The search for novel antitumour drugs has reached a plateau phase. The carcinomas remain almost as intractable as they did 40 years ago and the need for effective therapy is pressing. There is an argument that the current pharmacopoeia is sufficient but, to be effective, the biochemical mechanisms of drug resistance must be circumvented. In tackling the question of why certain cancer cells are resistant, the converse question of why others are sensitive still remains to be answered fully. Asking the fundamental question of why and how a cell dies may provide clues as to what avenues lie open for improved chemotherapy. In this review we survey the recent literature on cell death and we argue that it is possible that the outcome of chemotherapy may be determined by the response of the cell to the formation of the drug-target complex, and/or its sequellae, rather than to the biochemical changes brought about by the drug alone. One of these responses, determined by the phenotype of the cell, may be activation of a genetic programme for cell death.


Presently, health costs associated with nitrate in drinking water are uncertain and not quantified. This limits proper evaluation of current policies and measures for solving or preventing nitrate pollution of drinking water resources. The cost for society associated with nitrate is also relevant for integrated assessment of EU nitrogen policies taking a perspective of welfare optimization. The overarching question is at which nitrogen mitigation level the social cost of measures, including their consequence for availability of food and energy, matches the social benefit of these measures for human health and biodiversity.Epidemiological studies suggest colon cancer to be possibly associated with nitrate in drinking water. In this study risk increase for colon cancer is based on a case-control study for Iowa, which is extrapolated to assess the social cost for 11 EU member states by using data on cancer incidence, nitrogen leaching and drinking water supply in the EU. Health costs are provisionally compared with nitrate mitigation costs and social benefits of fertilizer use.3) for more than ten years. We estimate the associated increase of incidence of colon cancer from nitrate contamination of groundwater based drinking water in EU11 at 3%. This corresponds to a population-averaged health loss of 2.9 euro per capita or 0.7 euro per kg of nitrate-N leaching from fertilizer.For above median meat consumption the risk of colon cancer doubles when exposed to drinking water exceeding 25 mg/L of nitrate (NO3 are probably beneficial for society and that a stricter nitrate limit and additional measures may be justified. The present assessment of social cost is uncertain because it considers only one type of cancer, it is based on one epidemiological study in Iowa, and involves various assumptions regarding exposure. Our results highlight the need for improved epidemiological studies.Our cost estimates indicate that current measures to prevent exceedance of 50 mg/L NO Nitrogen is emitted to the environment by various sources in various forms that lead to a multitude of effects on human health, ecosystems and climate. On the other hand nitrogen is a key input for food production and deficient in many parts of the developing world. Therefore, Galloway et al.  concludeThe existence of adverse health impacts of nitrate via drinking water has been debated ,3. ThereThere is consensus that the role of nitrate exposure in causing methaemoglobinaemia is minor  and not Although there is evidence for both beneficial and adverse health effects of increased nitrate intake in drinking water, these effects are likely to be small and uncertain compared to other factors like life style and diet. Moreover, health effects may also results from other drinking water pollutants . Therefore, it is not surprising that epidemiological studies into the relation between nitrate in drinking water and cancers  often provide weak associations, both positive and negative . Ward et2O, NO2 and NH3 to the atmosphere [The present paper provides a method to assess the health costs by nitrates in drinking water. We compare the results with very rough estimates of the costs of improved water treatment and of reduced fertilizer use. However, the problem is more complex because of the multiple impacts and pathways of nitrogen in the environment. Nitrogen is a major factor for eutrophication and biodiversity loss and contributes to global warming. Even though most nitrates in drinking water come from fertilizer, a full analysis would have to consider more than just the passage from fertilizer to drinking water. Since part of the fertilizer nitrogen also ends up as emission of Nmosphere , the damFor our assessment we assume a link with colon cancer as working hypothesis to provide a tentative assessment of health loss and social cost of nitrate in drinking water in the European Union. Ward et al.  indicateData were taken from IARC , Michelihttp://www.ikcnet.nl/). Total prevalence of colon cancer in the Netherlands, 169 cases per 100,000, compares well to the average situation in the Europe, 176 cases per 100,000 [We inferred loss of healthy life years and life years due to premature death using data on colon cancer incidence and mortality per five year age class between 1989 and 2006 provided by the Dutch Cancer Registry , the odds ratio almost doubled as compared to the reference group that was not exposed to NO3-N levels exceeding 5 mg/L. This association was not found for rectum cancer.We derive the increased colon cancer risk from DeRoos et al. , a case-3-N exceeding 5 mg/L for more than ten years, but only the increased risk of approximately a factor 2 for the subpopulation with above median meat intake is statistically significant (95% CI). For the purpose of this assessment we assumed a doubling of colon cancer incidence for above median meat consumers exposed to a NO3-N concentration exceeding 5 mg/L (22.5 mg/L NO3). In an ecologic study, Gulis et al. [3-N concentrations of 4.5 mg/L in drinking water as compared to a reference group below 2.2 mg/L. These results are similar to those of DeRoos [3-N concentrations exceeding 2.2 mg/L, but did find nearly a doubling of the relative risk of mortality for stomach cancer. Gulis et al. [Figure s et al.  found anf DeRoos , for whif DeRoos  combinedf DeRoos  did not s et al.  state th2, with model estimates of the agricultural nitrogen leaching from the rooting zone by Velthof et al. [First, we investigated potential associations between observed nitrate concentrations in shallow aquifers and estimates of the nitrogen leaching from agricultural land. For this purpose we combined monitoring data reported by Zwart et al.  to the Ef et al. . Velthoff et al. . The med2 = 0.67) was found between the % of samples with exceedance and the mean nitrogen leaching intensity (kg/ha/yr)  Figure ; eq. 1.3 was exceeded and therefore also a proxy for the percentage of the population, using private wells or small communal supplies, that is exposed to drinking water exceeding 25 mg/L NO3. We did not consider temporal trends of nitrate in groundwater.where LAE = fraction of land area in country with exceedance and N-leach = mean nitrogen leaching intensity (kg/ha/yr). We assume the percentage of exceedance in monitoring to be proportional to the land area where 25 mg/L NO3 may result from incidental exceedance in public supplies or structural exceedance in small local facilities or private wells.The relationship between nitrate in groundwater and in drinking water depends on the drinking water infrastructure and water treatment in the different EU member states. Important variables are the percentage of the population connected to public supply, the presence of drinking water treatment in public supplies and the relative use of surface water and groundwater. These parameters vary considerably across the EU; for 12 member states, selected for data availability, connection to large public supplies (serving more than 5000 customers or delivering more than on million liters per day) ranged from 36 to 100% and use of groundwater for public supply from 25 to 99% (Table 3) or nitrite (0.5 mg/L NO2) between 1995 and 2000 occurred in 0 and 4.5% of the water samples for large supplies in 12 reporting EU member states [Non-compliance with EU legal limits in the EU drinking water directive  for eithr states . Non-comr states .It may be expected that exposure in eastern European countries is higher than in northern and western Europe in view of a lower access of the rural population to improved drinking water supply (10-70% ) and low3 in 2001 were reported [3. Data on exceedance of 25 mg/L NO3 for the other 11 member states were not available and estimated using the Dutch value and the ratio of exceedance of 25 mg/L NO3 in untreated groundwater. Next exposure to drinking water from groundwater in public and private supplies exceeding 25 mg/L NO3 and using groundwater can be calculated (eqs. 2 and 3).In the Netherlands, nitrate concentrations in drinking water exceeding 25 mg/L NOL = fraction (%) of population in country exposed through large public supply systems; Exc EU = sum of levels (%) of non compliance to 50 mg NO3 and 0.1 mg/L NO2 for drinking water samples between 1995 and 2000 as officially reported to EU; Exc 25 mg/L = estimated fraction (%) of drinking water with NO3 between 25 and 50 mg/L taking the exceedance value of 5.3% for the Netherlands (NL) and assuming that Exc 25 mg/L is proportional to LAEcountry/LAENL; Connect = fraction (%) of population connected to large public supply; and Grw = fraction (%) of drinking water production from groundwater.where PopS = fraction (%) of population in country exposed through private wells and small public supply systems.where Pop3 concentrations above 25 mg/L the half that consumes more than the median amount of meat, has a risk of colon that is twice as high as for the total population (eq. 4).First the total colon cancer incidence was taken from section 'Present incidence and prevalence of colon cancer in Europe'. Next the proportion of cases associated with nitrate was inferred from section 'Increased risk of colon cancer due to exceedance of the nitrate standard in drinking water', where we assume that of the population exposed to NO3 in groundwater based drinking water; Incidence = crude total incidence of colon cancer ; ΔR = increased risk of colon cancer for individuals that have consumed drinking water with NO3 in excess of 25 mg/L longer than 10 years and with above median meat consumption  and the number of years lived with disability (YLD), i.e. the loss of healthy life years. Thus we combine the nitrate related additional colon cases with the result of the section 'Loss of healthy life years and life years from premature death for colon cancer' to calculate the YLD and YLL both for individual member states and the EU (eq. 5).where Soc-Cost = social cost of loss of healthy life years and premature death from additional colon cancer; YLD = Years of life lived with disease (per colon cancer case); VYLD = value of a YLD = QALY score - VOLY; YLL = years of life lost (per colon cancer case); and VOLY = economic value of a life year.For the value of a life year (VOLY) we take 40,000 euro/YLL, as determined by a large contingent valuation study in nine EU countries by Desaigues et al. ; that vaFor the valuation of years lived with disability (YLD) we invoke the DALY and QALY scores that have been published for colon cancer. The DALY (Disability Adjusted Life Year) is an indicator for the severity of a health condition. Developed by the World Health Organization, the DALY is a number between 0  and 1 (death). The QALY  is a similar