Creutzfeldt-Jakob disease (CJD) is the most frequent human Prion-related disorder (PrD). The detection of 14-3-3 protein in the cerebrospinal fluid (CSF) is used as a molecular diagnostic criterion for patients clinically compatible with CJD. However, there is a pressing need for the identification of new reliable disease biomarkers. The pathological mechanisms leading to accumulation of 14-3-3 protein in CSF are not fully understood, however neuronal loss followed by cell lysis is assumed to cause the increase in 14-3-3 levels, which also occurs in conditions such as brain ischemia. Here we investigated the relation between the levels of 14-3-3 protein, Lactate dehydrogenase (LDH) activity and expression of the prion protein (PrP) in CSF of sporadic and familial CJD cases. Unexpectedly, we found normal levels of LDH activity in CJD cases with moderate levels of 14-3-3 protein. Increased LDH activity was only observed in a percentage of the CSF samples that also exhibited high 14-3-3 levels. Analysis of the PrP expression pattern in CSF revealed a reduction in PrP levels in all CJD cases, as well as marked changes in its glycosylation pattern. PrP present in CSF of CJD cases was sensitive to proteases. The alterations in PrP expression observed in CJD cases were not detected in other pathologies affecting the nervous system, including cases of dementia and tropical spastic paraparesis/HTLV-1 associated myelopathy (HAM/TSP). Time course analysis in several CJD patients revealed that 14-3-3 levels in CSF are dynamic and show a high degree of variability during the end stage of the disease. Post-mortem analysis of brain tissue also indicated that 14-3-3 protein is upregulated in neuronal cells, suggesting that its expression is modulated during the course of the disease. These results suggest that a combined analysis of 14-3-3 and PrP expression pattern in CSF is a reliable biomarker to confirm the clinical diagnosis of CJD patients and follow disease progression. PRNP) and represent in average 10% of all cases, whereas less than 1% of CJDs are infectious, highlighting new variant CJD, which is most likely associated with the consumption of cattle affected with bovine spongiform encephalopathy PRNPRES, a misfolded and protease-resistant form of the normal cellular prion protein (PrPC) RES in blood and CSF samples of CJD cases may represent a better indicator for diagnosis as previously reported based on the fact that alterations in this protein are the main cause of the disease C is present in high levels in CSF of healthy people, but lower concentrations of PrPC are found in CSF of CJD patients Protein misfolding is a common hallmark of several neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), as well as Prion-related disorders (PrDs), among other pathologies C levels in CSF in a large group of CJD patients. We found dramatic changes in the overall quantities and glycosylation pattern of PrP, which directly correlated with disease progression. Our data suggest that monitoring the pattern and levels of PrPC in combination with measurements of 14-3-3 level in CSF of CJD patients may be used as a predictive biomarker for CJD diagnosis and progression.In this study, we report the analysis of PrP14-3-3 levels were monitored in CSF samples from CJD patients and control subjects by Western blot analysis , followeC levels was observed in CJD-derived samples . Interestingly, this comparison revealed that the two bands present in CSF have a similar electrophoretic migration to the mono- and di-glycosylation PrP forms . SamplesRES in brain. We treated CSF samples from control and CJD patients with different concentrations of proteinase K (PK). With this method no PrPRES species were detected even with very low levels of PK  over time at different disease stages . The firOverall the analysis of the PrP expression pattern over time showed robust and sustained changes . In contTo confirm the CJD diagnosis, we performed autopsies on two patients (CJD-5 and CJD-6) with complete histological analysis of the brain. The areas usually affected in CJD  are shown in RES conformations in CSF samples of the CJD cases This work was performed as part of a coordinated effort to develop a centralized diagnosis and surveillance of Chilean patients suffering from a prevalent human PrD form. Here, we present a full study that examines potential CSF biomarkers for CJD that can be used in combination with 14-3-3 protein. We monitored the levels of 14-3-3 protein, LDH activity and different aspects of PrP biology in the CSF of patients with the clinical diagnosis of CJD. The general consensus in the field indicates that although CSF 14-3-3 levels are not specific, in concert with characteristic clinical features, it may be used for the diagnosis of CJD. Here we report that total PrP levels combined with changes in its glycosylation pattern mirrors the disease progression in CJD. This is a significant finding since altered physicochemical properties of PrP underlie all PrDs. It is possible that the PrP expression pattern in CSF reflects the molecular events affecting PrP structure in the brain. Of note, an inverse relationship between the amount of 14-3-3 protein and total levels of PrP in the CSF was observed. When PrP levels were monitored in the same CJD patient over time, we observed a progressive decline in PrP levels paralleled by changes in the glycosylation pattern. As previously observed, PrP in CSF of CJD patients and non-CJD patients is sensitive to PK digestion C and the cell types responsible for maintaining its levels in CSF are unknown. Some studies have shown that the soluble form of PrP participates in signal transduction, cell survival and protection against oxidative stress The biological function of soluble PrPDespite initial predictions, we found normal levels of LDH activity and total protein concentration in CSF from confirmed CJD cases. Interestingly, post-mortem analysis revealed that, although there was a marked reduction in neurons in affected areas in CJD patients, 14-3-3 protein expression was significantly upregulated in brain tissue. Taken together, our results support the new concept that increased CSF 14-3-3 levels may reflect the induction of a cellular response against prion rather than cell lysis. In agreement with this hypothesis, it has been reported that some 14-3-3 protein isoforms may play a role in synaptic function Changes in the glycosylation pattern of PrP in CSF may reflect alterations in the protein maturation pathways during its synthesis. PrP is generated and folded in the endoplasmic reticulum (ER) where it is subjected to several post-translational modifications, and then is transported to the Golgi apparatus to undergo maturation by glycosylations to be further exported to the plasma membrane To our knowledge, this study is the first report with comparative measurements of 14-3-3 protein, PrP levels and glycosylation pattern in CSF of CJD patients following these changes at different stages of disease progression. Our results suggest that analysis of 14-3-3 protein level and PrP expression pattern in CSF samples may be a reliable biomarker for diagnosis of CJD.CSF samples were collected from 46 patients diagnosed with CJD  and 16 anesthesia controls of healthy individuals . SamplesIn routine practice, a CSF analysis is performed in all patients with the suspected diagnosis of CJD. All patients gave written informed consent to participate in the study as part of a secondary analysis after 14-3-3 levels were tested as a diagnostic practice. The study was conducted according to the provisions of the Helsinki Declaration, and was designed in accordance with the relevant Chilean legislation and was carried out with the approval of the Ethics Committee of the El Salvador Hospital, Santiago, Chile. All samples were manipulated in a level II biosecurity facility. Histological analysis of human post mortem samples was approved by the Ethics Committee of the Faculty of medicine of the University of Chile and FONDECYT funding agency (protocol number CBA #0323 FMUCH).CSF samples were collected in polypropylene tubes, centrifuged at 2500 rpm for 5 min to eliminate possible contamination with cells or tissue, and the supernatant was stored at −80°C prior to analysis. For this study the presence of 14-3-3 protein, PrP and active LDH was analyzed. 