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Ludwig Boltzmann Institute for Research into Infectious Venerodermatological Diseases, Department of Dermatology, University of Vienna Medical School, Vienna, Austria
Prion diseases or transmissible spongiform encephalopathies are lethal neurodegenerative diseases caused by proteinaceous agents that consist of an abnormal form of a host protein designated PrP and are devoid of nucleic acids. In laboratory settings these diseases are usually transmitted by intracerebral or peripheral inoculation. In the field they have been shown to be transmitted by uptake of contaminated food but in most instances the route of transmission remains obscure. Both nervous and lymphatic tissues in peripheral organs have been implicated in the spread and propagation of prions. The exact sites of uptake and initial propagation of the infectious agents have not yet been determined, however. As the expression of PrPc is required for the propagation of the infectious agent the search for peripheral cells positive for PrPc may reveal potential routes of entry and transmission. Recently epidermal and mucosal keratinocytes have been found to express PrPc. These data together with the recent finding that epithelial cells are able to support prion replication in vitro suggest that keratinocytes might play a role in the pathogenesis and/or transmission of prion diseases.
) characterized by cerebral accumulation of the scrapie prion protein (PrPSc). These diseases include kuru, Creutzfeld–Jakob disease (CJD), and Gerstmann–Sträussler–Scheinker disease in humans, and scrapie and bovine spongiform encephalopathy (BSE) in animals. Recently, the emergence of a new human transmissible encephalopathy, i.e., new variant Creutzfeld–Jakob disease (nvCJD) attracted considerable attention. This disorder is presumably transmitted from cattle with BSE to humans (
) nvCJD is believed by some researchers to reach epidemic dimensions in the future.
The infectious cause of transmissible spongiform encephalopathies (TSE) are the so-called prions, which stands for proteinaceous infectious particles lacking nucleic acids. They consist mainly if not entirely of PrPSc (
). It is thought that the contact of prions with cell prion protein (PrPc) leads to a conformational change of the latter, transforming it into the PrPSc isoform, which is resistant to protease digestion and which accumulates in brain cells and cerebral interstitium. This conversion of PrPc to PrPSc is considered to be the central mechanism in the pathogenesis of prion diseases (
), it is accepted by most researchers and is widely used to explain the pathogenesis of TSE.
Consequently, expression of glycolipid-anchored PrPc on brain cells is the prerequisite for the manifestation of prion diseases, which are characterized by the cerebral accumulation of PrPSc (
). For the understanding of the pathobiology of TSEs it is important to identify cell types in peripheral tissues expressing PrPc that could be involved in propagating prions.
In addition to brain cells PrPc is expressed by a variety of peripheral tissues including lymphocytes, monocytes, and cells of different lineages (
). The deletion of the PrP gene in mice makes the animals resistant to the induction of TSE but does not lead to an overt phenotype indicative for a particular physiologic function of PrPc (
). In contrast, the routes of transmission in the field are still not elucidated and several different modes such as the ingestion of contaminated food and peripheral inoculation have been suggested for some of the TSEs (
). Ingestion of contaminated human tissue in the course of ritual funeral ceremonies is favored by most as the mode of transmission of kuru in the Fore people on Papua New Guinea. Similarly meat and bone meals derived from scrapie-infected carcasses is the most likely cause of transmission of BSE to cattle. A formal proof for these assumptions has not been provided so far, however. Supporting evidence for the involvement of the gastrointestinal tract in the transmission of prion diseases comes from the recent demonstration that keratinocytes of the tonsils and epithelia throughout the gastrointestinal tract of primates fed BSE-infected cattle brain stained for PrPSc (
Prion immunoreactivity in brain, tonsil, gastrointestinal epithelial cells, and blood and lymph vessels in lemurian zoo primates with spongiform encephalopathy.
) is of interest also in view of the hypothesis that nvCJD might be linked to BSE through oral exposure to the infectious agent.
Also in kuru, probably the best studied TSE, prion transmission is thought to occur by ingestion of tissue of deceased relatives. The high proportion of diseased women who are involved in preparing the ritual meals and the infection of small children who do not participate in the ritual meals but are with their mothers during meal preparation (
), however, suggest that ingestion is only one of the possible routes of kuru transmission and that alternative ways of infection must exist. As inflammatory skin diseases were frequent among the Fore people (
), an impaired skin barrier might have facilitated transmission of kuru by peripheral inoculation.