indicator, but its range is opposite, from 0 (death) to 1 . A DALY is roughly equivalent to 1 - QALY, although their precise definitions involve differences such as discounting and age-weighting (for DALY but not QALY). Such differences do not matter in view of the uncertainties, and we set the monetary value of a DALY or QALY equal to 1 VOLY = 40,000 euro/YLL. Mathers et al.  indicateNeedless to say, the uncertainties of the monetary valuation are large. In particular we note that often an alternative approach is used for the monetary valuation of fatal cancers, based on the value of a prevented fatality  for which the DG Environment of the European Commission assumes approximately one million euro ; some ecA unit N-cost or -benefit is defined as the monetary value of an effect  expressed per kg of pollutant or kg N in pollutant or per kg N in applied fertilizer. The unit N-cost approach allows a first comparison of the social benefit of less nitrate in drinking water to the social cost of measures to mitigate nitrate e.g. by water treatment or reduction of fertilizer use (eq. 6).where UCN = Unit damage cost (euro per kg of N leaching); and N-loss = nitrogen leaching loss from agricultural land (kg).3 ranged between 20% and 60% , to nearly 13% in Denmark. For the 12 EU member states, the total exposed population amounts to 23 million persons  of which 8 million persons (2.3%) were exposed through public supply.Connection to large public water supply varies considerably and is, among other factors, related to population density, cost for installing drinking water infrastructure and national policies. Also the use of groundwater for drinking water varies and is typically related to the presence of aquifers. Using Equation 1 Figure , the are3 for more than 10 years doubles the risk. The total loss for these 11 countries is 23,000 YLD and 18,000 YLL. Although this loss is modest, it represents a total social cost of 1.0 billion euro per year or 2.9 euro year per person averaged over the entire population , and to 150 euro per year for a person exposed to drinking water exceeding 25 mg/L NO3. Low values (less than one euro/capita) are found for the UK, Finland and Ireland and in part could be viewed as benefits of investments in a good drinking water infrastructure. The highest values (3-7 euro/capita) are found for Denmark, Italy, France and Germany, in part due to lower levels of connection to or availability of large high quality drinking water infrastructure, in combination with high nitrate leaching. Finally the unit cost is obtained by dividing the health cost by the total quantity of NO3-N leaching in each country. Unit damage cost for the 11 countries ranges between 0.1 and 2.4 euro per kg of N leaching, with an average of 0.7 euro/kg.In Table The results for social cost and unit cost of health loss due to nitrate in drinking water should be viewed as tentative values for comparative use against social costs or benefits of impacts for other nitrogen pollutants or against cost of measures. In fact, our assessment is based on just one epidemiological study in Iowa using a number of educated assumptions and guesses about exposure in the EU. The main sources of data uncertainty are discussed in Table In view of the uncertainty about the health impact itself, the lower limit of the health cost is zero, and in line with the lower limit of the 95% confidence interval of the risk increase inferred from DeRoos et al. . Using tAnother source of bias is that our analysis is based on data for 11 "old" EU member states with relatively high GDP and levels of implementation of environmental and drinking water policies. For the new central and east EU member states the social cost per capita is expected to be higher.Typical measures to prevent nitrate exceedance in drinking water are blending polluted water with clean water, biochemical water treatment and installing deeper extraction wells. Data on costs of these measures are scarce but the costs are expected to decrease with increasing scale of the drinking water production. Illustrative annual cost values are 0.5 euro/capita/yr for water treatment and mixing for the UK and the Netherlands where large aquifers are available ,33, 3 euAreas with aquifers suitable for groundwater extraction for drinking water production often are also areas suitable for agriculture. For this reason use of fertiliser or manures is a major source of nitrate pollution, and reduction of this use is a typical measure to prevent nitrate pollution of aquifers. Agricultural production clearly benefits from additional nitrogen input, but there is an optimum and for some crops  the yield diminishes when the nitrogen input is further increased (see for example Lord and Mitchell ). Althou2O, NO2 and NH3 to the atmosphere [x  and 9.5 euro/kg of ammonia . In ExternE the exposure-response functions for health are assumed to be linear without threshold. Health impacts of these compounds are mainly indirect and mediated through several steps, where NOx, and NH3 act as precursors for ozone and/or airborne particulate matter. The health impacts for airborne NH3-compounds are very uncertain and unit costs could be much smaller. Using typical emission factors [x-N from fertilizer (0.2-0.6%) and NH3-N  mean damage costs were derived for the EU per kg applied fertilizer N (Table 3 (0.7 euro/kg of N) is much lower than for NOx and NH3, health damage values expressed per kg of added fertilizer-N are comparable because of the relatively high emission factor for nitrate. Consideration of the health cost of nitrate leaching from fertilizer and manure in agriculture, is therefore a relevant N-related externality that needs to be considered when defining the socially optimal input level of nitrogen in agriculture [Since part of the fertilizer or manure nitrogen ends up as emission of Nmosphere , the dammosphere  and CAFEmosphere , in the mosphere , and by mosphere . These a factors  for NOx- N Table . This reiculture , in addi3-N leaching. The cost of water treatment to abate exceedance of 25 mg/L ranges between 0.5 and 3 euro/capita/yr, indicating that these measures are beneficial for society. Average costs to prevent nitrate exceedance by reduced fertilizer use range between 0.6 and 2.7 euro/kg of N-fertilizer, and tend to be lower in regions with intensive fertilizer use. These values indicate that in these regions, reduction of fertilizer use will also likely create net benefits for society, particularly when drinking water production is vulnerable to nitrate leaching.Health loss due to nitrate in drinking water is an issue under debate both in the scientific and policy arena. Estimates of associated health loss and potential welfare effects can help to evaluate current nitrate policies and measures. We derived a first and tentative estimate of a 3% increase of incidence of colon cancer for 11 EU member states due to nitrate in drinking water exceeding 25 mg/L, being half the legal US and EU limit of 50 mg/L. This health impact corresponds to an economic loss of 2.9 euro/capita/yr and of 0.7 euro per kg of NOHowever, the epidemiological evidence for increased risk of colon cancer is weak or absent. Clearer negative or positive answers about associations between nitrate in drinking water and disease are reasonably to be expected from prospective case-control studies. In view of the magnitude of the potential health gain (2.9 euro/capita/year) improved epidemiological studies would certainly be worthwhile. Then, integrated cost-benefit assessment of nitrogen management, including all relevant impacts and measures, including those debated for nitrate in drinking water, may help to further improve current EU nitrogen policies from a precautionary approach.CAFE: EU programme Clean Air for Europe; CBA: Cost Benefit Assessment; DALY: Disability Adjusted Life Year); ExternE: EU programme Externalities of Energy generation; EU: European Union ; GDP: Gross Domestic Product; NDMA: nitrosodimethylamine; QALY: Quality Adjusted Life Year; VYLD: economic Value of Life Year with Disability ; US EPA: USA Environmental Protection Agency; VOLY: economic Value Of a Life Year; YLD: number of Years Lived with Disability per cancer case; YLL: number of Years of Life Lost.The authors declare that they have no competing interests.HJMG is an agro-environmental scientist and did the actual assessment. AR is one of the lead scientists in ExternE and contributed to the social cost approach. TMK is a toxicologist and provided input on medical and physiological aspects of colon cancer incidence. All authors read and approved the final manuscript.


Monocytes have long been considered a heterogeneous group of cells both in terms of morphology and function. In humans, three distinct subsets have been described based on their differential expression of the cell surface markers CD14 and CD16. However, the relationship between these subsets and the production of cytokines has for the most part been based on ELISA measurements, making it difficult to draw conclusions as to their functional profile on the cellular level. In this study, we have investigated lipoteichoic acid (LTA)- and lipopolysaccharide (LPS)-induced cytokine secretion by monocytes using the FluoroSpot technique. This method measures the number of cytokine-secreting cells on the single-cell level and uses fluorescent detection, allowing for the simultaneous analysis of two cytokines from the same population of isolated cells. By this approach, human monocytes from healthy volunteers could be divided into several subgroups as IL-1β, IL-6, TNF-α and MIP-1β were secreted by larger populations of responding cells (25.9–39.2%) compared with the smaller populations of GM-CSF (9.1%), IL-10 (1.3%) and IL-12p40 (1.2%). Furthermore, when studying co-secretion in FluoroSpot, an intricate relationship between the monocytes secreting IL-1β and/or IL-6 and those secreting TNF-α, MIP-1β, GM-CSF, IL-10 and IL-12p40 was revealed. In this way, dissecting the secretion pattern of the monocytes in response to TLR-2 or TLR-4 stimulation, several subpopulations with distinct cytokine-secreting profiles could be identified. Monocytes and macrophages are key cells of the innate immune system. Here, they fulfil a series of important functions relating to their phagocytic capacity, their role as antigen-presenting cells and their ability to produce and secrete a large number of signalling molecules including pro- and anti-inflammatory cytokines . DevelophighCD16−) represent the great majority (90–95%) of all monocytes and are referred to as ‘classical’ monocytes. The remaining, ‘non-classical’ monocytes are CD16+ and have been further divided into two subsets based on their level of CD14 expression, CD14dimCD16+ and CD14highCD16+. These two minor subpopulations of cells are thought to represent more mature macrophage-like monocytes  and TLR-4 [ultrapure lipopolysaccharide (LPS) from E. coli K12] were all from InvivoGen . Red blood cell (RBC) lysis buffer was purchased from BioLegend . Anti-cytokine monoclonal antibodies (mAb) for ELISpot and FluoroSpot were obtained from Mabtech  for the detection of the following cytokines: IL-1β, IL-6, TNF-α, MIP-1β, GM-CSF, IL-10 and IL-12p40. Based on their reactivity with the p40 chain common to both IL-12 and IL-23, the IL-12p40 reagents also detected IL-23-secreting cells. Streptavidin-alkaline phosphatase (SA-ALP) and BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro-blue tetrazolium) substrate were both from Mabtech as were anti-FITC-Green, Streptavidin-Red and fluorescence enhancer. The CD16+ monocyte isolation kit was from Miltenyi Biotec . For flow cytometry, Phycoerythrin (PE)-conjugated anti-CD3 (clone HIT3a), anti-CD19 (clone HIB19), anti-CD56 (clone