Aerosols: An underestimated vehicle for transmission of prion diseases?

We and others have recently reported that prions can be transmitted to mice via aerosols. These reports spurred a lively public discussion on the possible public-health threats represented by prion-containing aerosols. Here we offer our view on the context in which these findings should be placed. On the one hand, the fact that nebulized prions can transmit disease cannot be taken to signify that prions are airborne under natural circumstances. On the other hand, it appears important to underscore the fact that aerosols can originate very easily in a broad variety of experimental and natural environmental conditions. Aerosols are a virtually unavoidable consequence of the handling of fluids; complete prevention of the generation of aerosols is very difficult. While prions have never been found to be transmissible via aerosols under natural conditions, it appears prudent to strive to minimize exposure to potentially prion-infected aerosols whenever the latter may arise—for example in scientific and diagnostic laboratories handling brain matter, cerebrospinal fluids, and other potentially contaminated materials, as well as abattoirs. Equally important is that prion biosafety training be focused on the control of, and protection from, prion-infected aerosols.Key words: prion, prion transmission, scrapie, chronic wasting diseases, CWD, Creutzfeldt-Jacob-disease, CJD, TSE, aerosol, pathogens, allergensPrions, the causative agents of transmissible spongiform encephalopathies, can be undoubtedly propagated from one individual organism to another. The specific routes of prion transmission have been subjected to intensive studies over the past two decades. Incidental and iatrogenic transmission has occurred through the intracerebral route in the case of Dura mater implants¹ and the parenteral route in the case of contaminated pituitary hormones.² In addition, the Bovine Spongiform Encephalopathy (BSE) disaster has provided grim evidence that prion can be transmitted enterally as well. Experimental transmission of prions has been routinely achieved via intraperitoneal and intravenous injection^3,4 but also through more exotic routes such as intralingual,⁵ intranerval⁶ and conjunctival inoculation⁷ and via the nasal cavity.⁸In all prion disease paradigms studied so far the propagation, accumulation and dissemination of the prion protein has been mostly shown to depend on a functional immune system.^9–12 This dependence of prion pathogenesis on the lymphoid compartment, however, is only true for peripheral routes of infection—whereas direct inoculation into the brain does not require any components of the adaptive or innate immune system.B cells in secondary lymphoid organs have been shown to be of importance for the neuroinvasion of the prion protein; in contrast, B lymphocytes in the blood do not appear to play a crucial role.^13–15A special role in prion pathogenesis can be assigned to follicular dendritic cells (FDC). The generation, maturation and function of FDC are dependent on cytokines and chemokines predominantly synthesized and secreted by B lymphocytes. Consistently with this role of B cells in prion pathogenesis, B cell deficient mice show a significantly impaired prion replication due to severely impaired maturation of FDCs.¹⁶ Other soluble and membrane-bound immune mediators such as lymphotoxin heterotrimers and TNFalpha^17,18 as well as components of the complement system^19,20 play an important role in prion pathogenesis.While prions mostly reside in tissues, prion infectivity has also been detected in a variety of body fluids including cerebrospinal fluid,²¹ blood,²² saliva,²³ milk²⁴ and urine.²⁵ Although shedding of prions may occur constitutively from these secretions and excretions, many of the latter phenomena are enhanced by chronic inflammatory processes such as granulomas²⁶ and follicular infiltrates,²⁷ which trigger the maturation of lymphotoxin-dependent, prion-replicating cells.²⁶ The presence of prions in fluids begs the question whether nebulization, and subsequent inhalation, of such fluids may trigger prion infections.Aerosols are finely dispersed particles originating from solid material or liquid using air or other gases as carriers. Natural examples of aerosols include dust (e.g., volcano ashes), smoke, haze and sprays (e.g., sneezing or sea water sprays from breaking waves). Aerosols might be formally categorized as primary or secondary, with primary aerosols being generated in mechanical or thermal processes e.g., by whirling up, impact on surfaces, or burning, whereas secondary aerosols are generated during chemical reactions or by using condensation nuclei.Primary aerosols play an important role in microbiology since they can act as efficacious vehicles for pollen, spores, algae, fungi, bacteria and viruses. Of medical importance are also dandruff, fragments of fur, hairs or skin and mites, which can all function as allergens and trigger e.g., allergic asthma.Moreover, aerosols are excellent vehicles for the transportation of drugs into the respiratory tract. The size of the individual droplets is crucial in specifying the target organs of aerosol. Particle sized 3–10 µm are generally deposited in the nasal cavity and in the throat, whereas smaller particles (e.g., 1 µm) tend to deposit within the lower airways. In rodents pulmonary deposition can reach 10%.