14-3-3 and PrP protein levels in CSF were measured after analysis of 20 µl of sample by Western blot analysis using standard methods Bacillus thermoproteolyticus rokko (Sigma–Aldrich). Proteolysis was stopped by adding SDS-sample buffer and heating for 5 min at 95°C. For deglycosylation, 20 µl of CSF samples were treated with N-glycosidase F (PNGase F) (Biolabs) following manufacturer's recommendations. Briefly, after addition of denaturation buffer CSF samples were incubated for 5 min at 95°C. Samples were cooled to 25°C, and then the reaction buffer and 10 U of N-glycosidase F (PNGaseF) (BioLabs) was added. After 1 h at 37°C, sample buffer was added and samples were heated for 5 min at 95°C. Samples were analyzed by Western blot. As a positive control, a human post mortem sample derived from brain cortex of a healthy control individual was analyzed. Frozen brain tissue was obtained from the Harvard Brain Tissue Resource Center  and then processed for biochemical analysis by homogenization of equivalent amounts of tissue in PBS containing protease and phosphatase inhibitors with further dilution in RIPA buffer.PK assays were performed using an adapted protocol we previously described 2O2 in methanol for 30 min at room temperature to inhibit endogenous peroxidase activity. After washing two times with TBS IHC wash buffer for 5 min, sections were blocked with 0,3% normal horse serum for 30 min at room temperature, followed by incubation with anti-14-3-3 (1∶1000) and mouse monoclonal anti-PrP (3F4) (1∶1000) in a humidified chamber at 4°C overnight. Negative control sections were incubated with a negative control reagent (Dako) instead of primary antibodies. After washing two times with TBS IHC wash buffer for a total of 5 min, the sections were incubated with biotinylated secondary antibody for 30 min at room temperature, and rinsed two times with TBS IHC wash buffer for a total of 5 min, followed by incubation with an ABC kit (Vector) for 30 min at room temperature. After rinsing with, TBS IHC wash buffer, peroxidase labeling was visualized with DAB  for 3 min at room temperature. Sections were rinsed in tap water for 10 min, dehydrated, cleared and mounted.Ten micrometer-thick sections were obtained from formalin fixed, paraffin embedded blocks of the brains of CJD and control patients. The paraffin-embedded sections were deparaffinized in xylene, followed by rehydration in a decreasing concentration of ethanol solutions. For routine pathological examination, deparaffinized sections from all blocks were stained with hematoxylin and eosin stains and Luxol Fast Blue. Sections for immunohistochemistry were incubated in 10 mM citrate sodium buffer (pH 6.0) and heated in a microwave oven three times for 5 min for antigen recovery. Washed in TBS IHC wash buffer and treated with formic acid for 5 min and washed again. Sections were pretreated with 0.3% Hp<0.05; ** p<0.01; *** p<0.005; n.s.: non significant. For the statistical analyses SigmaPlot and GraphPad software were employed.Data was analyzed by parametric t-test (two-tailed) and significance was expressed as follow: *


The alpha-radioactive polonium 210 (Po-210) is one of the most powerful carcinogenic agents of tobacco smoke and is responsible for the histotype shift of lung cancer from squamous cell type to adenocarcinoma. According to several studies, the principal source of Po-210 is the fertilizers used in tobacco plants, which are rich in polyphosphates containing radio (Ra-226) and its decay products, lead 210 (Pb-210) and Po-210. Tobacco leaves accumulate Pb-210 and Po-210 through their trichomes, and Pb-210 decays into Po-210 over time. With the combustion of the cigarette smoke becomes radioactive and Pb-210 and Po-210 reach the bronchopulmonary apparatus, especially in bifurcations of segmental bronchi. In this place, combined with other agents, it will manifest its carcinogenic activity, especially in patients with compromised mucous-ciliary clearance. Various studies have confirmed that the radiological risk from Po-210 in a smoker of 20 cigarettes per day for a year is equivalent to the one deriving from 300 chest X-rays, with an autonomous oncogenic capability of 4 lung cancers per 10000 smokers. Po-210 can also be found in passive smoke, since part of Po-210 spreads in the surrounding environment during tobacco combustion. Tobacco manufacturers have been aware of the alpha-radioactivity presence in tobacco smoke since the sixties. WHO has declared a tobacco epidemic, indicating the spread of smoking dependency, which affects 1.3 billion people worldwide and results in 5.4 million tobacco-related deaths each year. If this trend continues, there will have been 10 million deaths by 2030 [Every year, approximately 11 million people are diagnosed with cancer worldwide; 8 million of them will die . Cancer Tobacco smoking is a risk factor for six out of eight main death causes all over the world; with lung cancer being one of the six causes, tobacco represents the most important one , 7. EachAlmost 46% of new cases of nonsmall-cell lung cancer pertain to the IIIB and IV stages . In EuroMerck Manual, which was published in 1889, suggesting that smoking could be used for the treatment of bronchitis and asthma [Tobacco smoke has been known to be harmful for health since the fifties , 18. In d asthma .In 1912, the very first cause/effect hypothesis between lung cancer and tobacco smoking was made in a research monograph by Adler , 21. In In 1914, Kellogg stated in a public health report that cancer killed 75000 people in the US each year, corresponding to 1 out of 20 deaths, and he noted that domestic animals were affected by cancer more frequently than humans, probably because of the indoor pollution deriving from combustions and tobacco smoking .Almost two decades later, Dr. James Gilmore, a 48-year-old gynecologist from Pittsburgh, underwent the first successful left pneumonectomy for carcinoma. The operation was carried out by Dr. Evarts A. Graham, a pioneer in thoracic surgery –25. NearBefore the Second World War, experimental research on carcinogenesis from tar and polycyclic hydrocarbons was begun by an Argentinean researcher, Roffo –30. ManyJAMA [On May 27, 1950, Ernest L. Wynder from the Sloan Kettering Institute and Evarts A. Graham published the first scientific paper on tobacco smoking as a possible etiological factor for bronchogenic carcinoma in JAMA . 684 patBritish Medical Journal in 1961 and confirmed the relationship between smoking and lung cancer [In 1951, Richard Doll and Bradford Hill started the first extensive prospective epidemiological study, which was published in g cancer . The autReader's Digest, which had a large circulation at the time, featured an article with the provocative title “Cancer by the Carton,” in which the role of cigarette smoke in lung cancer was described as “a medical controversy… largely kept from public notice.” [Frank Statement,” which aimed at contrasting the evidence implicating smoking as a health issue [A year later, notice.”  