MEM-188) and Alexa Fluor 488-conjugated anti-CD16 (clone 3G8) mAb (including recommended isotype controls) were purchased from BioLegend. PE-conjugated anti-CD14 (clone M5E2) mAb and the recommended isotype control were from BD Biosciences  as were the BD Vacutainer® blood collection tubes containing sodium citrate.RPMI1640, penicillin/streptomycin, HEPES and low-endotoxin (<1 EU/ml) FCS were all purchased from Invitrogen Life Technologies . Ficoll-Paque™ PLUS was obtained from GE Healthcare Life-Sciences . The RosetteSep® Monocyte enrichment cocktail was purchased from Stemcell Technologies . Ligands for TLR-2 [purified lipoteichoic acid (LTA) from m EDTA (PBS/FCS/EDTA) and layered on top of 15 ml Ficoll-Paque™ PLUS. After centrifugation at 1200 g for 20 min, the enriched monocytes were collected, washed twice in PBS/FCS/EDTA and suspended in cell culture medium . The enriched monocytes, comprising the whole population of classical (CD14highCD16−), non-classical (CD14dimCD16+) and intermediate (CD14highCD16+) monocytes, were then counted and analysed for viability  using the Guava ViaCount assay and the EasyCyte Mini System . With this protocol, the average proportion of CD14-positive cells was 79%  including an average of 9% CD16+ monocytes. The level of contaminating T cells, B cells and NK cells in these monocyte preparations was found to be below 2% as assessed by flow cytometry.Blood was obtained from healthy volunteers after informed consent and with approval from the ethics committee at the Karolinska Institute, Stockholm, Sweden. The blood was collected in sodium citrate, and monocytes were enriched according to the manufacturer’s instructions using RosetteSep® (Monocyte enrichment cocktail). Briefly, 750 μl of RosetteSep cocktail was mixed with 15 ml of EDTA-treated whole blood and incubated for 20 min at room temperature (RT). The sample was then diluted 1:1 in PBS containing 2% FCS with 1 m+ monocytes were isolated using magnetic beads. For this purpose, one volume of blood was mixed with one volume of PBS and layered on top of Ficoll-Paque™ PLUS. After centrifugation at 400 g for 30 min at 22 °C, the peripheral blood mononuclear cells (PBMC) fraction was collected, washed twice and suspended in cell culture medium. Following depletion of granulocytes and NK cells, the CD16+ monocytes were then isolated using anti-human CD16 microbeads according to the manufacturer’s instructions . The average purity of the CD16+ monocyte preparations was 88% as assessed by flow cytometry.For one set of FluoroSpot experiments, CD162O. Capture anti-cytokine antibodies were diluted with sterile PBS to 15 μg/ml, and 100 μl was added to each well. After incubation overnight at +4 °C, the coated wells were washed five times with 200 μl/well of sterile PBS followed by blocking of the membrane for 30 min with 200 μl/well of cell culture medium. The medium was then removed, and 50 μl/well of the same culture medium with or without stimuli (LTA 1000 ng/ml or LPS 100 ng/ml) was added followed by the addition of 50 μl/well of cells (1000 or 3000 cells/well). Each sample was analysed in quadruplicates or triplicates. The plates were thereafter transferred to a 5%-CO2 incubator and incubated for 20 h at 37 °C. After incubation, cells were removed by washing five times with 200 μl/well of PBS using an automated ELISA washer . Detection antibodies conjugated with biotin or FITC were diluted with PBS with 0.5% FCS (PBS/FCS) to 1 μg/ml, and 100 μl was added to each well. After incubation for 2 h at RT, plates were washed as mentioned previously and wells with biotinylated detection antibodies were incubated for 1 h at RT with 100 μl/well of Streptavidin (SA) conjugated either with alkaline phosphatase (ALP) diluted 1:1000 in PBS/FCS for ELISpot or with red fluorophore, diluted to 0.5 μg/ml in PBS/FCS for FluoroSpot. At the same time, wells with FITC-labelled detection antibodies were incubated with anti-FITC-Green mAb (0.5 μg/ml in PBS/FCS). After incubation for 1 h at RT, plates were again washed five times with 200 μl/well of PBS. At this stage, the plastic underdrain of the FluoroSpot plates was removed (not for ELISpot) and the plates were incubated with either 100 μl/well of the substrate BCIP/NBT (ELISpot) or fluorescence enhancer (FluoroSpot). In ELISpot, the substrate reaction was stopped after 10 min by extensive washing in tap water and the plates were left to dry at RT. In FluoroSpot, the fluorescence enhancer was discarded after 10 min and plates were left to dry protected from light. Analysis and counting of spots were made in an ELISpot/FluoroSpot reader system  where fluorescent spots were counted utilizing separate filters for FITC and Cy3. Double-secreting cells were determined as spots having the same position (centre point) in an image overlay of FITC and Cy3 images (FITC+Cy3).Low-fluorescent 96-well PVDF membrane plates  were prewet with 20 μl 35% ethanol/well for 1 min and washed five times with 200 μl sterile H3 and 0.5% FCS (FACS buffer). Cells were then stained for expression of CD3, CD14, CD19, CD56 and CD16 by incubating 4 × 105 cells with anti-CD3-PE (10 μl), anti-CD14-PE (20 μl), anti-CD19-PE (20 μl), anti-CD56-PE (10 μl) and anti-CD16-Alexa488 (5 μl) for 15 min at 4°C in a total volume of 50 μl. After incubation, cells were washed twice in FACS buffer and analysed by flow cytometry in a Guava EasyCyte Mini System. Matched isotype controls were used to establish the background level of non-specific staining. Guava ExpressPro software (Guava Technologies) was used for data acquisition and analysis.Prior to examining the purity of our isolated monocytes, erythrocytes were lysed according to the manufacturer’s recommendations using RBC lysis buffer and suspended in cold PBS with 0.02% NaNspss 16.0 software . Differences were considered significant for *P < 0.05.Data are presented as boxplots or as means ± range. The number of individuals assessed is indicated in each figure legend. Statistical analysis was performed by applying the Wilcoxon signed-rank test using We have previously investigated monocyte-derived cytokine secretion in ELISpot using PBMC as the source of cells . In thisTo assure that the FluoroSpot assay was comparable in performance and sensitivity to the ELISpot method, enriched monocytes (1000 or 3000 cells per well) were incubated for 20 h with or without LPS (50 ng/ml) and the frequencies of cytokine-secreting cells were determined using the two methods in parallel. In the FluoroSpot assay, detection was performed either using FITC-labelled detection antibody  in combination with anti-FITC-Green or with biotinylated detection antibody  in combination with Streptavidin-Red. Both assays were performed in low-fluorescent PVDF membrane plates to allow optimal detection of fluorescent spots. As shown in To investigate the numbers of cytokine-secreting cells after TLR-2 and TLR-4 stimulation, enriched monocytes from six healthy donors were incubated for 20 h in the absence or presence of LTA (500 ng/ml) or LPS (50 ng/ml) and analysed in FluoroSpot for the secretion of seven monocyte-derived cytokines . As prevAfter having established the number of secreting cells for each cytokine and stimulus, we went on to investigate co-secretion among the most frequent cytokines secreted, that is IL-1β, IL-6, TNF-α and MIP-1β. For this purpose, plates were coated with anti-IL-1β or anti-IL-6 antibodies in combination with antibodies to either TNF-α or MIP-1β, and detection was performed using secondary reagents coupled with green fluorophore (IL-1β and IL-6) or red fluorophore . By this approach, three distinct subpopulations could potentially be revealed: FITC – single-secreting cells, FITC + Cy3 – double-secreting cells and Cy3 – single-secreting cells.As shown in GM-CSF-secreting monocytes, when combined with IL-1β, displayed a limited degree of co-secretion . ConsequCompared with the high frequencies of monocytes secreting IL-1β, IL-6, TNF-α and MIP-1β, (25.9–39.2%) only a minority secreted IL-10 and IL-12p40 (<2%). Lacking suitable reagents for the double staining, we could not establish to what extent IL-10 and IL-12p40 were produced by the same or different populations of cells. However, simultaneous staining for IL-12p40 and IL-6 showed that a majority of the IL-12p40-secreting monocytes were also positive for IL-6 (78% with LTA and 87% with LPS), and a similar but slightly lower overlap was observed for IL-10 and IL-6 . In cont+ monocytes are recognized as key producers of TNF-α, their production of other cytokines is less clear, and contradicting data exist as to their secretion of IL-10 [+ non-classical and intermediate monocytes (CD14dimCD16+ and CD14highCD16+) were separated from PBMC using anti-CD16 magnetic beads and analysed in FluoroSpot in the absence or presence of LPS (1000 cells per well).Although CD16of IL-10 –16. To e+ monocytes secreted TNF-α and of this population, approximately half co-secreted IL-6  . Apart fFor example, cytokine determinations based on ELISA and RT-PCR have only resulted in crude characterizations as neither assay permits resolution at the cellular level –16. FurtBy this approach, we were able to determine the numbers of monocytes secreting IL-1β, IL-6, TNF-α, MIP-1β, GM-CSF, IL-10 and IL-12p40 in response to stimulation. Of these, the first four were secreted by larger populations of the cells (25.9–39.2%), whereas GM-CSF was produced by a smaller population (9.1%) and IL-10 and IL-12p40 only by a few per cent (1.2–1.3%) of the monocytes. This pattern of secretion was surprisingly consistent between different donors and, apart from IL-1β and IL-12p40, very similar for both LTA and LPS stimulation .Given the similar and high numbers of monocytes secreting IL-1β, IL-6, TNF-α and MIP-1β, it was natural to assume that these cytokines were, in fact, all secreted by the same population of cells. However, once we were able to combine these cytokines in two-colour FluoroSpot, the results revealed a much more intricate, but consistent pattern of co-secretion. Thus, while the two cytokines IL-6 and MIP-1β showed a high degree of secretory overlap, co-secretion of IL-1β and MIP-1β was only observed in a minority of the cells . This loWhile our results provide further support for the heterogeneous nature of monocytes, they also demonstrate the possibility of dividing these cells into several subsets based on differences in cytokine-secreting capacity. A similar division of T cells into subpopulations, each with their own unique cytokine profile, has proven very useful and has allowed for the characterization of a number of T cell subsets with distinct functional properties . Althoug+ monocytes have been claimed to be the major source of TNF-α [+ population when compared to the number of cells secreting IL-6 or MIP-1β . Furthermore, it has been claimed that the monocytes secreting IL-10 are primarily found in the CD14highCD16+ subpopulation [dimCD16+ subset. However, in line with previous investigators [+ population (Differences in cytokine production by monocytes have been reported previously, and CD16of TNF-α –15. In lr MIP-1β . Howevertigators –15, we wpulation .Finally, while the results of this study add a further level of complexity to monocytes, we believe that they also provide a meaningful basis for further characterization of these cells from a functional perspective and may therefore serve as an important complement to the currently accepted subclassification of monocytes. We also think that the same approach may be used to study cytokine secretion by other members of the MPS, including macrophages, dendritic cells and bone marrow–derived precursors and that such characterization may offer new insights into their functional/developmental relationships.