^28,29 In humans, particles of 5 µm may reach the lung if inhaled orally, but deposition in the alveolar compartment after inhaling via the nose is highly unlikely.^28,29 For the reasons discussed above, we have become interested in exploring the transmission potential of aerosol-borne prions. Indeed, we found that mouse scrapie can be efficiently transmitted via aerosols.³⁰ In addition to results obtained by exposure to aerosols, we found that mice developed prion infections when inoculated intranasally.Interestingly, this route of transmission was entirely independent on immune cells as shown by challenging various transgenic mouse strains lacking defined functions of the immune system.Well-known examples of transmission of pathogens via aerosols are infections by respiratory viruses (e.g., influenza viruses, adenoviruses, rhinoviruses, coronaviruses) and bacterial diseases (e.g., legionellosis, pneumonic plague by Yersinia pestis, Q-fever by Coxiella burnettii, anthrax) and fungal diseases (particularly aspergillosis and candidosis). In stark contrast, aerosols have historically never been regarded as potential vectors for prion diseases—although very little data existed in favor or against this possibility. This attitude goes along with the implicit “conventional wisdom” that prions are not airborne diseases. However, the concept of “airborne disease” in all the bacterial, fungal and viral examples quoted above, encompasses three distinct phases: (1) release of the infectious agent into aerosols by an infected donor, (2) uptake by a healthy recipient and (3) establishment of disease. It is self-evident that little or no natural transmission between individuals will be observed if any one of these three steps is inefficient. The epidemiological evidence from human prion diseases seems to indicate, albeit indirectly, that step #1 does not occur in CJD patients—inter alia because there is a dearth of evidence of proximity clustering of sCJD.³¹ In the case of CWD the situation may be different since saliva and droppings, which might plausibly give rise to powerful aerosols under a variety of conditions, were found to harbor infectivity. Finally, milk from sheep affected by mastitis can carry scrapie infectivity and—again—could conceivably give rise to aerosols. Since both CWD and sheep scrapie can efficiently spread horizontally within animal collectives, it is extremely appealing to speculate whether aerosols may play a role in said transmission.In natural scrapie in sheep horizontal transmission of prion diseases has been long thought to arise from placental contamination. However, in mice suffering from nephritis prion infectivity is shed with the urine.²⁵ Furthermore, sheep having a mastitis can transmit infectious prions with milk.³²In Chronic Wasting disease (CWD) of deer several careful studies have been performed that, together with our present finding, depose in favor of airborne transmission in this naturally occurring disease. Indeed, CWD prions can be transmitted experimentally via aerosol and the nasal route to transgenic cervidized mice.³³ Although no anecdotal or epidemiological evidence has come forward that airborne transmission may be important for the spread of CWD, several lines of thought suggest that this possibility is not implausible. In deer, prions have been detected in urine, saliva, feces and blood of diseased animals. Moreover, it was claimed that pathological prion protein could be recovered from the environmental water in an endemic area.³⁴ Since all fluids can act as sources for the generation of aerosols, any of the body fluids mentioned above may represent the point of origin for airborne transmission of CWD prions.In this context, also the presence of infectious prions in blood of patients should be mentioned which was demonstrated by the transmission of vCJD by blood transfusions.