The artith issue . This doSurgeon General's report that addressed the consequences of tobacco smoke for public health was released [A decade later, the first released . By thenThe second part of the twentieth century saw a rapid increase in this disease leading to a lung cancer epidemic, especially in males of the developed countries , 37. In Even though the carcinogenetic mechanisms of tobacco smoke are not fully explored , only veBesides well-known organ-specific carcinogenic substances, such as polycyclic aromatic hydrocarbons, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 2-Naphthylaine, 4-aminobiphenyl, arsenic, and chromium, there is another one, which has recently been involved in the spy case of Litvinenko: Polonium 210 (Po-210).Polonium, also called “radium F,” was discovered by Marie and Piere Curie in 1898 and was named after the home land of Curie-Sklodowska. For the discovery of radium and polonium Marie Curie received a Nobel Prize in Chemistry in 1911 , 51. The−4% of the Earth's crust [α-particle emitter (5.3 MeV), but it can also emit gamma photons with energy 803 keV and emission probability of nearly 1 × 10−5 [Polonium is a fairly volatile metal, rarely found in nature in pitchblende containing rocks, and constitutes 2.1 × 10's crust . The maj's crust . This is's crust , and can1 × 10−5 , 56. It −6 μg [Polonium is a highly toxic element, with elevated specific radioactivity, and is dangerous to handle even in milligram amounts. The maximum allowable body burden for ingested Polonium is 1100 Bq, which is equivalent to a particle weighing only 6.6 × 10−6 μg .Alpha rays, which are formed by helium 4 (He-4) nucleus, are the least penetrating type of radiation and they manage to travel only a few centimeters in air. They can be easily stopped by obstacles, such as a sheet of paper, and they can penetrate living tissues by only a few microns , 58, 59.In addition, alpha rays are highly ionizing and, therefore, are particularly harmful for living tissues. 1 mg of polonium can emit as many alpha particles as 5 grams of radium. The impact on humans can be devastating, as it can cause considerable damage by causing cell death, promoting a massive, progressive, and rapid necrosis, and not allowing the organism enough time to replace the quantity of dead cells .Po-210 use is rather limited due to its high alpha radiation emissions and the difficult extraction process. The main uses are (a) as a resource of neutrons when it is mixed with beryllium, (b) as an energy resource for satellites and other space devices, (c) in antistatic devices of some precision instruments and in brushes that eliminate dust gathered on photographic film, and (d) in devices that eliminate static charges in textile mills, though less dangerous beta-ray sources are now more widely used .Traces of Po-210 can be found in many plants and foods and consequently, in human tissues as well , 61. TheThe discovery of the presence of Po-210 in tobacco smoke dates back to the early sixties, thanks to the work of Turner et al. , MarsdenThrough the plant's roots, directly from terrain that contains uranium –69.Coating on leaves as a result of meteorological events, rain, snow, and environmental dust. In fact, Radon-222, a product of U-238 decay, is a noble and volatile gas that can partially escape from terrain into the atmosphere and create Pb-210 and Po-210. These are absorbed by atmospheric dust, creating the Aitken particles that consequently lie on the leaves. The numerous trichomes of tobacco plants resemble filamentous pores and are metal accumulators, particularly of Pb-210 and Po-210. The quantity of the latter will then increase, as there is further Pb-210 decay , 71. FleOn the other hand, the majority of authors, such as Singh and Nilekani, have identified the importance of the fertilizers employed . Calcium−5), gamma, beta, and X emitter. All these inhaled particles are deposited in the broncho-pulmonary apparatus and particularly in segmental bronchi bifurcations, due to ciliary action. According to measurements by Cohen et al., radium and thorium are also present in cigarettes; however, 99% of the radioactivity comes from Po-210 [The journey of Po-210 and Pb-210 towards bronchopulmonary apparatus starts by lighting a cigarette. In this combustion chamber, tobacco burns, reaching 800–900°C when inhaling, and smoke is created, which is composed of a corpuscular (5%) and a gas phase (95%) . Po-210 m Po-210 , which rm Po-210 .All these particles have a different “destiny” based on the efficacy of the mucous-ciliary clearance. This mechanical purification is reduced gradually in smokers with COPD, resulting in the accumulation of insoluble Pb-210 particles, which decay to Po-210 over time , 77. In Subsequently, radioactive particles reach various organs and tissues through pulmonary and systemic circulation and cause mutations of the genetic cellular structure, deviations of the standard cellular characteristics, accelerated ageing, and quicker death due to a wide range of diseases , 79. In Polonium radiation in the bronchial epithelium depends not only on the particle concentration of these areas, but also on the time of their permanence. Half-life of polonium is 138.38 days and of lead 22 years, which decays afterwards into polonium. There is a significant cancer risk due to chronic exposure to low levels of insoluble alpha-emitting particles , 85, whiPo-210 alpha radioactivity in tobacco smoke depends on several variables: geographic region of tobacco growth, storage time and modality, presence of a filter, its length and composition, and the way of smoking . FurtherRadford and Hunt  and MussProfessor Gattavecchia from the Complex Unit of the Institute of Chemical, Radiochemical, and Metallurgic Sciences of University of Bologna (SMETEC), in association with ENEA  and the Italian Society of Tobaccology (SITAB), have conducted various studies on the alpha radioactivity of Po-210 in tobacco smoke. It has been confirmed that a cigarette with tobacco of Western origin emits 75 mBq of alpha radioactivity from Po-210, distributed in mainstream , sidestream , and ash  –100.Many studies have already reported that second-hand smoke is an important risk factor for lung cancer. After studying 91540 people for 14 years, in 1981 Hirayama demonstrated the lung cancer mortality of non-smoker wives or husbands was one-third higher compared to those with non-smoker partners .This increased risk was also confirmed by a vast analysis of two case-control studies conducted in the US and Europe, in which a dose-response relationship between lung cancer risk and prolonged exposure to second-hand smoking has been found among partners, in workplaces and in public places. Risk for one-off exposure to spousal smoking increased by 18% (95% CI = 1–37%) and for long-term exposure by 23% (95% CI = 1–51%). Augmented risk for long-term exposure was also found for the work place  and public places  .It should be considered that passive smokers are exposed to the same components as active smokers, including radioactive elements. As a matter of fact, Po-210 in second-hand smoke is 50–70% the quantity found in active smoke. Moreover, passive smokers are exposed to environmental pollution from radon, as well as from Po-210 of cigarette smoke, both of which increase lung cancer risk , 104.Po-210 is also present in narghilé smoke. An international multidisciplinary team , coordinated by Khater et al., has recently published a pioneering study on narghilé  tobacco radioactivity . Before The research was based on the measurement of some natural radionuclides activity and the estimation of the internal radiation dose due to narghile tobamel (moassel) smoking. Tobamel is a fashionable flavoured tobacco-molasses mixture (with added glycerol) currently used in narghilé. However, there are other forms such as jurak, similar to tobamel, but unflavoured, containing minced fruits and no glycerol . It is aμSv/y (average 79.7 μSv/y) for Ra226; 19.3 to 116.0 μSv/y (average 67.1 μSv/y) for Ra-228; 47.0 to 134.9 μSv/y (average 104.7 μSv/y) for Pb-210. In sum, the order of magnitude was the same for each radionuclide. The sum of effective doses of the three radionuclides varied from 151.9 to 401.3 μSv/y (average 251.5 μSv/y). Notably, the annual effective dose from Cs137 of Chernobyl origin was three orders of magnitude lower as it varied