Scrapie of sheep is one of the longest known prion-associated disorders; however, also for this disease the transmission routes have not yet been determined (
). Transmission of scrapie appears not to depend on the direct contact of noninfected and infected sheep as animals can acquire the disease by grazing on pastures that previously have been populated by infected animals (
). Interestingly, in contrast to experimentally infected sheep, naturally infected sheep develop a severe, itching skin disease leading to hair loss and skin ulcers (Figure 1). Therefore, despite the impressive progress that has been made in the elucidation of the pathogenesis of TSEs over the past decade, the initial steps in acquiring these diseases by infection remain obscure. This and recent reports on the transmission of prion diseases via inoculation into the skin of rodents (
In normal human skin only little expression of PrPc was detectable by immunohistochemistry. Apart from sporadic mononuclear cells within the dermis, weak staining was confined mainly to a minority of epithelial cells. By contrast, in squamous epithelium of mucous membranes of different sites, distinct constitutive PrPc expression was detected in basal but not suprabasal keratinocytes. This pattern of expression was consistently observed in pharynx, larynx, esophagus, vulva, and the ectocervix. In contrast to relatively low PrPc expression in normal epidermis this protein was strongly upregulated in basal and suprabasal keratinocytes up to the granular layer in eczema, psoriasis, and adjacent to skin ulcerations (
; Figure 2). In addition infiltrating inflammatory cells comprising T lymphocytes and macrophages were also distinctly positive for PrPc. In epithelial tumors PrPc expression was most pronounced in common warts and squamous cell carcinomas whereas basal cell carcinomas were mostly negative (
Figure 2PrPcis expressed by keratinocytes in inflammatory skin diseases. In contrast to regular human skin (panel A), keratinocytes are strongly positive for PrPc in inflammatory skin diseases such as psoriasis (panel B). In addition, mononuclear cells within the dermis and epidermis express PrPc. Immunohistochemical analysis in Figure 2 was performed as described previously (
The identity of PrPc expressed by keratinocytes was confirmed by Western blot analysis of cultured cells, which detected PrPc moieties in a range of 30–43 kDa (Figure 3, left lane) corresponding to the size reports for other tissues. The expression of PrPc by keratinocytes in tissue culture was upregulated by interferon-γ and transforming growth factor α (TGF-α) (Figure 3, right lane), but not by interleukin-1β or tumor necrosis factor α. In keeping with the protein data were the data obtained by northern hybridization with a PrPc cDNA probe. A specific band of around 2.3 kb was present in RNA from epithelial cells in primary culture and in SkMEL28 melanoma cells but not in A431 vulvar carcinoma cells (
Figure 3PrPcexpression by human foreskin keratinocytes (passage 2–5) is upregulated by TGF-α. Western blot analysis of keratinocytes demonstrates that addition of TGF-α to the culture medium upregulates PrPc expression. The band at around 26 kDa corresponds to the nonglycosylated form of PrPc. Culture of keratinocytes and Western blot analysis were performed as described previously (
). In regular skin staining for PrPc was observed on basal keratinocytes. Upregulation of PrPc expression was observed in acanthotic epidermis adjacent to skin ulcers. On the snout and to a lesser degree on the tongue also suprabasal keratinocytes were consistently stained (Figure 4). Whereas basal cells of the esophagus were focally positive for PrPc, epithelia of the forestomach did not stain above control levels.
Figure 4PrPc is expressed in bovine skin. PrPc expression is readily observed in basal and suprabasal keratinocytes of regular bovine snout skin (
To confirm the data obtained by immunohistochemistry we analyzed cultured bovine keratinocytes (2–5 passage) derived from the snout or ear by Western blotting. In five independent experiments we found that bovine keratinocytes similarly to brain tissue constitutively expressed PrPcin vitro (
In both human and bovine skin, parts of the hair follicle expressed PrPc distinctly. Both the inner part of the hair bulb around the dermal papilla and keratinocytes of the outer root sheath exhibited a weak to moderate staining for PrPc. Also the outer epithelial layer of the sweat glands as well as myoepithelial cells of the mammary gland were mostly weakly positive for PrPc. This staining was focally moderately enhanced in inflamed areas. Dermal fibroblasts that have been described to express PrPc in tissue culture (
) were negative by immunohistochemistry. PrPc was found also to be expressed in keratinocytes of ovine and murine origin (J. Pammer, unpublished observations). The expression of PrPc by keratinocytes was recently confirmed by others (
Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice.
Implications of PrPc expression by human keratinocytes
Whereas the expression of PrPc is a must for the reproduction of prions, it could be demonstrated that PrPc expression alone is not sufficient for this process but requires additional yet unknown cofactors (
); however, little is known about the role of other cell types.