^35,36 The growing body of evidence that prion transmission can be airborne—at least under certain conditions—dictates that the release of potentially contaminated aerosols should be avoided under all circumstances. In this context it is mandatory that reliable precautions be defined and followed in scientific and diagnostic laboratories. In particular, it is self-evident that safety cabinets should be used while processing brain and nerve tissue (or any other potentially contaminated tissue) of man and animals suspected with prion disease. Our experience shows that this necessity is generally very well-understood by prion scientists.A further stone of contention relates to the biosafety level of the laboratory environment. Because prions were hitherto considered not be airborne, so far no specific regulations have been implemented. As a consequence, prion laboratories have been mostly required to adhere to the category “BSL3**.” While it is understood that the airborne transmission of prions has thus far only been observed under extreme conditions, we feel that it is in order to critically reassess biosafety regulations in the light of the recent discoveries. In particular, one might consider implementing more stringent measures towards protecting workers within diagnostic and scientific laboratories from aerosols.The situation in slaughterhouses and plants handling potentially contaminated offal may be even more problematic. Although regulations in slaughterhouses dictate the use of protecting glasses and masks or, alternatively, visors the use of personal protecting equipment should be rigorously controlled. In addition, high-pressure cleaning devices produce massive aerosols and should be strictly avoided in areas of slaughterhouses where prion-containing material may be processed. Regulations concerning cleaning of heads from slaughtered animals do pay attention to aerosol avoidance, e.g., by allowing only water hoses without pressure.A case in point is the severe neurological syndrome arising in swine abattoir workers.³⁷ Here, an immune-mediated polyradiculoneuropathy was reported to be related to a process using high-pressure fluids to remove the brains of swine.³⁷ During this process, high amounts of swine brain tissue became aerosolized and were inhaled and/or gained access to the respiratory tract mucosa of abattoir workers, resulting in immunization with myelin constituents akin to experimental autoimmune encephalitis (EAE). Although significant physiological differences exist concerning breathing, where humans are regarded as mouth breathers and mice as nose breathers, many people indeed show nose breathing under no or only moderate body burden. Therefore, results obtained in mouse experiments might also be extrapolated to a considerable extent to the situation in man.In this context it is of importance to stress again that aerosols might be generated under various conditions and represent a normal entity of the environment in a variety of daily life situations.In our studies of airborne transmission of prion protein in mice³⁰ we took advantage of the fact that mice breathe exclusively through their nostrils^38,39 and therefore could be exposed in groups to aerosolized brain suspensions. Using this system, it was possible to vary both time of exposure as well as concentration of the prion load in the aerosol. We were surprised to discover that exposure times as short as 1 min were sufficient to achieve high attack rates. By extending the time of exposure it became obvious that incubation times were shortened. A possible alternative route of infection via the cornea or the conjunctiva was extremely unlikely, since newborn mice, whose eyelids were still closed, could also be infected. These findings show that the aerogenic transmission of prions is very efficient.But how do prions spread from the airways to the brain? Peripheral replication of prions in the lymphoid system—a characteristic of most other peripheral routes of transmission—appeared to be dispensable. Instead, the results argue for a direct pathway of brain invasion. One anatomical peculiarity of the nasal cavity is the “area cribriformis” of the olfactory epithelium. Here the olfactory bulb sprouts axons of olfactory receptor neurons passing through the cribriform plate of the ethmoidal bone to reach the olfactory mucosa where olfactory cilia extend representing non-myelinated nerve endings. Thus, open nerve endings are located in the nasal cavity through which aerosolized infectious prions might get access to the brain. In this context it is noteworthy that pathological prion protein was found in the olfactory cilia and basal cells of the olfactory mucosa of sCJD patients, as well as in the olfactory bulb and olfactory tract.^40,41 However, it was hitherto never clearly documented that olfactory receptor neurons represent an entry site for infectious prions; this might also be due to the sensitivity threshold of detection assays.In conclusion, aerosols can infect mice with a surprisingly high efficiency. Just how important a role is played by this newly recognized pathway of spread in natural transmission is, as of now, unclear and in need of further studies. Although it was not identified as a route of infection in epidemiological studies thus far, the worryingly high attack rate suggests that we would be well-advised to carefully avoid the inhalation of aerosols from prion-containing materials.     