A new variant of Creutzfeldt Jacob Disease (vCJD) was identified in humans and linked to the consumption of Bovine Spongiform Encephalopathy (BSE)-infected meat products. Recycling of ruminant tissue in meat and bone meal (MBM) has been proposed as origin of the BSE epidemic. During this epidemic, sheep and goats have been exposed to BSE-contaminated MBM. It is well known that sheep can be experimentally infected with BSE and two field BSE-like cases have been reported in goats. In this work we evaluated the human susceptibility to small ruminants-passaged BSE prions by inoculating two different transgenic mouse lines expressing the methionine (Met) allele of human PrP at codon 129 (tg650 and tg340) with several sheep and goat BSE isolates and compared their transmission characteristics with those of cattle BSE. While the molecular and neuropathological transmission features were undistinguishable and similar to those obtained after transmission of vCJD in both transgenic mouse lines, sheep and goat BSE isolates showed higher transmission efficiency on serial passaging compared to cattle BSE. We found that this higher transmission efficiency was strongly influenced by the ovine PrP sequence, rather than by other host species-specific factors. Although extrapolation of results from prion transmission studies by using transgenic mice has to be done very carefully, especially when human susceptibility to prions is analyzed, our results clearly indicate that Met129 homozygous individuals might be susceptible to a sheep or goat BSE agent at a higher degree than to cattle BSE, and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD. Our results suggest that the possibility of a small ruminant BSE prion as vCJD causal agent could not be ruled out, and that the risk for humans of a potential goat and/or sheep BSE agent should not be underestimated. Prion diseases, also referred as transmissible spongiform encephalopathies, are fatal neurodegenerative diseases caused by proteinaceous infectious particles denominated “prions.” Prion diseases acquired their first real public relevance with the outbreak of bovine spongiform encephalopathy (BSE) (“mad cow disease”) in the United Kingdom in the 80s and its link with the appearance of a new, variant form of Creutzfeldt-Jakob disease in humans. Recycling of ruminant tissues in meat and bone meal has been proposed as origin of the BSE epidemic. During this episode, sheep and goats have also been exposed to BSE-contaminated meal, so transmission to this species may have occurred. We analyzed the human susceptibility to sheep and goat passaged-BSE prions by using transgenic mice expressing human prion protein (PrP). When different sheep and goat BSE isolates were inoculated in these transgenic mice, higher susceptibility than that observed for cattle BSE was detected and the disease manifestation was similar to that observed in mice inoculated with the new variant of Creutzfeldt-Jakob disease. Our findings suggest that humans are at least equally, and might be even more, susceptible to a sheep or goat BSE agent compared to a cattle BSE one. Sc) of the cellular prion protein (PrPC) Sc is widely distributed in lymphoid tissues of experimentally BSE-infected sheep Transmissible Spongiform Encephalopathies (TSEs) are fatal neurodegenerative diseases which include Scrapie in sheep and goats, Bovine Spongiform Encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD) in humans. Prions, the causal agents of these diseases are thought to be infectious protein particles essentially composed of a misfolded isoform  without clear clinical signs. The remaining inoculated mice failed to develop a clinical disease or to accumulate detectable levels of PrPres in the brain up to ∼700 days after inoculation. On second passage performed with brain homogenate from a PrPres-negative mouse (succumbed at 576 dpi) from the first passage (BSE2 isolate), 3 out of 4 inoculated tg340 mice tested positive for brain PrPres by western blot with a survival time of 572±37 d.p.i. It is important to note that all the cattle BSE isolates tested in this study were transmitted as efficiently as vCJD isolates or other BSE-related sources to bovine PrP transgenic mice , respectively. These features were stable upon subpassaging, suggesting an absence of transmission barrier for this agent  and 3. Inic mice  and 3, tres detection in the brain) with almost all the sheep and goat BSE isolates used  contained higher PrPres levels in their brains than the sheep and goat BSE isolates  and prominent diglycosylated species was consistently observed in the challenged, PrPres-positive mice. This signature clearly differed from that observed after inoculation of mice with sporadic CJD (89WGQGG93 according to the human PrP sequence) is known to be poorly protected from proteinase K digestion 2) in tg340 mice, one of the three positive mice presented a brain PrPres profile clearly distinct from PrPvCJD, that was comparable to that of typeI sCJD-inoculated tg340 mice with predominantly monoglycosylated and higher size fragments (∼21 kDa for the aglycosyl band) and preserved detection by 12B2 antibody  and on 3rd passage in tg650. As illustrated in The regional distribution of PrPAt microscopic level, abundant amyloid-like plaques were present , as sugg2) was passaged into bovine (tg110) and ovine PrP transgenic mice  2/TgBov and Ca-BSE2/TgOv, 2/TgBov isolate did not induce a clinical disease nor PrPres accumulation in tg340 mice while an intermediate passage on the ovine PrPARQ sequence (Ca-BSE2/TgOv) restored the disease susceptibility, with survival times, biochemical and neuropathological features similar to those obtained with experimental sheep BSE isolates  produced the shortest disease in bovine PrP transgenic mice (0/TgOv isolate (which maintains its PrP ovine sequence) showed a full transmission rate in human transgenic mice with similar survival times as those of the original Sh-BSE0 isolate  in bovine PrP transgenic mice  in two different laboratories. In general, the transmission results obtained in both human-PrP transgenic mouse lines were very comparable. Some shortening in survival times was observed in tg650 mice (compared to the tg340 mice line), which was probably due to higher PrP expression levels in this line. Worryingly, our results support the view that an intermediate passage of BSE agent in small ruminants accelerates the appearance of a vCJD-like disease in human PrP mice or markedly increases its transmission efficiency. Because the apparent phenotype of cattle and sheep/goat BSE prions is conserved, these data also unravel an important role of PrPThe transmission efficiency of cattle BSE isolates in both human-PrP transgenic mouse models was apparently low. With all BSE isolates, whose high infectivity has been demonstrated in bovine-PrP transgenic mice  and 3, vSc type of cattle and sheep/goat BSE agents appeared indistinguishable from the vCJD agent propagated in these mice, as previously demonstrated in bovine transgenic mice res appeared similar in human-PrP transgenic mice, other assays are currently performed to further compare the biochemical or biophysical properties of the respective proteins are ongoing.Remarkably, a different picture emerged when the sheep and goat BSE isolates were inoculated to human PrP transgenic mouse models. Attack rates approaching 100% were observed from the primary passage onwards and mean incubation times were more consistent with those measured after transmission of vCJD. On further passaging, the neuropathological phenotype and PrPres content of these isolates. In addition, the data from inoculation to BoPrP-Tg reporter mice suggest that cattle BSE and sheep and goat-BSE isolates could have similar transmission efficiency , suggesting no infection, in accordance with the lack of epidemiological evidence linking scrapie with human TSE. Moreover, the low transmission efficiency observed for the cattle BSE agent is not exclusively linked to the bovine PrP sequence since other uncommon BSE strains (BSE-L) are efficiently transmitted to human-PrP mice C prefers a BSE PrPSc with conformational characteristics templated by the ovine sequence, to a bovine BSE PrPSc. Because a similar increased transmission efficiency of sheep/goat BSE has been reported in wild type mice Sc sequence may not be limited to human PrPC. One explanation might be an alteration in the quaternary structure (after passage into sheep/goat) generating PrPSc polymers less degraded or more rapidly/easily amplified favouring or enhancing the initial conversion. This question is currently being addressed by sedimentation velocity The PrP primary sequence influence seems to depend strongly on the strain involved, since no PrPAlthough extrapolation of results from prion transmission studies by using transgenic mice has to be done very carefully, especially when human susceptibility to prions is analyzed, our results clearly indicate that Met129 homozygous individuals might be susceptible to a sheep or goat BSE agent at a higher degree than to cattle BSE, and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD. Although no vCJD cases have been described in Val129 homozygous individuals so far it is relevant to analyze if similar results will be observed in this genotype. This issue is currently being addressed in transmission experiments using transgenic mice expressing Val129 human PrP.res detected in brains of our sheep and goat BSE-inoculated mice seem to be indistinguishable from that observed in vCJD. Considering the similarity in clinical manifestation of BSE- and scrapie-affected sheep Sc have been detected in many peripheral organs, small ruminant-passaged BSE prions might be a more widespread source of BSE infectivity compared to cattle Taken all together, our results suggest that the possibility of a small ruminant BSE prion as vCJD causal agent could not be ruled out, which has important implications on public and animal health policies. On one hand, although the exact magnitude and characteristic of the vCJD epidemic is still unclear, its link with cattle BSE is supported by strong epidemiological ground and several experimental data. On the other hand, the molecular typing performed in our studies, indicates that the biochemical characteristics of the PrPAnimal experiments were carried out in strict accordance with the recommendations in the guidelines of the Code for Methods and Welfare Considerations in Behavioural Research with Animals (Directive 86/609EC) and all efforts were made to minimize suffering. Experiments were approved by the Committee on the Ethics of Animal Experiments of the author's institutions (INRA and INIA); Permit Number: RTA06-091 and CT05-036353.The isolates used in this study are described in The tg650 transgenic mouse line over expresses human PrP M129 at a 6-fold level on a mouse PrP null background res) by immunohistochemistry (IHQ) or histoblotting and the other was frozen at −20°C to determine presence of PrPres by Western blot (WB). In all cases, survival time and attack rate were calculated for each isolate. Survival time was expressed as the mean of the survival days post inoculation (d.p.i.) of all the mice scored positive for PrPres, with its correspondent standard error. Attack rate was determined as the proportion of mice scored positive for PrPres from all the mice inoculated. When all mice were scored negative for PrPres, the survival time range was shown. Brain homogenates from PrPres positive mice, when available, were used for further passaging. When all mice were scored negative for PrPres on primary passage, PrPres-negative brain homogenates were used for second passage.All inocula were prepared from brain tissues as 10% (w/v) homogenates. Individually identified 6–10 week-old mice were anesthetized and inoculated with 2 mg of brain homogenate in the right parietal lobe using a 25-gauge disposable hypodermic needle. Mice were observed daily and the neurological status was assessed weekly. When progression of a TSE disease was evident or at the end of lifespan, animals were euthanized because of ethical reasons. Once euthanized, necropsy was performed and brain was taken. A part of the brain was fixed by immersion in 10% formol to quantify spongiform degeneration by histopathology and PK resistant PrP accumulation . 