Christine Farquhar

Migrating intestinal dendritic cells transport PrPSc from the gut

Bovine spongiform encephalopathy, variant Creutzfeldt-Jakob disease (vCJD) and possibly also sheep scrapie are orally acquired transmissible spongiform encephalopathies (TSEs). TSE agents usually replicate in lymphoid tissues before they spread into the central nervous system. In mouse TSE models PrP(c)-expressing follicular dendritic cells (FDCs) resident in lymphoid germinal centres are essential for replication, and in their absence neuroinvasion is impaired. Disease-associated forms of PrP (PrP(Sc)), a biochemical marker for TSE infection, also accumulate on FDCs in the lymphoid tissues of patients with vCJD and sheep with natural scrapie. TSE transport mechanisms between gut lumen and germinal centres are unknown. Migratory bone marrow-derived dendritic cells (DCs), entering the intestinal wall from blood, sample antigens from the gut lumen and carry them to mesenteric lymph nodes. Here we show that DCs acquire PrP(Sc) in vitro, and transport intestinally administered PrP(Sc) directly into lymphoid tissues in vivo. These studies suggest that DCs are a cellular bridge between the gut lumen and the lymphoid TSE replicative machinery.

Tumor Necrosis Factor Alpha-Deficient, but Not Interleukin-6-Deficient, Mice Resist Peripheral Infection with Scrapie

ABSTRACT In most peripheral infections of rodents and sheep with scrapie, infectivity is found first in lymphoid tissues and later in the central nervous system (CNS). Cells within the germinal centers (GCs) of the spleen and lymph nodes are important sites of extraneural replication, from which infection is likely to spread to the CNS along peripheral nerves. Here, using immunodeficient mice, we investigate the identity of the cells in the spleen that are important for disease propagation. Despite possessing functional T and B lymphocytes, tumor necrosis factor alpha-deficient (TNF-α−/−) mice lack GCs and follicular dendritic cell (FDC) networks in lymphoid tissues. In contrast, lymphoid tissues of interleukin-6-deficient (IL-6−/−) mice possess FDC networks but have impaired GCs. When the CNSs of TNF-α−/−, IL-6−/−, and wild-type mice were directly challenged with the ME7 scrapie strain, 100% of the mice were susceptible, developing disease after closely similar incubation periods. However, when challenged peripherally (intraperitoneally), most TNF-α−/− mice failed to develop scrapie up to 503 days postinjection. All wild-type and IL-6−/− mice succumbed to disease approximately 300 days after the peripheral challenge. High levels of scrapie infection and the disease-specific isomer of the prion protein, PrPSc, were detectable in spleens from challenged wild-type and IL-6−/− mice but not from TNF-α−/−mice. Histopathological analysis of spleen tissue demonstrated heavy PrP accumulations in direct association with FDCs in challenged wild-type and IL-6−/− mice. No PrPScaccumulation was detected in spleens from TNF-α−/−mice. We conclude that, for the ME7 scrapie strain, mature FDCs are critical for replication in lymphoid tissues and that in their absence, neuroinvasion following peripheral challenge is impaired.

Prophylactic potential of pentosan polysulphate in transmissible spongiform encephalopathies

Variant Creutzfeldt-Jakob disease (CJD) probably results from exposure to the bovine spongiform encephalopathy agent. There is no treatment to slow or halt variant CJD, and the diagnosis is usually made when patients are terminally ill. During the long incubation period when there are no symptoms, there is a potential but unknown risk of transfer of infection by blood transfusion, treatment with blood products, transplantation, or reuse of surgical instruments. Scrapie was the first described transmissible spongiform encephalopathy (TSE). After peripheral infection with scrapie, titre rises in lymph nodes and spleen before transfer to the central nervous system (CNS). A fraction of the host’s “prion protein” (PrP) aggregates into a protease-resistant isoform (PrP), and this marker enables tracking of infectious spread throughout the host. From mouse models we know that the timing of these events depends on the infecting TSE strain and host PrP genotype. PrP aggregates have been identified in lymphoid tissue removed from a patient long before variant CJD was diagnosed. Dextran sulphate 500 (DS500) prolongs scrapie incubation period in mice and reduces disease susceptibility. Another polyanion and heparin analogue, pentosan polysulphate (PS syn SP54, Tavan, Elmiron) is less toxic in vivo. It is also more effective in lengthening incubation and reducing susceptibility when given at high dose (3310 mg) weeks before scrapie inoculation. In a model in which infectivity enters the CNS very quickly, however, PS is only effective if given close to scrapie injection. We report results of a single administration of low-dose PS given intraperitoneally after a normally 100% lethal intraperitoneal scrapie injection in mice. Two mousepassaged scrapie strains were inoculated into six different inbred mouse strains. PS (sodium salt, Sigma) was given intraperitoneally or orally after scrapie, as either 250 mg or 1 mg in saline. Controls matched for age and sex were given saline. Individual incubation periods and scrapie diagnosis were by standard methods. Intraperitoneal PS given 7 h after scrapie prolonged survival significantly in all cases (table). Depending on mouse strain, 250 mg of PS increased the mean incubation period of the ME7 scrapie strain by up to 66%. 1 mg of PS protected VM mice completely from the 22A scrapie strain; animals died from unrelated causes between 500 and 707 days after inoculation whereas controls succumbed to scrapie by 428 days (mean 376); this represents at least a hundredfold reduction in the sensitivity of these mice to infection. After ME7 infection, there were survivors in treated groups. For the CBA mouse strain, 1 mg PS was completely protective. Treated animals dying of unrelated causes up to 526 days after inoculation displayed neither clinical symptoms of scrapie nor pathological changes: untreated CBA mice all died from scrapie by 310 days (mean 283). Oral PS was ineffective in delaying disease. Multiple high doses of PS are licensed in the USA for the treatment of interstitial cystitis. Although direct testing of the efficacy of PS in reducing susceptibility to variant CJD is not possible, further animal studies are essential to examine the possible use of PS for risk reduction.