The prion organotypic slice culture assay--POSCA

Methods enabling prion replication ex vivo are important for advancing prion science. However, few such technologies exist and many prion strains are intractable with them. Here, we describe a prion organotypic slice culture assay (POSCA), which allows for prion amplification and titration ex vivo under conditions that closely resemble intracerebral infection. Organotypic slices are incubated with infectious inoculum as free-floating sections, washed and cultured for up to 8 weeks. Slice cultures are a rich source of protein or RNA and allow for stringent comparisons between uninfected and prion-infected samples generated from the same mouse. Thirty-five days after contact with prions, cerebellar slices have amplified PrP(Sc) quantitatively similar to that seen in vivo, but accelerated fivefold. The POSCA detects replication of specific prion strains from disparate sources, including bovines and ovines, with variable efficiency. The culture procedure and prion infection can be performed in 8 h.     

Protocol for aerosol-free recombinant production and NMR analysis of prion proteins

The central hallmark of prion diseases is the misfolding of cellular prion protein (PrP^C) into a disease-associated aggregated isoform known as scrapie prion protein (PrP^Sc). NMR spectroscopy has made many essential contributions to the characterization of recombinant PrP in its folded, unfolded and aggregated states. Recent studies reporting on de novo generation of prions from recombinant PrP and infection of animals using prion aerosols suggest that adjustment of current biosafety measures may be necessary, particularly given the relatively high protein concentrations required for NMR applications that favor aggregation. We here present a protocol for the production of recombinant PrP under biosafety level 2 conditions that avoids entirely exposure of the experimenter to aerosols that might contain harmful PrP aggregates. In addition, we introduce an NMR sample tube setup that allows for safe handling of PrP samples at the spectrometer that usually is not part of a dedicated biosafety level 2 laboratory.     

Cultured cell sublines highly susceptible to prion infection

Cultured cell lines infected with prions produce an abnormal isoform of the prion protein (PrP(Sc)). In order to derive cell lines producing sufficient quantities of PrP(Sc) for most studies, it has been necessary to subclone infected cultures and select the subclones producing the largest amounts of PrP(Sc). Since postinfection cloning can introduce differences between infected and uninfected cell lines, we sought an approach to generate prion-infected cell lines that would avoid clonal artifacts. Using an improved cell blot technique, which permits sensitive and rapid comparison of PrP(Sc) levels in multiple independent cell cultures, we discovered marked heterogeneity with regard to prion susceptibility in tumor cell sublines. We exploited this heterogeneity to derive sublines which are highly susceptible to prion infection and used these cells to generate prion-infected lines without further subcloning. These infected sublines can be compared to the cognate uninfected cultures without interference from cloning artifacts. We also used susceptible cell lines and our modified cell blot procedure to develop a sensitive and reproducible quantitative cell culture bioassay for prions. We found that the sublines were at least 100-fold more susceptible to strain RML prions than to strain ME7 prions. Comparisons between scrapie-susceptible and -resistant cell lines may reveal factors that modulate prion propagation.     

Saccharomyces cerevisiae

Yeast prions are infectious proteins that spread exclusively by mating. The frequency of prions in the wild therefore largely reflects the rate of spread by mating counterbalanced by prion growth slowing effects in the host. We recently showed that the frequency of outcross mating is about 1% of mitotic doublings with 23–46% of total matings being outcrosses. These findings imply that even the mildest forms of the [PSI+], [URE3] and [PIN+] prions impart > 1% growth/survival detriment on their hosts. Our estimate of outcrossing suggests that Saccharomyces cerevisiae is far more sexual than previously thought and would therefore be more responsive to the adaptive effects of natural selection compared with a strictly asexual yeast. Further, given its large effective population size, a growth/survival detriment of > 1% for yeast prions should strongly select against prion-infected strains in wild populations of Saccharomyces cerevisiae.     