10–50 µl of a 10% (w/v) brain homogenate were diluted in a 10% (w/v) negative sheep brain homogenate, to obtain a 200 µl final volume. Homogenates were incubated for 10 min at 37°C with 200 µl of a 2% proteinase K solution (in buffer A). PrPres was recovered as a pellet after addition of 200 µl of buffer B and a centrifugation at 15,000× g for 7 min at 20°C. Supernatants were discarded and pellets were dried inverted over absorbent paper for 5 min. Pellets were solubilised in Laemmli buffer and samples were incubated for 5 min at room temperature, solubilised, and heated at 100°C for 5 min. Samples were centrifuged at 20,000× g for 15 min at 20°C and supernatants were recovered and loaded on a 12% Bis-Tris Gel . Proteins were electrophoretically transferred onto PVDF or nitrocellulose membranes (Millipore). Membranes were blocked O/N with 2% BSA blocking buffer. For immunoblotting, membranes were incubated with either Sha 31 175±20 mg of frozen brain tissue were homogenized in 5% glucose in distilled water in grinding tubes (Bio-Rad) adjusted to 10% (w/v) using a TeSeE™ Precess 48™ homogenizer (Bio-Rad) following manufacturer instructions. Presence of PrPAll procedures involving mice brains were performed as previously described nd and 3rd passage, using the 3F4 anti-PrP antibody as previously described Brains were rapidly removed from euthanised mice and frozen on dry ice. Thick 8–10 µm cryostat sections were cut, transferred onto Superfrost slides and kept at −20°C until use. Histoblot analyses were performed on 3 brains per infection at 2XhoI restriction enzyme site adjacent to the translation start and stop sites of the human PrP ORF . The PCR fragments obtained were sub cloned into a pGEM-T Easy Vector System (Promega) following manufacturer instructions, and inserts were sequenced to confirm no differences in the inferred amino acid sequence with respect to previously sequenced human PrP genes (GenBank accession number NM_183079) and to confirm the presence of the consequent codon 129 nucleotide variant (MetATG). The human PrP ORF was excised from the cloning vector using the restriction enzyme XhoI and inserted into the expression vector MoPrP.Xho XhoI restriction sites but could be distinguished from the wild type murine PrP gene because of the absence of intron 2. The vector was also digested with XhoI to excise the murine PrP ORF and the correspondent human PrP ORF were inserted by ligation, obtaining the plasmid pMo-huPrP129M.Xho.Tg340 mouse line expressing about 4-fold level of human PrP M129 on a mouse PrP null background has been generated following a similar procedure previously describe for the generation of other transgenic mouse line expressing different species PrP Not I leading to DNA fragments of approximately 12 Kb. Finally, the DNAs were purified and dissolved in TE at a final concentration of 2 to 6 µg/ml and microinjected into pronuclear stage ova collected from super-ovulated B6CBAf1 females mated with 129/Ola males carrying a null mutation in endogenous PrP The human transgene was excised from the plasmid vector using the restriction endonuclease 5′- TAGATGTCAAGGACCTTCAGCC- 3′ and 5′- GTTCCACTGATTATGGGTACC -3′.DNA from founders' tails biopsies was extracted using a Extract-N-Amp Tissue PCR kit (Sigma-Aldrich) following manufacturer instructions. The presence of the human transgene in these founders was identified by PCR amplification using specific primers for the mouse PrP exon 2 and human PrP open reading frame. The absence of the murine PrP ORF in the transgenic mice generated was confirmed by PCR amplification using the primers: C (huPrP) and murine PrPC (muPrP) heterozygous transgenic mice (PrP mu+/− hu+/−) were obtained. The expression of human PrPC in brain of these mouse lines was analyzed and compared with PrPC content in human brain homogenate by western blot using mAb 3F4 which recognizes the 109MKHM112 epitope (numbered according to the human PrP sequence). Human PrPC was detected in 100% of the tested lines (data not shown). From the initial 8 different mouse lines heterozygous for both murine and human PrP genes (PrP mu+/− hu+/−), the mouse line named as tg340 was selected for further experiments on the basis of the level of PrPC expression.Eight different lines (founders) of human PrP−/− (Prnp−/−) to obtain a null murine PrP background (PrP mu−/− hu+/−). Interbreeding within these animals was performed to obtain homozygosis for the human PrP transgen within a murine PrP background (PrP mu−/− hu+/+). The absence of murine PrP gene was determined by PCR using specific primers. Human PrPC expression levels, determined more accurately in brain from homozygous tg340 animals was about 4-fold higher than PrPC levels in human brain homogenates as determined by dilution experiments in western blot (Homozygous Tg340 mouse line was established backcrossing these animals with homozygous null animals MuPrPern blot .Prnp gene used in this paper is NM_183079.The GenBank accession number for the human


BSE can infect small ruminants and could be misdiagnosed as scrapie. Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that include variant Creutzfeldt-Jakob disease in humans, scrapie in small ruminants, and bovine spongiform encephalopathy (BSE) in cattle. Scrapie is not considered a public health risk, but BSE has been linked to variant Creutzfeldt-Jakob disease. Small ruminants are susceptible to BSE, and in 2005 BSE was identified in a farmed goat in France. We confirm another BSE case in a goat in which scrapie was originally diagnosed and retrospectively identified as suspected BSE. The prion strain in this case was further characterized by mouse bioassay after extraction from formaldehyde-fixed brain tissue embedded in paraffin blocks. Our data show that BSE can infect small ruminants under natural conditions and could be misdiagnosed as scrapie. Surveillance should continue so that another outbreak of this zoonotic transmissible spongiform encephalopathy can be prevented and public health safeguarded. Sc) of a naturally occurring host-encoded protein (PrPC)  are fatal diseases characterized by neurodegenerative changes in the central nervous system that include vacuolation, gliosis, and accumulation of an abnormal isoform , a TSE of cattle, was first detected in 1986  before homogenization. Unfixed samples were kept frozen at −80°C. After thawing, they were suspended in normal saline (10% wt/vol) before homogenization. All homogenates were examined microbiologically and treated with antimicrobial drugs as required. Only microbiologically cleared inocula were used to challenge animals.,The only tissue available from the 1990 suspected UK case . ParaffiSc distribution, which was verified by IHC analysis of the adjacent rostral and caudal coronal levels of the selected sample, we assumed that titer did not vary substantially on either side of the midline. Therefore, the obex was cut sagitally in half. Half was processed histologically and was subsequently recovered and rehydrated to replicate the process applied in the fixed samples; the other half was kept frozen. Each half was homogenized and inoculated into mice.Additional controls of fixed and frozen brain tissues from the same source were used to assess the effect of fixation, processing, and retrieval on the biological properties of the TSE agents present. All samples included in this study were from animals showing clinical signs of TSE. These came from animals with confirmed TSE sourced through passive surveillance schemes, with the exception of an ovine BSE case that was produced experimentally  and a transgenic mouse line . C57/BL6 and RIII mice share the same PrP sequence (PrP-a), but it is believed that RIII alone could be used to discriminate BSE from other TSEs on the basis of lesion profile (LP) data on first passage  were performed  of 4 different coronal levels  were stained with hematoxylin and eosin according to standard methods  according to established methods  and12B2 , epitope 93WGQGG97 (0.2 µg/mL) and 12% BisS/Tris (Criterion) acrylamide gels (Bio-Rad Laboratories) in 3-(N-morpholino)propanesulfonic acid buffer. Relative band mass was measured by using Quantity One software (Bio-Rad Laboratories).Western blotting (WB) was applied only for PrP-a mice (C57/BL6 and RIII) because PrP-b mice inoculated with either scrapie or BSE produce similar banding profiles and cannot be distinguished by this approach .Clinical-stage TSE  did not Sc bands that were indistinguishable from those of mice inoculated with the various BSE sources .After 1 serial passage in PrP-b (VM) mice, the sample from the goat with suspected BSE generated IP of 128 ± 4 (mean ± SD) days postinoculation similar to serially passaged ovine BSE (109 ± 4) and the 301V mouse-adapted BSE strain (107 ± 6). The comparatively longer IP generated by that goat sample relative to these mouse-adapted BSE isolates is a common observation at second passage; for example, the IP of the serially passaged ovine BSE at second passage was 148 ± 3 days postinoculation. In these mice, the LPs were indistinguishable from those produced by serially passaged experimental ovine BSE and similar to the 301V strain . After sSc mapping in the brain, and WB of PrPSc with those of mice inoculated with BSE from various ovine, caprine, and bovine sources.We confirmed that the agent responsible for TSE in a UK goat, which was initially reported as scrapie in 1990 and subsequently as suspected BSE in 2006 (Sc deposits can be identified, and the distribution of each deposit in the brain can be mapped (,,From a method perspective, the data suggest that AR, IP, and LP are not optimal bioassay parameters for differentiating TSE sources during first passage because they represent mean values derived from a group of animals that have been inoculated with a specific source. Therefore, a substantial number of animals must die of clinical TSE for these parameters to be meaningful. This finding is a limiting factor in instances in which TSE is diagnosed in only a few animals because of low titer, restricted permissiveness of specific TSE strains in certain laboratory animals, or both. These limitations can be overcome by application of IHC and WB to differentiate BSE from scrapie confidently in individual mice on first passage. Use of IHC has shown that different PrPSc patterning, WB can also be applied to diagnose BSE. In contrast, its application in PrP-b mice is less informative (The data show that the TSE agents in this study were not altered by the adverse conditions applied to them during histologic procedures. However, titer may decrease, suggesting that the effect of histologic processing is quantitative not qualitative. Therefore, bioassay is a valid approach for identifying BSE in archived histologic material when other techniques are not applicable, as in the current study. Regarding the suitability of different mouse lines for confirming BSE, our data show that any mouse line in which the agent can propagate sufficiently is suitable. An additional requirement at a practical level is the ability to characterize the agent on first passage. In this respect, use of PrP-a mice is preferable because in addition to AR, IP, histopathologic analysis, and PrP,These methods can also be applied to analyze bioassay data derived from validated transgenic mouse lines that offer the advantage of higher AR and decreased IP, provided that appropriate transgenic lines are selected and the TSE source and the donor species under investigation are taken into consideration. In this particular instance, our first choices would have been the use of a mouse line overexpressing a bovine transgene in combination with 1 that overexpresses a caprine transgene. At initiation of the study, an established bovinised line was not available to us, and the data generated from the wild-type mice were considered sufficient to identify unequivocally the agent strain. Caprine transgenic mouse lines are still under development and not characterized or widely available. Instead, we used tg338 mice although they show <100% AR and extended IP when inoculated with BSE (The 2 cases of naturally occurring BSE in small ruminants—the 1 reported here and the 1 identified in France (,,,The BSE case we have confirmed was 1 of 26 historic goat samples examined in the United Kingdom collected during 1984–2002 (Because TSEs in goats are still a problem, particularly in Mediterranean countries, our data suggest that extensive surveillance and breeding schemes must remain in place to prevent a BSE outbreak in small ruminants and to safeguard public health. This report also highlights several issues regarding the use of mouse bioassay to identify TSE strains. As governing bodies seek confirmation of equivocal cases that are identified worldwide, they must be aware of the limitations, cost, and timescale demands of confirming such cases.