Deposition of PrPSc and distribution of infectivity in peripheral organs show marked variation, which may be attributed to different qualities of both prion strains and infected hosts. This divergence can be significant in a variety of TSEs. Depending on the strain PrPSc was found in macrophages (
Further studies of blood infectivity in an experimental model of transmissible spongiform encephalopathy, with an explanation of why blood components do not transmit Creutzfeldt–Jakob disease in humans.
Prion immunoreactivity in brain, tonsil, gastrointestinal epithelial cells, and blood and lymph vessels in lemurian zoo primates with spongiform encephalopathy.
Depending on the amount of infectivity in some peripheral organs like the gingiva, not only is infectious material transported by inflammatory cells or possibly nerves to these organs, but the infectious agents can replicate extraneurally (
) demonstrating the importance of identifying any cell types expressing PrPc in peripheral tissues that could be involved in propagating prion infectivity.
In a recent study, lemurs fed over a number of years with food containing cattle meat developed neurologic symptoms and the presence of PrPSc was demonstrated on epithelia throughout the digestive tract indicating their infection (
). Both basal cells and a number of flattened cells of the superficial zone of the stratified epithelium of the tonsils and the esophagus exhibited reactivity for PrPSc pointing to a possible role of keratinocytes in TSE transmission. In this context it is important that it was recently demonstrated that active prion replication can take place in cells of an epithelial type, i.e., a rabbit kidney epithelial cell line expressing ovine PrPc (
) in tissue culture support the view that cofactors necessary for prion replication are not restricted to neuronal and hematopoietic cells. Thus it has been speculated that some features shared by neurons and epithelial cells might be involved in the replication of prions (
) and thus argues against a continuous prion replication in keratinocytes. As the efficiency of scrapie agent replication depends on the level of PrPc expression (
), however, failure to detect PrPSc in regular human skin samples may be due to a low expression of the target protein PrPc in this organ under normal conditions. It would be interesting to study the presence of prions in the skin in inflammatory skin diseases associated with an upregulation of PrPc. The absence of PrPSc from the normal skin of patients with nvCJD does not preclude a potential role of keratinocytes as port of entry for prions. Expression of PrPc by mucous membrane and skin keratinocytes suggests that they represent a possible first target for prions. Although it is highly improbable that prions are able to cross an intact stratum corneum, there are many instances where this outer barrier is defective either as a consequence of microtrauma or in the course of various dermatoses. In particular inflammatory skin diseases such as eczema or inflamed ulcerations could provide ideal conditions for prion entry and replication: the infectious agent could pass through the breaks in the barrier (Figure 5) and then either bypass epithelial cells and infect the lymphoid tissue via the inflammatory leukocytes (
). Both the lymphoid system and nerves have been found to contain PrPSc in prion diseases. Alternatively the increased production of PrPc by keratinocytes provides a substrate for a first round of prion replication followed by the spread of prions to the immune system and the central nervous system (Figure 5).
Figure 5Possible steps in prion invasion and spread in the skin. (1) Breakdown of the cornified layer in inflammatory skin diseases and simultaneous upregulation of PrPc expression by epidermal keratinocytes could allow entry of prions and infection of keratinocytes. (2) Spread of prions to the central nervous system via intraepidermal sensory skin nerves. (3) Spread of prions to the lymphatic system by passenger leukocytes. (4) Transport of prions to the epidermis by infected leukocytes in the course of inflammatory skin diseases.
Another potential port of entry relates to the use of in vitro reconstructed skin for transplantion on burn wounds or chronic ulcers. Constituents of bovine origin are frequently added to the tissue culture medium used to grow keratinocytes. As in vitro cultivated keratinocytes express high amounts of PrPc (
). Whereas it is generally believed that at least some of the prion diseases are transmitted via the gastrointestinal tract by ingestion of contaminated material, the exact routes have not yet been elucidated. It is therefore reasonable to keep in mind also the possibility that inoculation via the skin represents a possible route for prion transmission and that epidermal and mucosal keratinocytes are potential target cells for this infectious agent.
References
Adams D.H.
Edgar W.M.
et al.
Transmission of agent of Creutzfeldt–Jakob disease [letter].
Prion immunoreactivity in brain, tonsil, gastrointestinal epithelial cells, and blood and lymph vessels in lemurian zoo primates with spongiform encephalopathy.
Further studies of blood infectivity in an experimental model of transmissible spongiform encephalopathy, with an explanation of why blood components do not transmit Creutzfeldt–Jakob disease in humans.
Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice.
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