Lichens

The prion diseases sheep scrapie and cervid chronic wasting disease are transmitted, in part, via an environmental reservoir of infectivity; prions released from infected animals persist in the environment and can cause disease years later. Central to controlling disease transmission is the identification of methods capable of inactivating these agents on the landscape. We have found that certain lichens, common, ubiquitous, symbiotic organisms, possess a serine protease capable of degrading prion protein (PrP) from prion-infected animals. The protease functions against a range of prion strains from various hosts and reduces levels of abnormal PrP by at least two logs. We have now tested more than 20 lichen species from several geographical locations and from various taxa and found that approximately half of these species degrade PrP. Critical next steps include examining the effect of lichens on prion infectivity and cloning the protease responsible for PrP degradation. The impact of lichens on prions in the environment remains unknown. We speculate that lichens could have the potential to degrade prions when they are shed from infected animals onto lichens or into environments where lichens are abundant. In addition, lichens are frequently consumed by cervids and many other animals and the effect of dietary lichens on prion disease transmission should also be considered.     

Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes

The pathological prion protein PrP(Sc) is the only known component of the infectious prion. In cells infected with prions, PrP(Sc) is formed posttranslationally by the refolding of the benign cell surface glycoprotein PrP(C) into an aberrant conformation. The two PrP isoforms possess very different properties, as PrP(Sc) has a protease-resistant core, forms very large amyloidic aggregates in detergents, and is only weakly immunoreactive in its native form. We now show that prion-infected rodent brains and cultured cells contain previously unrecognized protease-sensitive PrP(Sc) varieties. In both ionic (Sarkosyl) and nonionic (n-octyl beta-D-glucopyranoside) detergents, the novel protease-sensitive PrP(Sc) species formed aggregates as small as 600 kDa, as measured by gel filtration. The denaturation dependence of PrP(Sc) immunoreactivity correlated with the size of the aggregate. The small PrP(Sc) aggregates described here are consistent with the previous demonstration of scrapie infectivity in brain fractions with a sedimentation coefficient as small as 40 S [Prusiner et al. (1980) J. Neurochem. 35, 574-582]. Our results demonstrate for the first time that prion-infected tissues contain protease-sensitive PrP(Sc) molecules that form low MW aggregates. Whether these new PrP(Sc) species play a role in the biogenesis or the pathogenesis of prions remains to be established.     

In Vitro Detection of prionemia in TSE-Infected Cervids and Hamsters

Blood-borne transmission of infectious prions during the symptomatic and asymptomatic stages of disease occurs for both human and animal transmissible spongiform encephalopathies (TSEs). The geographical distribution of the cervid TSE, chronic wasting disease (CWD), continues to spread across North America and the prospective number of individuals harboring an asymptomatic infection of human variant Creutzfeldt-Jakob Disease (vCJD) in the United Kingdom has been projected to be ~1 in 3000 residents. Thus, it is important to monitor cervid and human blood products to ensure herd health and human safety. Current methods for detecting blood-associated prions rely primarily upon bioassay in laboratory animals. While bioassay provides high sensitivity and specificity, it requires many months, animals, and it is costly. Here we report modification of the real time quaking-induced conversion (RT-QuIC) assay to detect blood-borne prions in whole blood from prion-infected preclinical white-tailed deer, muntjac deer, and Syrian hamsters, attaining sensitivity of >90% while maintaining 100% specificity. Our results indicate that RT-QuIC methodology as modified can provide consistent and reliable detection of blood-borne prions in preclinical and symptomatic stages of two animal TSEs, offering promise for prionemia detection in other species, including humans.     