We compared transmission characteristics for prions from L-type bovine spongiform encephalopathy and MM2-cortical sporadic Creutzfeldt-Jakob disease in the Syrian golden hamster and an ovine prion protein–transgenic mouse line and isolated distinct prion strains. Our findings suggest the absence of a causal relationship between these diseases, but further investigation is warranted.  Microcebus murinus) , the L-type bovine spongiform encephalopathy (L-BSE) in cattle requires particular attention for public health. L-BSE is transmitted more efficiently than is classical BSE among primates  expressing ovine PrP . Both of these species are susceptible to L-BSE prions from cattle ; a lemur injected intracerebrally (i.c.) with the 02-2528 L-BSE cattle isolate (res). At the terminal stage of the disease, animals were euthanized, and their brains and spleens were collected for PrPres analyses by Western blot and for histopathologic studies  brain homogenates in 5% sterile glucose. Serial passages were performed in TgOvPrP4 mice by i.c. inoculation of 1% (wt/vol) homogenates from mice positive for protease-resistant PrP (PrPd) in brain samples was not detected by paraffin-embedded tissue blot (PET-blot)  , and disET-blot) , panel AET-blot) , panel Cern blot . res distribution in the brain; immunohistochemical analysis  associated with a lack of reactivity toward the N terminal 12B2 antibody, in contrast to that for the control animal with scrapie  . PET-bloanalysis , panel Din lemur . Brain Pd accumulation in the mouse brains   compared with L-BSE–infected mice . PrPres was readily identified in the spleens of TgOvPrP4 mice at the second passage for sCJD and L-BSE from cattle and at the first passage for L-BSE from lemur  , panel Achloride . Howevernd L-BSE , panel Com lemur . No sign spleens , panel Dres occurred in the frontal parts of the brain, but the intensity and appearance of PrPres in the cortex, thalamus, and hippocampus were distinctly different. Immunohistochemical analyses of the hippocampus showed PrPd deposition in the dentate gyrus in sCJD-infected mice, in contrast to a lack of deposition in lemur-passaged L-BSE–infected mice.Histopathologic analysis showed severe vacuolar lesions in TgOvPrP4 mice infected at second passage with sCJD and lemur-passaged L-BSE . Howeverres electrophoretic mobility and the conformational stability of PrPd, sCJD and L-BSE differed in PrPres glycosylation for the mouse brains and gel migrations for the mouse spleens. Mice infected with MM2-cortical sCJD versus those infected with L-BSE also showed distinct lesion profiles and PrPd distribution, which confirms clear biologic differences between these diseases.We report the isolation of 2 prion strains derived from L-BSE and MM2-cortical sCJD after transmission in Syrian hamsters and ovine PrP–transgenic mice. In hamsters, we did not transmit any disease with sCJD, but the transmission of L-BSE from lemur was efficient, as previously reported for L-BSE from cattle , L-type bovine spongiform encephalopathy (L-BSE) from lemur, L-BSE from cattle, and classical BSE; conformational stability assay of disease-associated prion protein in brains of TgOvPrP4 mice at second passage; and histopathological features of MM2-cortical sporadic sCJD and L-BSE from lemur transmitted to ovine prion protein–transgenic mice at second passage.


C) in comparison with other non-BSE related prions from the same species. After these passages, most features of the BSE agent remained unchanged. BSE-derived agents only showed slight modifications in the biochemical properties of the accumulated PrPSc, which were demonstrated to be reversible upon re-inoculation into transgenic mice expressing bovine-PrPC. Transmission experiments in transgenic mice expressing bovine, porcine or human-PrP revealed that all BSE-derived agents were transmitted with no or a weak transmission barrier. In contrast, a high species barrier was observed for the non-BSE related prions that harboured an identical PrP amino acid sequence, supporting the theory that the prion transmission barrier is modulated by strain properties (presumably conformation-dependent) rather than by PrP amino acid sequence differences between host and donor.The specific characteristics of Transmissible Spongiform Encephalopathy (TSE) strains may be altered during passage across a species barrier. In this study we investigated the biochemical and biological characteristics of Bovine Spongiform Encephalopathy (BSE) after transmission in both natural host species  and in transgenic mice overexpressing the corresponding cellular prion protein (PrPC sequence and not influenced by other host genetic factors. The results presented herein reinforce the idea that the BSE agent is highly promiscuous, infecting other species, maintaining its properties in the new species, and even increasing its capabilities to jump to other species including humans. These data are essential for the development of an accurate risk assessment for BSE.As identical results were observed with prions propagated either in natural hosts or in transgenic mouse models, we postulate that the species barrier and its passage consequences are uniquely governed by the host PrP C, which is encoded by the prnp gene) into an abnormal disease-associated isoform (PrPSc) in tissues of infected individuals. Conversion of PrPC into PrPSc is a post-translational process involving structural modifications of the protein and resulting in a higher β-sheet content C is completely degraded after controlled digestion with proteinase K (PK) in the presence of detergents. In contrast, PrPSc is N-terminally truncated under such conditions, resulting in a PK resistant core, termed PrPresres, also named PrP 27–30, is a disease marker for TSE and the presence of PrPSc seems to associate with infectivity Sc represents the infectious TSE agent itself Bovine spongiform encephalopathy (BSE) in cattle, like scrapie in sheep and goats, is a transmissible spongiform encephalopathy (TSE). The key event in TSE infection is the conversion of the normal cellular prion protein  Interestingly, after passage in different host species, the BSE agent conserved its strain specific signature as assessed by bioassay in RIII mice C amino acid sequences. The properties of these BSE-derived prions were characterized in comparison with other non-BSE related prions with the aim of gaining a better understanding of the species barrier phenomenon and the consequences of passage across a species barrier.In this study we investigated the biochemical and biological characteristics of BSE after transmission in both natural host species  and in transgenic mice overexpressing the corresponding PrPInstituto Nacional de Investigación y Tecnología Agraría y Alimentaria (INIA); Permit Number: CEEA2012/024 and CEEA2009/004.Animal experiments were carried out in strict accordance with the recommendations in the guidelines of the Code for Methods and Welfare Considerations in Behavioural Research with Animals (Directive 86/609EC) and all efforts were made to minimize suffering. Experiments were approved by the Committee on the Ethics of Animal Experiments (CEEA) of the Spanish Cattle-BSE material was obtained from the brainstem of one BSE-affected cow (RQ 225:PG817/00), and supplied by the Animal Health and Veterinary Laboratories Agency (AHVLA), New Haw, Addlestone, Surrey, UK.BoTg-BSE was obtained from the brain of terminally ill BSE-inoculated transgenic mice expressing bovine PrP (BoPrP-Tg110) Sheep-BSE was obtained from the brain of a terminally ill ARQ/ARQ sheep intracerebrally inoculated with BSE, and provided by the French National Institute for Agricultural Research (INRA), Nouzilly, France.OvTg-BSE was obtained from the brain of terminally diseased BSE-inoculated transgenic mice expressing ARQ-ovine PrP (Tg IX) Pig-BSE was obtained from the brain of one BSE-affected pig (PG 33/03) intracerebrally inoculated with BSE as previously described PoTg-BSE was obtained from the brain of terminally diseased BSE-inoculated transgenic mice expressing porcine PrP (PoPrP-Tg001) Mouse-BSE was obtained from the brain of terminally diseased BSE-inoculated C57/Bl6 mice.MoTga20-BSE was obtained from the brain of terminally diseased BSE-inoculated transgenic mice expressing mouse PrP (Tga20) Human-vCJD (met 129) isolate (Ref.: NHBY0/0014) was obtained from the National Institute for Biological Standards and Control, Potters Bar, United Kingdom.HuTg-BSE was obtained from the brain of terminally diseased BSE-inoculated transgenic mice expressing M129-Human PrP (HuPrP-Tg340) Atypical Cattle-BSE H isolate was obtained from the brainstem of one naturally affected cow, diagnosed as atypical H-type BSE (French case 03-2095), and provided by the Agence Française de Sécurité Sanitaire des Aliments (AFSSA-France).Sheep-scrapie isolate SC-UCD-99 was obtained from the brain of an Irish ARQ/ARQ sheep naturally infected with scrapie .Mouse-RML scrapie was obtained from the brain of terminally diseased C57/Bl6 mice inoculated with Rocky Mountain Laboratory (RML) scrapie.Human-sCJD (met 129) isolate (Ref.: NHBX0/0001) was obtained from the National Institute for Biological Standards and Control, Potters Bar, United Kingdom.All inocula were prepared from brain tissues as 10% (w/v) homogenates in 5% glucose. To minimize the risk of bacterial infection, inocula were preheated for 10 min at 70°C before inoculation in mice.C at a 8-fold level C at a 4-fold level C at a 4-fold level All the isolates were inoculated in three different mouse models: i) BoPrP-Tg110 transgenic mouse line expressing bovine PrPIndividually identified 6–10 weeks-old mice were anesthetized with isoflurane and inoculated with 2 mg of brain homogenate in the right parietal lobe using a 25-gauge disposable hypodermic needle. Mice were examined twice weekly for neurological signs of prion disease and were euthanized by cervical dislocation when progression of the disease was evident or at the end of the study (650–700 dpi). The animals were humanely euthanized once a definitive diagnosis had been made or earlier if showing signs of distress or loss of up to 20% of body weight. A mouse was considered positive for prion disease when it showed two or three of 10 signs of neurological dysfunction previously described res) by immunohistochemistry (IHC) or histoblotting and the rest was frozen at −20°C before determining the presence of PrPres by Western blot (WB). In all cases, survival time and attack rate were calculated for each isolate. Survival time was expressed as the mean of the survival days post inoculation (dpi) of all the mice scored positive for PrPres, with its correspondent standard error. Attack rate was determined as the proportion of mice scored positive for PrPres from all the mice inoculated. Brain homogenates from PrPres positive mice, when available, were used for further passaging. When all mice were scored negative for PrPres on primary passage, PrPres-negative brain homogenates were used for second passage.Once euthanized, a necropsy was performed and the brain was taken. A part of the brain was fixed by immersion in neutral-buffered 10% formalin  to quantify spongiform degeneration by histopathology and PK resistant PrP accumulation . 