Immunodetection of PrPSc in spleens of some scrapie-infected sheep but not BSE-infected cows

The development of diagnostic tools for transmissible spongiform encephalopathies (TSEs) would greatly assist their study and may provide assistance in controlling the disease. The detection of an abnormal form of the host protein PrP in noncentral nervous system tissues may form the basis for diagnosis of TSEs. Using a new antibody reagent to PrP produced in chickens, PrP can be readily detected in crude tissue extracts. PrP from uninfected spleen had a lower molecular mass range than PrP from brain, suggesting a lower degree of glycosylation. A simple method for detecting the abnormal form of the protein, PrPSc, in ruminant brain and spleen has been developed. PrPSc was detected in sheep spleen extracts from a flock affected by natural scrapie and was also found in spleens from some, but not all, experimental TSE cases. In spleens from cattle with bovine spongiform encephalopathy (BSE) no PrPSc was detected. It is therefore suggested that there is differential targeting of PrPSc deposition between organs in these different types of TSE infection which, with other factors, depends on strain of infecting agent.

Morphogenesis of amyloid plaques in 87V murine scrapie

Amyloid plaques of scrapie–infected mouse brains are composed of fibrillar forms of a host coded, cell surface sialoglycoprotein called PrP (prion protein). Serial ultrastructural immunogold staining was performed on plaques identified by light microscopic immunocytochemistry of brains of VM mice infected with the 8 7V strain of scrapie. Classical plaques, of a kuru–type morphology, were composed of a central core of bundles of amyloid fibrils. Amyloid fibrils of classical plaques were immunoreactive for PrP. In addition, PrP was also found at the plaque periphery, in the absence of fibrils, at the plasmalemma of cell processes and in the associated extracellular spaces. Frequent microglial cells and occasional astrocytes contained PrP within lysosomes. Other plaques with few or no recognizable amyloid fibrils were frequent and were termed primitive plaques. PrP could be demonstrated in a non–fibrillar form at the plasmalemma and in the extracellular spaces between neurites of such plaques. Many primitive plaques showed little or no sub–cellular pathology associated with the PrP accumulation. PrP was closely associated with the plasma–lemma of occasional dendrites passing towards the centre of primitive plaques. These results suggest that plaques are formed around one or more PrP releasing dendrites. PrP accumulates in the extracellular spaces adjacent to such processes prior to its spontaneous aggregation into fibrils. Lysosomal accumulation of PrP in microglia and astrocytes located at the periphery of plaques suggest that these cells are involved in the phagocytosis of excess or abnormal PrP.

Straining the prion hypothesis

Aguzzi and Weissmann in their News and Views feature correctly state that research on the molecular genetics of PrP protein has contributed greatly to our knowledge of the transmissible spongiform encephalopathies (TSEs). But their firm belief that these diseases are caused by rogue proteins (‘prions’) leads them to misrepresent alternative hypotheses of the nature of the agent, dismissing all non-believers as “the die-hard pro-virus faction”. In fact, the prion hypothesis is far from proven: the precise nature of a prion still eludes identification and the prion hypothesis has yet to explain satisfactorily the many strains of TSE. The alternative ‘virino’ hypothesis is not a conventional virus hypothesis, but it addresses the diversity of biological properties of the TSEs. It proposes an agent-specific replicable informational molecule, yet to be identified, bound to a protective host protein, PrP.

Effect of Sinc genotype, agent isolate and route of infection on the accumulation of protease-resistant PrP in non-central nervous system tissues during the development of murine scrapie.

Mice congenic for the Sinc gene were infected intracerebrally with two scrapie strains, ME7 and 22A. At various times during the incubation period tissues were monitored for the infection-specific form of PrP (PrPSc). PrPSc was found in brain, spleen, lymph nodes, pancreas, submaxillary gland and thymus. After intraperitoneal inoculation PrPSc was found in spleen, lymph nodes, pancreas and submaxillary glands prior to its detection in brain. The kinetics of accumulation of PrPSc in these tissues was dependent on the infecting strain of agent, on the mouse Sinc genotype and on the route of infection. This study supports using the presence of PrPSc as an indicator of infectivity in brain and extraneural tissues and defines some of the parameters which influence when and where PrPSc is first found.

Follicular dendritic cells in TSE pathogenesis

The pathogenesis of transmissible spongiform encephalopathies (TSEs) often includes a replication phase in lymphoid tissues before infection spreads to the central nervous system. Recent studies show that the follicular dendritic cells of the germinal centres are critical for this replication. These cells are therefore potential targets for therapy or prophylaxis in natural TSEs, such as variant Creutzfeldt-Jakob disease.

Involvement of the immune system in TSE pathogenesis

Abstract The transmissible spongiform encephalopathies (TSEs) are continuing to attract both scientific interest and public concern. Here, Neil Mabbott and colleagues review current understanding of the peripheral pathogenesis of TSE diseases.

Comparative analysis of normal prion protein expression on human, rodent, and ruminant blood cells by using a panel of prion antibodies.

BACKGROUND: It is not known whether variant CJD can be transmitted within the human population by blood transfusion. The expression of normal cellular prion protein (PrPC) by different blood cell types may permit selective uptake and dissemination of infectivity.