from 70.4 to 410.4 nSv/y (average 199.3 nSv/y) [Another recent study on the radioactivity of Greek tobacco leaves used for cigarettes showed that the annual effective dose due to inhalation by adult smokers varied from 42.5 to 178.6 3 nSv/y) .The results of Khater et al., found that the radioactivity concentration in tobacco products basically depends on the tobacco content itself, not on other ingredients such as molasses, glycerol, or fruits. Interestingly, the lower yield of Po-210 in jurak might be in relation with the Indian origin of this smoking paste. The reason might be that Po-210 alpha-radioactivity of Indian tobacco would be several times lower than that of Western tobacco .Eighty-five to ninety out of a hundred lung cancers are caused by tobacco smoke; nevertheless, less than 20% of smokers get lung cancer . If indiMany factors could influence individual susceptibility to lung cancer in smokers. Polonium is among them, albeit it is still less considered or even ignored as a carcinogenic substance, which is also due to years of concealing by tobacco manufacturers . As a maPolonium 210 emits alpha particles, which have a penetration limit of about 40 microns or less in animal tissue, but a very high damaging effect , 58, 59.DNA chromosome damage by exposure to alpha radiation is 100-times greater than the one caused by other types of radiation . Little Black and Bretthauer reported that Po-210 radiation dose in heavy smokers was up to 82.5 mrad (0.83 mSv) per day . RadfordMany lung cancers are adenocarcinomas, a type of lung cancer that Po-210 inhalation can induce in laboratory animals . KennedyBoffetta et al. recently reviewed seven case-control studies and estimated that the odds ratio of BAC for smoking at all was 2.47 (95% CI = 2.08–2.93). The authors also reported that the risk increased linearly with duration, amount, and cumulative cigarette smoking and persisted long after smoking cessation .In a recent study, Prueitt et al. tried to explain the way alpha radiations affect DNA . Ionizin−4 year−1 . This estimate does not take into account the promoter role of Po-210 (cocarcinogen) in the bronchopulmonary cancer and the overall carcinogenic activity of all substances [But what is the level of biological damage caused by tobacco smoke Po-210? Estimating the damage is a very difficult and complicated task. Using the 1990 ENEA data on the average time of Po-210 presence in lungs, which is 53 days , the datbstances .To render the biological harm deriving from Po-210 in smoke more comprehensible, it has been compared to the damage caused by radiation in conventional chest X-rays. Since the dose of a modern chest radiograph is 0.034 mSv , 126, a There is evidence that in the last 40 years a histotype change of lung cancers has been noticed, shifting from squamous cell carcinoma to adenocarcinoma, in which the bronchial-alveolar (BAC) subtype is also included . The aboThe factors that have induced this shift are various and perhaps not all known. Nevertheless, almost all of them are linked to the tobacco cultivation and cigarette manufacture changes since the fifties. The most common are as follows.The utilization of different varieties of tobacco in the US cigarette blends. This change reduced benzopyrenes in smoke, but produced an increase of nearly 50% in nicotine-derived nitrosaminoketone (NNK) in the last quarter of the twentieth century , 134.The introduction of low-tar, low-nicotine, filtered cigarettes since the mid fifties, which seems to have contributed to the overall decline in lung cancer and the upward trend in the incidence of adenocarcinoma –138. Somμm, in a year's time [Interestingly, in the fifties the race for the safer filter led to a severely dangerous incident. Lorillard produced 13 billion cigarettes from 1952 through 1956 based on a filter composed of asbestos and cotton fibers. Each filter contained 10 mg of crocidolite, the fibers of which could be found in the mainstream smoke from the first two puffs. Consequently, a person smoking a 20-cigarette pack each day could inhale more than 131 million crocidolite fibers, which were longer than 5 r's time . When prThe massive introduction of polyphosphate fertilizers in tobacco cultivations, contributing alpha radiation (from Pb-210 and Po-210) and TSNAs significantly , especiaRegulating and reducing this harmful radiation, which comes from fertilizers, could help reduce lung cancer incidence . TobaccoUse of alternative polyphosphate sources, such as organic fertilizers from animals .Use of ammonium phosphate as a fertilizer, instead of calcium phosphate .Different storage methods. A study proved that Po-210 radioactivity of tobacco rose over time while in storage . As a coGenetic modifications of tobacco plants with significant reduction of trichomes concentration on the leaves, on which Pb-210 and Po-210 accumulate .Resin filters may decrease lung exposure to alpha radiation . On the LaRock et al. recommended a biological way to remove Po-210 by treating polyphosphate rocks with bacteria capable of reducing sulphates .Perhaps the simplest and most applicable solutions would be the quantitative decrease in polyphosphates use in tobacco cultivations and the regulation of the maximum acceptable level of alpha radiation of cigarettes, which should also be clearly indicated on the packet .waking a sleeping giant” [While multinational tobacco manufacturers have been aware of the alpha-radioactivity presence in tobacco smoke since the sixties, they have covered it up strategically. Not by chance, the polonium dossier was symbolically entitled “g giant” .www.pmdocs.com, one can find the lawsuit of the State of Minnesota against Philip Morris Incorporated, et al., in which there are 481 confidential documents and memorandums on the alpha-radioactivity from Po-210 in tobacco smoke (still available on 1 January 2011). The archives bring out the fact that Philip Morris has been aware of the lead and polonium existence in cigarettes since the sixties [waking a sleeping giant” [Among the 37 million documents that were released through the site  sixties , as was  sixties , Radford sixties , and rec sixties . In thes sixties . In factg giant” .In 1980, one confidential memorandum revealed that the issue was mainly caused by calcium phosphates fertilizers employed in tobacco cultivations. Moreover, cigarette manufacturers knew about the studies conducted by Martell , regardiSo far, the majority of public opinion still ignores the presence of polonium radioactivity in tobacco smoke and the serious public health threat that it represents. Yet, from a communicative and motivational point of view, it could become a great opportunity for prevention and smoking cessation. For now, it seems that something has changed in the media and scientific world since the widely covered Litvinenko case and the paper of Muggli et al. . In factPolonium-210 represents one of the principal causes of lung cancer and its shift from squamous cell carcinoma to adenocarcinoma. Provided that it is true that tobacco manufacturers have been aware of the presence of Po-210 in smoke since the early sixties and concealed its existence intentionally in various ways, it is likely that the medical and scientific sector is guilty of having ignored it.It is necessary that the medical and scientific world becomes aware and conscious of this problem, creating systematic educational programs of tobaccology in the university curricula of the medical sciences courses. Likewise, governments should force manufacturers to introduce cigarettes with low Po-210 concentration and place a clear indication about this on the packet in order to reduce smokers' risk.Finally, since people fear everything that is radioactive, perhaps it would be useful to create an adequate information campaign so as to enable and accelerate smokers' motivational pathways and increase the efficacy of anti-smoking programs .