10–100 µl of a 10% (w/v) brain homogenate were diluted in a 10% (w/v) negative sheep brain homogenate, to obtain a 200 µl final volume. Homogenates were incubated for 10 min at 37°C with 200 µl of a 2% proteinase K solution (in buffer A). PrPres was recovered as a pellet after addition of 200 µl of buffer B and centrifugation at 15,000× g for 7 min at 20°C. Supernatants were discarded and pellets were dried inverted over absorbent paper for 5 min. Pellets were solubilised in Laemmli buffer and samples were incubated for 5 min at room temperature, solubilised, and heated at 100°C for 5 min. Samples were centrifuged at 20,000× g for 15 min at 20°C and supernatants were recovered and loaded on a 12% Bis-Tris Gel . Proteins were electrophoretically transferred onto PVDF or nitrocellulose membranes (Millipore). Membranes were blocked O/N with 2% BSA blocking buffer. For immunoblotting, monoclonal antibodies Sha31 156YEDRYYRE163 epitope of the bovine-PrP sequence while 12B2 recognizes the 101WGQGG105 epitope of the bovine-PrP sequence. Immunocomplexes were detected by incubating the membranes for 1 hour with horseradish peroxidase conjugated anti mouse IgG (Amersham Pharmacia Biotech). Immunoblots were developed with enhanced chemiluminescence using ECL Plus reagent .175±20 mg of frozen brain tissue were homogenized in 5% glucose in distilled water in grinding tubes (Bio-Rad) adjusted to 10% (w/v) using a TeSeE Precess 48 homogenizer (Bio-Rad) following the manufacturer's instructions. The presence of PrPSc detection was carried out using two sandwich ELISA tests  following the manufacturer's instructions. The assay protocol includes a purification of PrPSc (TeSeE purification kit) consisting of (i) digestion of PrPC with PK, (ii) precipitation of PrPSc by centrifugation and (iii) denaturation of PrPSc at 100°C, before immuno-enzymatic detection. In this ELISA, the capture antibody recognizes the octarepeat region of PrP PrPSc portion recognized in the ELISA test was determined by measurement of the ELISA specific signal recovered after digestion with different concentrations of PK in ‘buffer A’ reagent . Each sample was first diluted in normal brain homogenate (between 100 and 10 000 fold) until a signal between 1.5 and 2 absorbance units was obtained after digestion with 50 µg/ml of PK. Triplicates of equilibrated samples were then submitted to a PK digestion with concentrations ranging from 50 to 300 µg/ml, before PrPSc precipitation and ELISA detection. Results were expressed as the percentage of residual signal as compared with the signal after 50 µg/ml PK digestion (lowest PK concentration). In each assay, two standardized controls (scrapie and BSE from sheep) were used as an internal standard.PK resistance of the PrPAll analyses of mouse brains were performed as previously described The PK resistance profiles were compared by Wilcoxon matched-pairs signed-rank test. Two-tailed P-values <0.05 were considered statistically significant.Nonparametric Mann-Whitney-U test was applied to establish statistically significant differences in survival times of the different mouse models inoculated with the different BSE-derived prions. A difference of P<0.05 was considered significant. Statistical analysis was performed using PAST software .C amino acid sequences. These BSE-derived prions have been characterized in comparison with the original cattle-BSE prions, as well as with other non-BSE related prion strains as controls. Human-vCJD was included as another BSE-derived prion In this work we investigated the biochemical and biological characteristics of a panel of BSE-derived prions corresponding to BSE passaged in both natural host species  and in transgenic mice overexpressing the corresponding PrPres was analysed using Sha31 mAb (res electromobility of cattle-BSE compared to the different BSE-derived prion isolates (res glycoforms was observed in the different BSE-derived prion isolates (res fraction (>50%) and a weak unglycolysated PrPres fraction (<20%). In contrast, PoTg-BSE showed a profile dominated by the monoglycosylated PrPres fraction (∼40%). In all cases, the glycoprofiles observed in the natural hosts were similar to those observed in the corresponding transgenic mouse models.The Western blot pattern of BSE-derived PrPha31 mAb  binding isolates . In all isolates . A certaisolates . BoTg-BSres from atypical cattle-BSE H, sheep-scrapie, mouse-RML and human-sCJD isolates was recognized equally well by both Sha31 and 12B2 mAbs. Conversely, PrPres from BSE in all investigated hosts was not recognized by 12B2 mAb  expressing bovine, porcine or human PrP, respectively In BoPrP-Tg110 mice, the different BSE-derived prions were fully transmitted (with a 100% attack rate) in primary passage, irrespective of their producing host . Either C. For BSE prions propagated in bovine, porcine, murine or human-PrP hosts, the transmission efficiency in both mouse models was similar to that observed for the cattle-BSE isolate. However, the OvTg-BSE isolate showed higher transmission efficiency on primary passage , as compared to the cattle-BSE isolate, in the three mouse models used. Similar increased transmission efficiency was previously reported for sheep-BSE in these mouse models C sequences , a low infectivity titer for these isolates cannot be considered to be the cause of their impaired transmission.A different scenario was observed when non-BSE related prions were inoculated into the same three mouse models. While all BSE-derived prions were transmitted with a 100% attack rate irrespective of their producing host, the sheep-scrapie SC-UCD-99 and the mouse-RML isolates showed prolonged incubation times and reduced attack rates on primary passage to BoPrP-Tg110 mice; and the human-sCJD isolate was not transmitted to BoPrP-Tg110 . Moreoveres biochemical signatures  in the brains of infected mice  were indistinguishable from those previously reported for both cattle-BSE and sheep-BSE isolates in this porcine-PrP mouse model Similarly, in PoPrP-Tg001 we found a similar and unique neuropathological phenotype for all BSE-derived agents irrespective of their producing host. In all cases, the vacuolation profile and the PET blot PrPC amino acid sequence or to other host factors. Through the biochemical and biological characterization of the BSE-derived prions tested herein  in comparison to other non-BSE related prions sharing the same PrPSc amino acid sequences, we can try to determine the role of these elements  in either the species barrier phenomenon or the species barrier passage consequences.Once the species barrier is crossed, both biochemical and biological properties of prion strains can change Sc variability Sc depending on the host PrP sequence in which the BSE agent was propagated. The only biochemical feature that was apparently conserved in all BSE-derived prions after passage in the different species was the lack of reactivity of PrPres to the N-terminal binding antibody 12B2 . These results support the statement that the alteration of the PrPSc biochemical features induced by trans-species transmission is only dependent on the host PrPC amino acid sequence and not influenced by the PrPC expression level or other host genetic factors.Similar changes of the abnormal PrPSc sequence. In contrast, a clear transmission barrier was observed with non-BSE related isolates propagated in hosts with an identical PrPC sequence. Moreover, these prion isolates, sharing identical PrPSc but from different strains, also showed a different transmission ability in the other two mouse models (PoPrP-Tg001 and HuPrP-Tg340) tested herein  increased its transmission efficiency in the three mouse models tested herein  compared to other species-PrPBSE including the bovine, murine or porcine one. Nevertheless, the PrPSc primary sequence influence seems to depend strongly on the strain involved, since the sheep-scrapie isolate was not transmitted to HuPrP-Tg340 or PoPrP-Tg001 mice and it was poorly transmitted to BoPrP-Tg110 mice  their transmission efficiency to several species could impact on the trans-species transmission risk under natural exposure conditions. This should always be kept in mind for risk assessment of the potential spread of non-bovine BSE in field cases, since there is a risk that species other than cows with no, or a low, transmission barrier will propagate BSE infection in several other species, including humans.


