor ͉ tissue culture infection

Membranous epithelial cells (M cells) of the follicle-associated epithelium in Peyer's patches have a high capacity for transcytosis of several viruses and microorganisms. Here, we report that we have successfully established a bovine intestinal epithelial cell line (BIE cells) and developed an in vitro M cell model. BIE cells have a cobblestone morphology and microvilli-like structures, and strongly express cell-to-cell junctional proteins and cytokeratin, which is a specific intermediate filament protein of epithelial cells. After co-culture with murine intestinal lymphocytes or treatment with supernatant from bovine PBMC cultured with IL-2, BIE cells acquired the ability of transcytosis. Therefore, BIE cells have typical characteristics of bovine intestinal epithelial cells and also have the ability to differentiate into an M cell like linage. In addition, our results indicate that contact between immune cells and epithelial cells may not be absolutely required for the differentiation of M cells. We think that BIE cells will be useful for studying the transport mechanisms of various pathogens and also the evaluation of drug delivery via M cells.

Rat septal cells, induced to enter a terminal differentiation-like state by temperature shift, produce prion protein (PrP) levels 7x higher than their proliferative counterparts. Host PrP accumulates on the plasma membrane, newly elaborated nanotubes, and cell-to-cell junctions, important conduits for viral spread. To find if elevated PrP increased susceptibility to FU-CJD infection, we determined agent titers under both proliferating and arresting conditions. A short 5 day arrest and a prolonged 140 day arrest increased infectivity by 5x and 122x (>2 logs) respectively as compared to proliferating cells. Total PrP rapidly increased 7x and was even more elevated in proliferating cells that escaped chronic arrest conditions. Amyloid generating PrP (PrP-res), the “infectious prion” form, present at ∼100,000 copies per infectious particle, also increased proportionately by 140 days. However, when these highly infectious cells were switched back to proliferative conditions for 60 days, abundant PrP-res continued to be generated even though 4 logs of titer was lost. An identical 4 log loss was found with maximal PrP and PrP-res production in parallel cells under arresting conditions. While host PrP is essential for TSE agent spread and replication, excessive production of all forms of PrP can be inappropriately perpetuated by living cells, even after the initiating infectious agent is eliminated. Host PrP changes can start as a protective innate immune response that ultimately escapes control. A subset of other neurodegenerative and amyloid diseases, including non-transmissible AD, may be initiated by environmental infectious agents that are no longer present.

The hypothesis that host prion protein (PrP) converts into an infectious prion form rests on the observation that infectivity progressively decreases in direct proportion to the decrease of PrP with proteinase K (PK) treatment. PrP that resists limited PK digestion (PrP-res, PrPsc) has been assumed to be the infectious form, with speculative types of misfolding encoding the many unique TSE agent strains. Recently, a PK sensitive form of PrP has been proposed as the prion. Thus we re-evaluated total PrP (sensitive and resistant) and used a cell-based assay for titration of infectious particles. A keratinase (NAP) known to effectively digest PrP was compared to PK. Total PrP in FU-CJD infected brain was reduced to ≤0.3% in a 2hr PK digest, yet there was no reduction in titer. Remaining non-PrP proteins were easily visualized with colloidal gold in this highly infectious homogenate. In contrast to PK, NAP digestion left 0.8% residual PrP after 2hr, yet decreased titer by >2.5logs; few residual protein bands remained. FU-CJD infected cells with 10x the infectivity of brain by both animal and cell culture assays were also evaluated. NAP again significantly reduced cell infectivity (>3.5 logs). Extreme PK digestions were needed to reduce cell PrP to <0.2%, yet a very high titer of ≥7.8 logs remained. Our FU-CJD brain results are in good accord with the only other report on maximal PrP digestion and titer. It is likely that one or more residual non-PrP proteins may protect agent nucleic acids in infectious particles.