Purity of spiking agent affects partitioning of prions in plasma protein purification.

Prions are not detectable in the blood or plasma of persons afflicted with classical or variant Creutzfeldt-Jakob disease, and they have never been shown to be transmitted by blood or plasma products. Despite the uncertainty as to the presence and biophysical properties of prions in plasma, prion removal studies have been conducted using brain homogenate or microsomes prepared from prion-infected rodent brains as model prions. In this study, we compare the partitioning of different prion spiking agents, having different biophysical properties, in the processes used for plasma protein purification. We have found that membrane-bound prion spiking agents partition similarly, whereas purified, unbound pathogenic prion proteins can have significantly different partitioning properties depending on the conditions in the production process. We conclude that prion spiking studies for the evaluation of prion reduction in plasma protein purification should employ spiking agents with different biophysical properties to mimic partitioning of the theoretical prion contaminant. This will give greater assurance as to the prion safety margins of the life-saving plasma protein therapeutics and excipients.     

Novel rapid method for visualization of extent and location of aerosol contamination during high-speed sorting of potentially biohazardous samples

BACKGROUND: Containment of potentially biohazardous aerosols that result from high-speed sorting of human cells has been an increasingly important problem in analytical cytometry. The current method for assessing the efficiency of aerosol containment involves detection of aerosols containing sorted T4 bacteriophage on lawns of T4-susceptible Escherichia coli on plates that are placed in and around the sort area. Although this method is sensitive, it is time consuming and involves maintenance and handling of bacteria and sorting of bacteriophage that may themselves serve as sources of contamination for sorted viable human cells. METHODS: Glo Germ (5-microm melamine copolymer resin beads), which are fluorescent under black light illumination, were sorted on a Beckman-Coulter Elite ESP sorter in order to visualize deposition of aerosols under normal and mock failure modes. RESULTS: Glo Germ was successfully used under both normal sorting conditions, as well as mock failure mode, to visualize aerosol formation. CONCLUSIONS: We have developed a method to examine aerosol containment using modified Glo Germ, a product used for teaching aseptic technique in hospitals, industry, restaurants, and schools. Use of this technique represents a rapid, inexpensive, qualitative analysis of the extent and location of aerosol contamination from cell sorters.     

Rapid Transepithelial Transport of Prions following Inhalation

Prion infection and pathogenesis are dependent on the agent crossing an epithelial barrier to gain access to the recipient nervous system. Several routes of infection have been identified, but the mechanism(s) and timing of in vivo prion transport across an epithelium have not been determined. The hamster model of nasal cavity infection was used to determine the temporal and spatial parameters of prion-infected brain homogenate uptake following inhalation and to test the hypothesis that prions cross the nasal mucosa via M cells. A small drop of infected or uninfected brain homogenate was placed below each nostril, where it was immediately inhaled into the nasal cavity. Regularly spaced tissue sections through the entire extent of the nasal cavity were processed immunohistochemically to identify brain homogenate and the disease-associated isoform of the prion protein (PrP^d). Infected or uninfected brain homogenate was identified adhering to M cells, passing between cells of the nasal mucosa, and within lymphatic vessels of the nasal cavity at all time points examined. PrP^d was identified within a limited number of M cells 15 to 180 min following inoculation, but not in the adjacent nasal mucosa-associated lymphoid tissue (NALT). While these results support M cell transport of prions, larger amounts of infected brain homogenate were transported paracellularly across the respiratory, olfactory, and follicle-associated epithelia of the nasal cavity. These results indicate that prions can immediately cross the nasal mucosa via multiple routes and quickly enter lymphatics, where they can spread systemically via lymph draining the nasal cavity.     