TOC Summary: An epidemic agent could have originated from such a cattle prion. res), named high-type (BSE-H) and low-type (BSE-L), that also differed from classical BSE. We characterized 5 atypical BSE-H isolates by analyzing their molecular and neuropathologic properties during transmission in transgenic mice expressing homologous bovine prion protein. Unexpectedly, in several inoculated animals, strain features emerged that were highly similar to those of classical BSE agent. These findings demonstrate the capability of an atypical bovine prion to acquire classical BSE–like properties during propagation in a homologous bovine prion protein context and support the view that the epidemic BSE agent could have originated from such a cattle prion.Bovine spongiform encephalopathy (BSE) and BSE-related disorders have been associated with a single major prion strain. Recently, 2 atypical, presumably sporadic forms of BSE have been associated with 2 distinct prion strains that are characterized mainly by distinct Western blot profiles of abnormal protease-resistant prion protein (PrP Sc) of the host-encoded physiologic prion protein (PrPC) in the central nervous system. PrPSc but not PrPC is partially resistant to digestion by proteinase K, resulting in an N terminally truncated prion protein termed PrPres that can be detected by Western blot and showing a characteristic banding pattern that reflects the 3 PrPres glycoforms. The apparent molecular masses and relative quantities of these glycoforms are used in biochemical PrPres typing as the criteria to differentiate between prion diseases.Transmissible spongiform encephalopathies, or prion diseases, are a group of neurodegenerative disorders that include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and goats, and bovine spongiform encephalopaty (BSE) in cattle. Prion diseases are characterized by specific histopathologic lesions and deposits of an abnormal conformational isoform  and low-type (L-type) according to the electrophoretic migration of the unglycosylated PrPres, which is higher (BSE-H) or lower (BSE-L) than classical BSE (BSE-C)  PrPres, more obvious in L-type BSE  led to emergence of a clearly distinct prion with strain features similar to those of the BSE-C agent and that such similarities were maintained on subsequent passages. These observations provide new insights into the nature of the events that could have led to the BSE epizootic.Tg110 transgenic mice in all inoculation experiments. This mouse line expresses bovine PrPC (≈8× that of the level of PrPC in cattle brain) under the control of the mouse prnp gene promoter in a mouse PrP0/0 background  were inoculated with 20 µL of the appropriate sample in the right parietal lobe by using 25-gauge disposable hypodermic syringes. UNO MICRO ID-8 ISO transponders  were used for individual identification of mice. After inoculation, mice were observed daily and their neurologic status were assessed 2×/wk. When progression of the disease was evident, animals were euthanized. All animals were housed in accordance with guidelines of the Code for Methods and Welfare Considerations in Behavioral Research with Animals of the European Union directive 86/609EC). Necropsy was performed, and brain and spleen were taken. Part of the sample was frozen at −20°C for biochemical analysis, and the remaining part was fixed for histopathologic studies.Survival times were calculated for each inoculum as the time between inoculation and euthanasia in days and expressed as the mean of the survival days postinoculation (dpi) of all the inoculated mice with its correspondent standard error of the mean. Data were processed by using SigmaPlot 2001 software .156YEDRYYRE163 epitope, and Saf84 recognizes 171QVYYRPVDQYS181 epitope and 12B2 recognizes 101WGQGG105 epitope of the bovine PrP sequence. Immunocomplexes were detected by horseradish peroxidase–conjugated antimouse immunoglobulin G . Immunoreactivity was visualized by chemiluminescence (Amersham Pharmacia Biotech) and obtained after exposition with medical radiographic film .Frozen mouse brain samples were prepared as 10% (wt/vol) homogenates in 5% glucose in distilled water in grinding tubes  by using a TeSeE Precess 48 homogenizer (Bio-Rad) following the manufacturer’s instructions. All samples were analyzed by Western blot by using the kit TeSeE Western Blot 355 1169 (Bio-Rad) but with some adjustments for the different amounts of samples used. To achieve the volume proposed in the manufacturer’s recommendations, 100 μL of the brain homogenates to be tested were supplemented with 100 μL of a 10% brain homogenate from PrP null mice  induced a typical neurologic disease on primary transmission, with a 100% attack rate . Moreover, the survival time of mice infected with these 2 isolates was reduced on subpassage, approaching that for BSE-C or BSE-H isolates of presumably higher titer .The transmission dynamic of BSE-H agent into ack rate . Remarkares by Western blot analysis with Sha31 mAb. Consistent with the efficient transmission observed, PrPres was readily detected from the first passage in all Tg110 mice inoculated with the different BSE-H isolates. In 3 BSE-H isolates , the totality (100%) of the inoculated Tg110 mice produced a PrPres profile similar to that in cattle (H-type PrPres) but clearly distinct from that produced by BSE-C agent (C-type PrPres) in cattle  apparent molecular mass of the 3 PrPres glycoforms, which was indistinguishable from that produced by BSE-C agent in these mice (res with other antibodies showed that the PrPres produced by these mice was not recognized by 12B2 mAb (res produced by other mice (inoculated at the same time with the same isolates) remains essentially similar to that in cattle BSE-H  of cattle BSE-H but showed a PrPres profile (3 bands) similar to that of BSE-C in Tg110 mice (We observed a different situation for the other 2 BSE-H isolates (02-2695 from France and 45 from Poland), where 3 and 2, respectively, of infected mice  showed an cattle , panel B12B2 mAb . Howeverle BSE-H . Further110 mice .res increased in comparison with the mice with H-type features, as shown by using Sha31 mAb  PrPres molecular profile to obtain equivalent PrPres signals  or when compared with second passages of BSE-C in these mice. All the mice inoculated with the H-type brain homogenates showed H-type PrPres features, whereas all the mice inoculated with the C-like brain homogenates exhibited C-type PrPres molecular features indistinguishable from that of BSE-C (data not shown).For these 2 isolates, a second passage was performed in e PrPres . SurvivaSc distribution in the brain, which are known to vary by strain  apparent molecular mass of PrPres, 2) PrPres glycosylation pattern, 3) immunoreactivity with 12B2 mAb, and 4) pattern of labeling with Saf84 antibody. Second, the vacuolation profile essentially overlapped that in mice infected with BSE-C, with slight differences only in the mesencephalic tegmentum area. Third, the spatial distribution of PrPres in the brain was clearly similar to that of mice infected with BSE-C. Fourth, PrPSc was consistently detected in the spleen, similar to mice infected with BSE-C. These similarities with BSE-C were fully retained after a second passage by using brain homogenate from mice with C-like features, whereas a BSE-H strain phenotype was maintained in mice inoculated with mouse brains homogenates containing H-type PrPres.Although all BSE-H–inoculated mice showed homogeneous survival times, a phenotypic divergence was observed in a few animals infected with 2 of the BSE-H isolates. Surprisingly, these few mice showed phenotypic features clearly distinct from those in most of the BSE-H–infected mice but similar to those of BSE-C propagated onto the same mice, according to various criteria. First, a PrPres profiles of >100 Tg110 mice inoculated with 4 different BSE-C isolates , the lack of such observation in the other 3 isolates, and in 2 other independent studies of 3 BSE-H isolates in different bovine transgenic mouse lines  amount or regions of cattle brain tissue taken for inoculum preparation, 2) physicochemical treatment during inoculum preparation , 3) the precise site of mouse inoculation, 4) the infectious titer of the inoculum, and 5) others unknown mouse factor affecting prion propagation and disease evolution. Because samples used in this study were prepared from the same region (brainstem) following the same precise protocol and under identical conditions, differences in inoculum preparation and conditions are unlikely. However, the possibility that the observations might be influenced by the precise neuroanatomic origin of the inoculated bovine brainstem homogenate or by other mouse bioassay–related factors cannot be excluded.Sc in the BSE-H inocula used for transmissions as deduced by Western blot analysis; and 3) 2 independent transmission experiments, involving separate batches of both incriminated isolates, all produced consistent results.The possible cross-contamination of the BSE-H isolates material (02-2695 and 45 from 2 laboratories in different countries) by a BSE-C infectious source was judged highly improbable for several reasons. These reasons are 1) the strict biosafety procedures followed for sample collection, preparation of the inocula, inoculation scheme, and care of mice; 2) the absence of C-type PrPTg110 mice. Second, a new strain component has been generated during propagation of BSE-H agent in Tg110. In both instances, emergence of the new strain, either in the original cattle or during propagation in Tg110 mice, could be promoted by specific propagation conditions or by physicochemical treatment of the inoculum. In this regard, acquisition of novel properties by a sporadic cattle transmissible spongiform encephalopathy agent by a physicochemical treatment, such as that applied to carcass-derived products, has been invoked as a possible origin for the BSE epidemic (Together, these observations support 2 possible hypotheses. First, a minor strain component might be present in BSE-H isolates that could emerge on subsequent transmission in –Contrary to BSE-H, the atypical BSE-L agent retained unique and distinct phenotypic features, compared with BSE-C agent, on transmission to both bovine and human PrP transgenic mice (In contrast, our results suggest that prion strain divergence might occur on propagation of atypical BSE-H in a homologous bovine PrP context and that this strain divergence could result from a permanent strain shift of the BSE-H agent toward a C-like agent that is stable in subsequent passages. These findings emphasize the potential capacity of prion diversification during propagation, even in the absence of any species barrier, and represent an experimental demonstration of the capability of an atypical, presumably sporadic, bovine prion to acquire C-like properties during propagation in a homologous bovine PrP context.Results in transgenic mouse models cannot be directly extrapolated to the natural host. However, our observations are consistent with the view that the BSE agent could have originated from a cattle prion, such as BSE-H, and provide new insights into the nature of the events that could have led to the appearance of this agent.