Magnetic resonance imaging (MRI) cardiac gated phase contrast (PC) cine techniques have non-invasively shown the effect of the cardiac pulse on cerebrospinal fluid (CSF) movement. Echo planar imaging (EPI) has shown CSF movement as influenced by both cardiac pulsation and respiration. Previously, it has not been possible to visualize CSF movement in response to respiration non-invasively. The present study was undertaken to do so.The effect of respiration on CSF movement was investigated using a non-contrast time-spatial labeling inversion pulse (Time-SLIP) with balanced steady-state free precession (bSSFP) readout. CSF movement was observed in the intracranial compartment in response to respirations in ten normal volunteers. To elucidate the respiration effect, the acquisition was triggered at the beginning of deep inhalation, deep exhalation and breath holding.By employing this respiration-induced spin labeling bSSFP cine method, we were able to visualize CSF movement induced by respiratory excursions. CSF moved cephalad (16.4 ± 7.7 mm) during deep inhalation and caudad (11.6 ± 3.0 mm) during deep exhalation in the prepontine cisternal area. Small but rapid cephalad (3.0 ± 0.4 mm) and caudad (3.0 ± 0.5 mm) movement was observed in the same region during breath holding and is thought to reflect cardiac pulsations.The Time-SLIP bSSFP cine technique allows for non-invasive visualization of CSF movement associated with respiration to a degree not previously reported. For quite some time, it has been known that CSF movement results from the formation of new CSF and motion of cilia on the surface of the choroid plexus and ependyma lining the ventricles. In the last three decades, MRI cardiac-gated PC cine techniques have been widely applied to study CSF movement non-invasively in the human brain. Using PC cine, CSF “to and fro” movement can be observed by acquiring multiple-cine phases during a cardiac-gated acquisition -7. SinceA relationship between the cardiac and the respiratory influence on CSF flow has been investigated using echo planar imaging (EPI) -10 and oThe cardiac-gated, non-contrast MRI spin labeling, time-spatial labeling inversion pulse (Time-SLIP) with single-shot fast spin echo (SS-FSE) technique has been used to non-invasively observe CSF movement . The TimWe have developed a Time-SLIP method with bSSFP to acquire cine images with adequate spatial and temporal resolution to observe rapid changes in CSF movement. The fast bSSFP readout enables the technique to capture continuous movement of CSF in a shorter temporal resolution time than the previously reported single-shot fast spin echo (SS-FSE) readout .The institutional Review Board at Toshiba Rinkan Hospital, Kanagawa, Japan gave approval for this study and informed consent was obtained from the volunteers. The study group consisted of 10 normal healthy volunteers , who were each imaged in a supine position. The volunteers were instructed to perform maximum deep inhalation, exhalation and breath holding  during which the images were acquired. Respirations or lack thereof were monitored with a chest band detector. The respiratory and cardiac cycle was recorded and the obtained images provided the triggering phase times. Each acquisition was about 6 seconds with the subjects lying supine at rest. Each experiment was repeated three times to observe the reproducibility. Due to the magnet configuration, we were not able to conduct these studies with the subjects being in either a sitting or standing position. None of the volunteers had known pulmonary disease. MRI images of the head and spine were normal in all subjects.A flow-out technique was used in which a non-selective pulse (A) and a selective labeling (tag) pulse (B) with bSSFP cine were applied. Figure 2, parallel imaging factor = 3, and Time-SLIP tag pulse width = 10 – 30 mm. The acquired spatial resolution of 2.5 mm × 1.35 mm was interpolated to 0.68 mm × 0.68 mm. Segmentation of one means acquiring a whole 96 phase encode lines per shot. In our experiments, we achieved a temporal resolution of 134.4 ms with 40 cine phases within a single respiration in a total acquisition window of about 6 sec . Thus, each volunteer was instructed to start deep inhalation, exhalation, or breath holding for an acquisition period of about 6 seconds directed by voice instruction. This allowed the observation of tagged CSF movement over a period from TI of 230 ms to 5.376 ms during cine acquisition. With these parameters we were able to obtain 40 images during the 6 second acquisition time. The tagged plane was placed perpendicular to the image plane allowing observation of tagged CSF movement along the image plane.The Time-SLIP bSSFP cine examinations were performed at Toshiba Rinkan Hospital, Kanagawa, using a 1.5-T MRI scanner , equipped with quadrature-detected -Head SPEEDER phased array coils (5 channels). The bSSFP sequence was acquired in a cine mode with adequate spatial and temporal resolution to observe motion changes of CSF movement. Typical deep respiration  and breath holding acquisition parameters were repetition time (TR) = 4.2 ms, echo spacing = 2.1 ms, segmentation = 1, inversion time (TI) = 230 ms, matrix = 96 (phase encoded) × 192 (read out) (352 × 384 after interpolation), slice thickness =7 mm, FOV = 24 × 26 cm2, and a parallel imaging factor of 2. The acquisition window per image was 528 ms and the experiment was repeated five times. The water velocities obtained using Time-SLIP agreed with the calculated velocities (ground truth) with errors less than 1% (R2 = 0.9966).In order to validate the CSF flow measurement, we conducted a flow phantom study. A 4.5-mm inner diameter tube was used for the phantom and a syringe flow pump maintained a constant volumetric water flow in the tube of 0 to 9 ml/min. The flow velocity was calculated by assuming laminar flow in the tube. The tagging slab was placed perpendicular to the flow to label water spins in the tube. A continuous cine bSSFP sequence was implemented for acquisition. The entire tube was imaged in the slice direction and thus the tagged region and the movement of water was traced in the tube. From each cine image, the movement of water was calculated by a regression calculation. The movement of water using Time-SLIP cine bSSFP was plotted with the peak velocity of the laminar flow. The acquisition parameters for the flow measurement were: TR = 6.6 ms, echo spacing = 3.3 ms, matrix = 160 × 224 (320 × 448 after interpolation), slice thickness = 5 mm, FOV = 18 × 25 cmThe cine acquisition using Time-SLIP with bSSFP cine Figure  allowed Figure p values (student’s t-test) for maximum CSF movement with deep inhalation and deep exhalation are significant when compared to breath holding, p < 0.001 and p < 0.001, respectively.The Our results demonstrate that a substantial amount of CSF movement occurs with deep respiration. During the deep inhalation, significant cephalad CSF movement was observed; whereas, a corresponding caudad CSF movement was noted during deep exhalation. The breath holding data showed a small but rapid to and fro CSF movement. From these observations, there is no reason to believe that “normal breathing” would show CSF movement anything but somewhere between the deep inhalation/exhalation and breath holding.Recently, cardiac-gated PC with bSSFP has been reported to provide better CSF flow quantification by overcoming the intrinsic issues of lower SNR and temporal resolution PC with incoherent gradient echoes . The carThe limitation of this technique is in the temporal resolution or acquisition window of 6 seconds required for either deep inhalation or exhalation, whereas, “normal breathing” only requires about 2 to 3 seconds. This difference reflects an intrinsic delay of several seconds between respiration and CSF movement. Therefore, this method is limited to the deep respirations. Further improvement in cine temporal resolution are required to record in real time the effect of various breathing patterns on CSF movement.A finding of this study was the significant caudad CSF movement during deep exhalation with a corresponding cephalad CSF movement during deep inhalation that visually confirms the Monro-Kellie principle. During deep inhalation, thoracic pressure diminishes followed by negative atrial pressure, resulting in increased return of venous blood from the brain. Cephalad CSF movement may occur to compensate for the blood volume reduction in the brain. Cephalad CSF movement during deep inhalation was observed in both the ventricular system and cranial and spinal subarachnoid spaces. During deep exhalation, intrathoracic pressure increases, followed by positive atrial pressure, resulting in a decrease in the venous return from the brain. Caudad CSF flow may occur to compensate for the blood volume increase in the brain. No CSF pressure measurement was performed in this study to confirm the cause of CSF movement. An advantage of this technique is that by tagging CSF it becomes an endogenous tracer allowing non-invasive visualization of CSF movement under undisturbed conditions. The relationship between brain and spinal venous volume changes during deep respiration and its influence on CSF movement need to be investigated in future studies ,11.et al. in 1966 conducted CSF flow observations using oil contrast medium during pneumoencephalography in humans [Du Boulay n humans . They ton humans . CSF floCSF flow studies in animals are usually conducted with the animal in a prone position under general anesthesia. Likewise, MRI CSF flow studies in humans are done in a supine position, the one exception being a few sitting position studies using a unique MRI machine . Our stuIn summary, we have successfully shown significant CSF movement with deep respiratory excursions. The respiration-induced