Many mammalian species can be afflicted with prion diseases or transmissible spongiform encephalopathies. Prion diseases are neurodegenerative, transmissible, untreatable, and fatal (reviewed in references 1 and 2). The underlying pathogenesis of prion diseases involves the conversion of the host's normal prion protein, PrP C , to abnormal forms that are usually more protease resistant, multimeric, and insoluble. The multimeric and insoluble forms have been generically called PrP Sc (PrP-scrapie), or more functionally PrP Res (protease resistant) and PrP D (disease associated). At least some of these forms comprise the transmissible agent or prion (3-10). Multiple prion strains can be propagated within a given host species, giving consistently different clinical, pathological, and molecular phenotypes (11-16).The first prion disease to be recognized in cattle was classical bovine spongiform encephalopathy (C-BSE). C-BSE was likely caused primarily by widespread prion contamination of cattle feed (17). After peaking in the early 1990s, the incidence of C-BSE has now been greatly reduced by regulatory measures that limit its horizontal spread. C-BSE is the only known zoonotic prion disease, having caused variant Creutzfeldt-Jakob disease (vCJD) in humans who presumably consumed contaminated beef. Although new clinical cases of vCJD are rare (http://www.cjd.ed.ac .uk/documents/figs.pdf), a recent survey of appendices in the United Kingdom suggests a high incidence of subclinical vCJD infections, at ϳ1:2,000 of the population born between 1941 and 1985 (18).Since the C-BSE epidemic, two atypical strains of BSE, H-type BSE (H-BSE) and L-type BSE (L-BSE), have also been identified in cattle. PrPRes is usually composed of a mixture of glycosylated and unglycosylated molecules, and the various BSE strains can be differentiated biochemically using Western blotting of postmortem brain tissue samples by comparing the proteinase K (PK)-treated PrPRes banding patterns (19)(20)(21)(22). The H and L types of BSE are classified by their respective high and low apparent molecular masses of the unglycosylated PrP Res band. These atypical BSE strains, which are rare (Ͻ100 cases identified worldwide), tend to affect older animals (23) and appear to represent sporadic forms of bovine prion diseases (24). Despite the apparent rarity of the various types of BSE, the facts that H-and L-BSE appear to arise spontaneously and have distinct transmissibilities (20, 25-32) make it important to be able to detect and differentiate them to reduce the risk of transmission to cattle or other species, such as humans.Several in vitro methods have been developed to detect C-, L-, and H-BSE. Commercially available rapid immunochemical tests for PrP D can, in the best cases, give positive responses from 10 Ϫ3 to 10 Ϫ4 dilutions of postmortem brain tissues with high levels of PrP D (33, 34). Immunoblotting for PrP Res can detect BSE-infected tissues with similar sensitivity and also discriminate between the bovine strains based on the relative electro...

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There are two independent serotonin (5-HT) systems of organization: one in the central nervous system and the other in the periphery. 5-HT affects feeding behavior and obesity in the central nervous system. On the other hand, peripheral 5-HT also may play an important role in obesity, as it has been reported that 5-HT regulates glucose and lipid metabolism. Here we show that the intraperitoneal injection of 5-HT to mice inhibits weight gain, hyperglycemia and insulin resistance and completely prevented the enlargement of intra-abdominal adipocytes without having any effect on food intake when on a high fat diet, but not on a chow diet. 5-HT increased energy expenditure, O2 consumption and CO2 production. This novel metabolic effect of peripheral 5-HT is critically related to a shift in the profile of muscle fiber type from fast/glycolytic to slow/oxidative in soleus muscle. Additionally, 5-HT dramatically induced an increase in the mRNA expression of peroxisome proliferator-activated receptor coactivator 1α (PGC-1α)-b and PGC-1α-c in soleus muscle. The elevation of these gene mRNA expressions by 5-HT injection was inhibited by treatment with 5-HT receptor (5HTR) 2A or 7 antagonists. Our results demonstrate that peripheral 5-HT may play an important role in the relief of obesity and other metabolic disorders by accelerating energy consumption in skeletal muscle.