Analytical Background and Discussion of the Chaperone Model of Prion Diseases

It is generally accepted that prion infection is due solely to a protein i.e. the protein-only hypothesis. The essential constituent of infectious prions is the scrapie prion protein (PrP^Sc) which is chemically indistinguishable from the normal, cellular protein (PrP^C) but exhibits distinct secondary and tertiary structure. This very unusual feature seems to be in contradiction with a major paradigm of present structural biology stated by Anfinsen: a protein folds to the most stable conformation, this means only one structure.In order to reconcile the results obtained on prions with the biophysics of protein folding, a model is proposed. It is based on the hypothesis that a thermodynamically irreversible step is involved in protein folding. The model is then extended to chaperone-assisted protein folding. It is shown that, under certain conditions, the transitory secondary structure formed during the earlier step of folding could interact with chaperone. Analysis shows that chaperone may help the protein to find correct conformation. On the other hand, analysis reveals the possibility that more than one structure may form from a single polypeptide chain. Under these conditions, the behaviour of chaperones resembles the characteristics of prion diseases.     

Lichens: Unexpected anti-prion agents?

The prion diseases sheep scrapie and cervid chronic wasting disease are transmitted, in part, via an environmental reservoir of infectivity; prions released from infected animals persist in the environment and can cause disease years later. Central to controlling disease transmission is the identification of methods capable of inactivating these agents on the landscape. We have found that certain lichens, common, ubiquitous, symbiotic organisms, possess a serine protease capable of degrading prion protein (PrP) from prion-infected animals. The protease functions against a range of prion strains from various hosts and reduces levels of abnormal PrP by at least two logs. We have now tested more than twenty lichen species from several geographical locations and from various taxa and found that approximately half of these species degrade PrP. Critical next steps include examining the effect of lichens on prion infectivity and cloning the protease responsible for PrP degradation. The impact of lichens on prions in the environment remains unknown. We speculate that lichens could have the potential to degrade prions when they are shed from infected animals onto lichens or into environments where lichens are abundant. In addition, lichens are frequently consumed by cervids and many other animals and the effect of dietary lichens on prion disease transmission should also be considered.Key words: prion, lichen, degradation, environment, Chronic Wasting disease, protease, transmissible spongiform encephalopathy     

Direct detection of soil-bound prions

Scrapie and chronic wasting disease are contagious prion diseases affecting sheep and cervids, respectively. Studies have indicated that horizontal transmission is important in sustaining these epidemics, and that environmental contamination plays an important role in this. In the perspective of detecting prions in soil samples from the field by more direct methods than animal-based bioassays, we have developed a novel immuno-based approach that visualises in situ the major component (PrP(Sc)) of prions sorbed onto agricultural soil particles. Importantly, the protocol needs no extraction of the protein from soil. Using a cell-based assay of infectivity, we also report that samples of agricultural soil, or quartz sand, acquire prion infectivity after exposure to whole brain homogenates from prion-infected mice. Our data provide further support to the notion that prion-exposed soils retain infectivity, as recently determined in Syrian hamsters intracerebrally or orally challenged with contaminated soils. The cell approach of the potential infectivity of contaminated soil is faster and cheaper than classical animal-based bioassays. Although it suffers from limitations, e.g. it can currently test only a few mouse prion strains, the cell model can nevertheless be applied in its present form to understand how soil composition influences infectivity, and to test prion-inactivating procedures.     

Antioxidant functions of carotenoids

Carotenoid pigments, including hydrocarbons such as β-carotene or xanthophylls such as lutein and zeaxanthin, are very widely distributed in nature, where they play an important role in protecting cells and organisms against the harmful effects of light, air, and sensitizer pigments. This process has been demonstrated in bacteria, algae, plants, animals, and even in humans in the light-sensitive disease, erythropoietic protoporphyria. The primary mechanism of action of this phenomenon appears to be the ability of carotenoids to quench excited sensitizer molecules as well as quench ¹O₂.