non-contrast MRI spin labeling, Time-SLIP with bSSFP cine provides non-invasive observation of continuous CSF movement in both the intracranial and intraspinal compartments in response to deep respirations and breath holding in normal volunteers. A significant movement of CSF was observed cephalad during deep inhalation and caudad during deep exhalation. The visualization of CSF dynamics in sequential cine views may provide valuable information and management guidance for patients with pathophysiologic conditions.PC: Phase contrast; RACE: Real time acquisition and evaluation of motion; Time-SLIP: Time-spatial labeling inversion pulse; SS-FSE: Single-shot fast spin echo; bSSFP: balanced steady-state free precession; EPI: Echo planar imaging; SNR: Signal to noise ratio; CSF: Cerebrospinal fluid, IR: Inversion recovery; FOV: Field of view; ROI: Region of interest; T1: Spin lattice relaxation; TI: Inversion time; TR: Repetition time.There are no financial disclosures or ethnical adherences. Seven authors  are employees of Toshiba Medical Systems Corporation. The other authors, who are not employees of Toshiba Medical Systems Corporation, had sole control over the inclusion and evaluation of the study data and any other information that might pose conflict of interest.SY, MM and YM conducted study concept and design. SY, MM, YY, CO, MY, MN, SS, IA, MY, and JGM performed data acquisition. SY, YY, and JGM analyzed and interpreted data result. SY, MM, and JGM wrote the manuscript. All authors revised the manuscript and approved the final manuscript.CSF flow in a sagittal view during deep inhalation. During the early phase (up to 2.5 seconds) of deep inhalation, cephalad CSF flow occurs in the ventricular system and in the subarachnoid space. In the subarachnoid space, cephalad CSF flow was also observed in the prepontine cistern toward the suprasellar cisterns.Click here for fileCSF flow in a sagittal view during deep exhalation. Caudad CSF flow occurs in the ventricular system and in the subarachnoid space during the early phase of exhalation. A substantial volume of CSF moves from the prepontine cisterns toward the ventral spinal subarachnoid space.Click here for fileMid sagittal view during breath holding. Small but rapid cephalad and caudad CSF volume movements were observed during breath holding.Click here for file


Lastly, strong differences appeared between the populations, leisure horses being evaluated as having sounder spines, exhibiting lower sEMG measures and rounder neck than the riding school horses. sEMG measures and neck “roundness” seemed therefore to be reliable indicators of back disorders, easy to evaluate in field conditions. This highlights the accuracy of using postural elements to evaluate the animals’ general state and has important implications for animals’ welfare evaluations.Postures have long been used and proved useful to describe animals’ behaviours and emotional states, but remains difficult to assess objectively in field conditions. A recent study performed on horses using geometric morphometrics revealed important postural differences between 2 horse populations differing in management conditions . It was suggested that these postural differences may reflect chronic effects of riding techniques on the horses’ kinematics and muscular development. In the present study, we tried to evaluate the interest of postural measures to assess welfare in horses. This study was separated into 2 parts. First, 18 horses coming from these 2 types of populations (leisure/riding school horses) were submitted to 2 back evaluations by 1) manual examination (experienced practitioner) and 2) sEMG measures along the spine. We then measured neck roundness on 16 of these 18 horses. The results highlighted high correlations between manual and sEMG examinations over the spine. sEMG measures at the different locations were strongly correlated all over the spine. Moreover, neck postures and muscular activities were strongly correlated, horses with concave necks having higher sEMG measures both at precise locations ( More ree.g. head and tail) and only evaluated by mere visual inspection. The use of anatomical landmarks has made objective and reproducible measures possible but most such studies require highly standardized and artificial situations One main problem however to make postures reliable tools for such an assessment is the difficulty to develop repeatable, objective and comparable measures. Postures are generally characterized on the basis of a few elements  validating the use of sEMG as an alternative to manual evaluation of potential vertebral disorders throughout the axial skeleton, 2) relying neck postures to sEMG measures as reflecting back problems . Two populations, “extremes” in terms of management and riding techniques, were compared as in Fureix Experiments complied with current French laws  related to animal experimentation and were in accordance with the European directive 86/609/CEE. No licence/permit/institutional ethical approval was needed. Animal husbandry and care were under the management of a private owner (study 1) or the riding school staff (study 2). This experiment involved only horses in the “field” . We studied two samples of horses kept under different conditions to investigate the reliability of sEMG measures in reflecting back disorders in study 1 and the relation between sEMG measures and neck postures in study 2.The evaluations were performed on horses, distributed into two groups Fig. 1.The first group corresponded to 9 domestic horses  kept under natural conditions in stable social groups for several years, in 1–2 ha natural pastures, fed grass and hay ad libitum during winter  and used for occasional leisure outdoor “relaxed” riding (with long reins). They lived in 3 groups in the same site (Group 1).The second group corresponded to 9 riding school horses . These horses were kept in 3×3 m individual straw-bedded boxes, fed industrial pellets three times a day and hay once a day, exercised in riding lessons for 4–12 h per week with more constraining techniques. All horses were in the same riding school and had at least one free day per week(Group 2).The evaluation of the study horses’ spine was performed by a 20 years experienced licensed chiropractor who was totally blind to the results of the electromyogram and did not know the horses beforehand. Examination was based on bony and soft tissue manual palpation for localised regions of vertebral stiffness based on spinal mobilisation and palpable areas of muscle hypertonicity Data reliability was assessed by a second evaluation performed respectively by a veterinarian specialized in osteopathy in Group 1 and a second chiropractor in Group 2 whose techniques of detection  were similar in the present study. The Kappa agreement . The two joysticks were placed at the level of C2, C6, T3, T9, T17, and L6  one on each side of the spine, and electrodes gave the muscular activities at the level of C1, C3, C5, C7, T1, T3, T8, T10, T16, T18, L5 and S1. Thus we obtained muscular activity all along the neck, at the level of the shoulder, at the basis of the withers, at the level of the thoracolumbar joint and at the level of the lumbosacral joint, which are reported in the literature as very likely to be affected by musculoskeletal lesions (e.g.see.). The curve of sEMG values and number of sites tested (.) revealed a threshold at 10 µV which therefore was considered a value from which the tested site (taking both sides into account: Left and Right ≥10 µV) was considered as “hyperactive” . Manual palpation only allowed categorical classification of horses  as it was impossible for the practitioners to have precise comparative evaluations . Therefore results of sEMG values were replaced in the same categories: totally exempt (The sEMG examinations were conducted by a second experimenter (CL), blind to the results of the manual palpation , using a wire free device (Myovision®). The experimenter had 2 joysticks with 5 electrodes on each, designed to record muscle activities at the level of the vertebrae before and at the vertebrae after the joystick location. Muscular activities recorded were sent to a receptor related to a computer Fig. 2.ons e.g.. The rawons e.g.. Most mu. Horses were kept motionless, slightly restrained with a rope, by a second experimenter.In the two groups, examinations were performed on a flat ground, outside the pasture in the first group, and in the corridor of the stable in front of each horse’s box in the second group, outside any noise or disturbance . The experimenter  paid attention to the horses’ feet positions: anterior and posterior feet were on a lineSeven of the 9 group 1 horses and all group 2 horses were also involved in posture measurements.Horses were observed while interacting with an experimenter: walking and standing motionless near the experimenter . The experimenter did not talk to the horse, stayed on its left side and held the rope slackly at a predefined distance from the horse’s head (1 m), so that the experimenter never pulled the rope or the horse’s head. Horses’ postures were recorded using photographs taken perpendicularly 10±1 m from the horse . All data were recorded by the same experimenters . Data recording took place between 08.00 AM and 06.00 PM during a two days period in both groups . Leisure horses were photographed 10 times when standing motionless near the experimenter, and 20 times when walking. Horses in riding schools were less available (involved in riding lessons) and could be photographed on average 2.4±0.8 times when standing and 4.5±0.9 times walking.Five markers  were stuck onto the horses’ right side, in accordance to Fureix et al. In order to quantitatively evaluate neck height and roundness, different angles were measured, using usual trigonometrical