In addition to this protection, and potentially of even greater biological importance, is the fact that carotenoids can also serve as antioxidants under conditions other than photosensitization. This review presents the data available indicating the extent of this important function. Antioxidant action can be documented in both enzymic and nonenzymic systems, and has been reported in subcellular, cellular, and animal studies. In fact, the many reports indicating that carotenoids may possess some anticarcinogenic properties may well be related to their ability to interact with and quench various radical species that can be generated within cells.     

A Bovine Cell Line That Can Be Infected by Natural Sheep Scrapie Prions

Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrP^res, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice). We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases.     

The battle of the fold: chaperones take on prions

Protein conformational diseases, such as Alzheimer's, Parkinson's and Huntington's, affect a large portion of our aging population. Cells have evolved mechanisms for rescuing and recycling misfolded proteins, but these systems are not perfect. Chaperones can rescue misfolded proteins by breaking up aggregates and assisting in the refolding process. Proteins that cannot be rescued by refolding can be delivered to the proteasome by chaperones to be recycled. One class of 'misfolded' proteins, prions, appears to evade detection by this machinery and persist in a misfolded state. In fact, it seems that the prions usurp the refolding machinery and actually employ chaperones to propagate the prion state. Recent data has begun to uncover the mechanism behind this unique relationship.     

Life cycle of cytosolic prions

Prions are self-templating protein aggregates that were originally identified as the causative agent of prion diseases in mammals, but have since been discovered in other kingdoms. Mammalian prions represent a unique class of infectious agents that are composed of misfolded prion protein. Prion proteins usually exist as soluble proteins but can refold and assemble into highly ordered, self-propagating prion polymers. The prion concept is also applicable to a growing number of non-Mendelian elements of inheritance in lower eukaryotes. While prions identified in mammals are clearly pathogens, prions in lower eukaryotes can be either detrimental or beneficial to the host. Prion phenotypes in fungi are transmitted vertically from mother to daughter cells during cell division and horizontally during mating or abortive mating, but extracellular phases have not been reported. Recent findings now demonstrate that in a mammalian cell environment, protein aggregates derived from yeast prion domains exhibit a prion life cycle similar to mammalian prions propagated ex vivo. This life cycle includes a soluble state of the protein, an induction phase by exogenous prion fibrils, stable replication of prion entities, vertical transmission to progeny and natural horizontal transmission to neighboring cells. Our data reveal that mammalian cells contain all co-factors required for cytosolic prion propagation and dissemination. This has important implications for understanding prion-like properties of disease-related protein aggregates. In light of the growing number of identified functional amyloids, cell-to-cell propagation of cytosolic protein conformers might not only be relevant for the spreading of disease-associated proteins, but might also be of more general relevance under non-disease conditions.     

Pollen limitation and fruiting failure related to canopy closure in Calypso bulbosa (Orchidaceae), a northern food‐deceptive orchid with a single flower

Natural fruit set is constrained by pollen limitation and fruiting failure, and pollen limitation is expected to be especially severe in deceptive orchids. We performed hand cross‐pollinations in ten populations of a food‐deceptive orchid, Calypso bulbosa, under sparse and dense canopies in three non‐consecutive years. We explored the relationships between natural fruit set, pollen limitation and fruiting failure. Mean natural fruit set over the years was 60%, which is exceptionally high for a deceptive orchid. On average, hand cross‐pollination increased fruit set by 23%. Among open‐pollinated plants that did not set a fruit, 55.5% were estimated to be pollen limited and 44.5% to be limited by fruiting failure, i.e. inability to set a fruit after pollination. In species with high natural fruit set, hand cross‐pollination experiments may not always detect statistically significant pollen limitation. In our case, pollen limitation tended to become significant when the natural fruit set dropped below 60%. Canopy cover had a significant effect on fruiting failure, which was more severe under a dense canopy. Although our results demonstrate pollen limitation in many cases, they also highlight the fact that food deception can be a very effective pollination strategy. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013 , 171 , 744–750.