rules  Fig. 6:Angle α: Formed by the segment (M1–M2) and the horizontal plan running by the withers’ basis . It represents the neck’s elevation Angle β: Formed by the segment (M1–M2) and the segment (M2–M3). It represents the neck’s curve: the more concave the neck was, the more the angle was positive, and the rounder the neck was , the angle was negative.Angle σ: Formed by the segment (M2–M3) and the segment (M3–M4).Angle δ: Formed by the segment (M3–M4) and the segment (M4–M5).The last two angles were pooled (angle γ) to represent the M3–M5 angle . The narrower the head-jaw angle was, the more the angle was negative.s<0.94, p<0.00001 for each angle).The angles measurements required the horses to be exactly perpendicular to the camera. In order to have the same number of photographs per horse, 2 photographs were analysed for each horse while it was standing and 4 while walking . Photographs were taken in series of burst-shots. The experimenter (CL) studied all the photographs for each horse and kept the 2 where the horse had the most and the two where the horse had the less elevated neck. In each situation , the values were averaged for each angle. To assess the repeatability of the measures, the angles were measured twice for each photograph. In each case, the angle values were strongly correlated  or round .According to the β angle values, the horses’ neck shape will be called As data were not normally distributed, we used non-parametric statistical tests for the analyses. Vertebral sites were separated into 2 independent categories: sound or affected . sEMG values between these 2 categories were assessed using Mann-Whitney U-tests. Spearman correlation tests were used to assess whether chiropractic, sEMG and angle data were related to age, to detect the relations between chiropractic and sEMG evaluations, as well as the relations between sEMG measures and angle measurements of neck postures. As the different areas of the horses’ spine work together, the correlations between sEMG values at the different places along the spine were also assessed using Spearman correlation tests Finally, Chi square and Mann-Whitney U tests were used to evaluate the differences between the 2 horses’ populations. These analyses were conducted using Statistica© 10.0 software (accepted p level at 0.05).The chiropractic evaluation indicated that 55% (N = 10) of our horses were severely affected, 6% (N = 1) were slightly affected and the last 39% (N = 7) were totally exempt of back disorders. The sEMG evaluation indicated that 50% (N = 9) of horses were severely affected, 11% (N = 2) were considered as slightly affected, and the last 39% (N = 7) horses were totally exempt.s = 0.82, p = 0.001)  were also those with more tested sites affected according to sEMG evaluation (% of tested sites above 10 µV)  Fig. 7. s = −0.43, p>0.05, sEMG: Spearman correlation test, rs = −0.45, p>0.05).None of both evaluations found any correlation between potential back disorders and age   Fig. 8.2 = 14.4 and χ2 = 10.89 for chiropractic and sEMG evaluations respectively, p<0.001in both cases)  Fig. 9.On the whole, horses had a more elevated neck  when standing , whereas it varied more  when walking, whether the lowest  or highest  points were considered. Horses’ neck was rounder when it was at the low walking point , than when it was at the high walking point  or when standing . Concerning the γ angle, it was narrower when standing  and at the high walking point , than when at the low walking point .s = −0.5, p = 0.05; Walking: rs = −0.65, p = 0.007). Thus, horses with more elevated neck postures had also more vertical heads.Interestingly, the neck elevation during standing and walking was negatively correlated with the horses’ head-jaw angle , but larger variations occurred along the spine (from 41.82±14.61 µV at T1 level to 10.48±2.33 µV at L5 level).sEMG values are described in e.g. C1/T3: rs = 0.57 p = 0.02; C3/T1, T3, T10, T16, L5; C5/T1, T16, L5; C7/T3, T10; p<0.05 in all cases) (See .).As the horse’s spine areas cannot be considered independently, we investigated the relations between muscular activities all along the spine. The sEMG values were correlated for all cervical sites  and for most back sites . More interestingly still was the finding that the cervical values were correlated to those observed along the back  or walking . Moreover, a wider head-jaw angle was correlated with higher sEMG values at T18 level  (See .).A concave neck (β angle positive) was correlated with higher sEMG values  whether the horses were standing  were not correlated .No differences emerged between both populations concerning neck’s elevation or head-neck angle . However, significant differences were observed between the 2 populations in terms of neck roundness, with group 1 horses presenting an angle β more negative (meaning a rounder neck) than group 2 horses, both when standing , and when at the high  and low  walking points..).Overall, these differences in neck postures seemed to reliably reflect differences between populations in terms of muscular activity Fig. 10.The purposes of this study were to 1) assess sEMG as a useful method for the detection of back disorders, and 2) to assess correlations between sEMG and chronic neck postures (outside working time) so as to propose neck posture as a potential visible indicator of back disorders. Elevated and concave neck postures were associated with higher sEMG values, reflecting muscular activities that correlated with back disorders, as shown by the practitioners’ evaluations. In the cases of “affected” horses, sEMG measures were higher both at the exact location of the vertebral dysfunction (assessed by the practitioner) and all along the spine. sEMG measures and neck postures therefore appeared as potentially fruitful indicators of back disorders, a major issue in this species submitted to different types of riding and management styles. Thus, comparisons of horses living in two extreme types of domestic life (including different types of work) revealed that in one (riding school), horses were more prone to have concave necks and back disorders than in the other (leisure horses).e.g.e.g.In humans, patients with back pain or lesions present higher EMG and a more important muscular fatigue than healthy people e.g.. If EMG e.g. Children at school: longissimus dorsi and of the entire spine, and to a global “round” posture of the horse Some authors investigated the existing links between postural control/equilibrium and muscular activity in humans. Caneiro et al. Several factors, such as age, body fat, skin resistance or fear can modulate sEMG results. In this study, horses all presented the same corporal state , measure were conducted outside any disturbances and no fear reactions were observed see also  & 5. MorThis study led to the identification of key postural elements, allowing indirectly the detection of potential back disorders. The importance of the muscular activity at the level of C3 and of the β angle in neck shape points out neck “roundness” (position of M2 compared with the line between the neck basis and the head/neck joint) as a reliable indicator of back disorders, easy to evaluate in field conditions. This is of a considerable interest in a fundamental point of view, highlighting the accuracy of using postural elements to evaluate the animals’ general state and has important implications as a tool for animals’ welfare evaluation.


The correlation of hypertension with vascular dementia has long been established and it is becoming increasingly clear that hypertension is also a reversible risk factor in the development of Alzheimer’s dementia (AD). Aging, AD and hypertension are major determinants of cognitive dysfunction which are associated with profound alterations in the structure and function of cerebral blood vessels. The aim of the present work is to analyze cerebrovascular blood flow in the middle cerebral artery (MCA), the integrity of blood to CSF barrier (BCB), choroid plexus (CP) structure and protein secretion as well as cerebrospinal fluid (CSF) protein composition.Choroid plexus (CP), CSF and serum from hypertensive (SHR) and control (WKY) rats of 8, 26 and 52 weeks of age were used. Blood flow was measured by transcranial Doppler sonography on MCA. Brain sections containing CP were processed by immunohistochemistry using antibodies against transthyretin (TTR), caspase-3 (CAS-3), proliferating cell nuclear antigen (PCNA) and immunoglobulin G (IGG). CSF from 26 weeks of age was used by 2D electrophoresis and western-blot.CP immunohistochemistry: TTR increased during aging in SHR with respect to WKY. CAS-3 was undetectable at 8 weeks in SHR and at 8 and 26 weeks in WKY, at 26 weeks the SHR showed a slight reaction which was increased at 52 weeks of age, a soft reaction was also observed at 52 weeks in WKY rats, SHR also expressed a significant number of cells marked by CAS-3. PCNA results were similar to CAS-3; IGG was mainly expressed in the basolateral membrane of the CP and in CP cells at 52 weeks of age in SHR. 2D electrophoresis and Western-blot: By 2D, we found differences in the CSF protein profile of the SHR when compared to CSF of the WKY, these variations were similar to variations found by other authors in AD. By Western-blot (WB) the TTR was found to be higher in blood and lower in the CSF of SHR with respect to WKY groups.Long-term effects of hypertension cause a degeneration and hypertrophy of the CP producing variations in protein secretion to the CSF that could influence the maintenance of neurons and functions in the brain. Variations in CSF proteins in AD could similarly cause a CP disruption and support the close connection between cerebrovascular risk factors and AD in light of the fact that they could share common pathogenic etiological processes.


Bone marrow mononuclear cell (BMMC) transplantation is a promising therapy for cerebral ischemia; however, little is known if its therapeutic efficacy may be improved by co-administration of potential modulatory factors in vivo. To explore this possibility, the present study examined the effect of BMMCs and G-CSF on cell proliferation, early neuronal development and neurological function recovery in experimental cerebral ischemia relative to controls that received neither treatment.Ischemia/infarct area was significantly reduced in BMMCs+G-CSF group relative to animal groups treated with BMMCs only, G-CSF only or saline. Transplanted BMMCs were found to colocalize with the proliferative cell nuclear antigen (PCNA) and the immature neuronal marker doublecortin (DCX). The BMMCs+G-CSF group showed increased numerical density of cells expressing PCNA and DCX, improved performance in adhesive sticker removal test and reduced neurological function severity scores relative to other groups in a time-dependent manner.BMMCs and G-CSF co-administration exhibits synergistic beneficial effect over time. This effect could be at least partially related to increased proliferation and differentiation of bone marrow stem cells and enhanced host brain regeneration and functional recovery. The results suggest that G-CSF can increase the therapeutic efficacy of BMMCs transplantation in an experimental mouse model of cerebral ischemia. Bone marrow mononuclear cells (BMMCs) are a population of pluripotent progenitor cells with potential therapeutic value to boost cell repair and regeneration in some disease conditions. In the context of neurology, BMMCs have been shown to promote neuronal regeneration in ischemic cerebrovascular diseases ,2. BMMCsThe BMMC fraction of bone marrow can be a mix of different cellular populations. We characterized BMMCs harvested from the donor mice to obtain their surface antigen profile by fluorescence-activated cell sorting (FACS). The marrow cell markers CD34, CD44, CD45 and Sca-1 were used to label BMMCs, and the percentage of each type were estimated. In average, donor BMMCs exhibited CD34, CD44, CD45 and Sca-1 antigen signals at 93.5%, 45.8%, 80.9% and 1.9%, respectively (Table 3) in the BMMCs+G-CSF group was significantly smaller than that in the BMMCs (143.5 ± 1.5 mm3), G-CSF (148.2 ± 2.1 mm3) and saline (186.7 ± 1.8 mm3) groups   and without (BMMCs group) G-CSF co-administration. Two other groups of MCA occluded animals were not given BMMC transplantation but received either G-CSF (G-CSF group) or saline  injection to serve as non-transplant controls S-Figure . We assePKH26-labeled cells emit red fluorescence under 551 nm light excitation, whereas the blue fluorescence of DAPI is detectable using 405 nm light excitation. Thus, donor BMMCs double-labeled in vitro by PKH26 and DAPI can be reliably located in the host brain. Overall, no any PKH26 or DAPI-labeled cells were detected in the brains of mice that did not receive BMMC tail infusion  in the 4 groups of animals at day 1, 7, 14 and 28 post MCAO Figure . The denOverall, the time a mouse spent to remove the adhesive paper was dramatically increased after relative to before MCAO. Thus, at 1 day after MCAO, the adhesive removal times was longer but comparable among the 4 groups of occluded animals relative to pre-surgical basal scores Figure . During The neurological severity scores (NSS) were high but similar (p > 0.05) among the 4 animal groups as assessed at day 1 after MCAO Figure . During Bone marrow stem cell therapy has merged as a promising treatment for acute brain trauma. In practice, donor bone marrow cells are a mix of cellular components, with the BMMC fraction being considered a key player in the beneficial therapeutic efficacy in neuronal recovery. Our fluorescence activated cell sorting analysis suggests a phenotype of the transplanted cells being hematopoietic stem cells in nature, based on their expression of CD34 (93. 5%), CD44 (45.8%), CD45 (80.9%) and Sca-1(1.9%) . The useImproving the therapeutic efficacy of BMMC transplant may be of future clinical relevance. Data from the present study support that G-CSF, a biological regulator of BMMCs in vivo, may exhibit beneficial modulatory effect on both the transplanted bone marrow cells as well as the histological and neurological outcome in the host animals in an acute model of cerebral ischemia or infarction. Densitometric analyses show that the amount of PCNA and DCX-expressing cells are increased in the cerebral cortex in G-CSF-treated recipient mice relative to the BMMCs and G-CSF groups, in addition to the saline group. Therefore, it appears that BMMCs transplantation and G-CSF administration each could promote cell proliferation and DCX expression in the ischemic cortex , while BG-CSF administration appears to also reduce the cerebral infarct size according to our assessment on brain slices. In fact, this effect is most noticeable in animals received BMMC transplant as compared to ischemic animals without BMMC treatment. Thus, BMMC infusion and G-CSF treatment appear to exhibit synergeric protective effect in lesioned host animals. In parallel with the improved histological outcome, animals received G-CSF treatment show better neurological function recovery.The precise mechanism by which G-CSF may influence transplanted and host BMMCs, or other cellular components, in the ischemic mouse brain remains to be elucidated. However, a large body of basic and clinical studies suggest that G-CSF can mobilize hemopoietic stem cells, therefore might improve their delivery and differentiation in bodily tissue -14. It iIn summary, this work demonstrates that G-CSF administration facilitates the therapeutic efficacy of BMMC transplantation in histological and neurological outcomes in a mouse model of focal cerebral ischemia. Because autologous BMMC transplantation may be useful in the treatment of cerebral injury including ischemia, co-administration of G-CSF may be considered a promising adjunct therapy in clinical practice. Further basic and clinical investigations are warranted to explore the molecular mechanism underlying the effect of combined G-CSF administration and BMMCs transplantation in cerebral ischemia.The present study shows that G-CSF may promote the homing, proliferation and potentially neuronal differentiation of transplanted BMMCs in ischemic mouse cerebral cortex. G-CSF administration also appears to improve the efficacy of BMMC transplantation by reducing cerebral infarct size and enhancing neurology function recovery. The data raise a possibility of using BMMC transplantation together with G-CSF administration in clinical treatment of cerebral ischemia.Inbred BALB/c mice at 8 to 10 weeks of age, of male or female gender, with a bodyweight ranging between 18 to 20 g, were purchased from the Animal Center of the Second Affiliated Hospital of Harbin Medical University. The mice were freely accessible to food and water before and after the experimental procedures. Primary antibodies and secondary detection systems, as well as major reagents, used in the present study are listed in Table To evaluate the effect of G-CSF administration and BMMC transplantation, mice were divided into donor group  and recipient group with focal cerebral ischemia. The recipient group was then sub-divided. Thus, Group #1 animals (BMMCs+G-CSF) received BMMCs transplantation combined with G-CSF injection . Group #2 animals (BMMCs) received BMMCs without G-CSF injection. Group #3 animals (G-CSF) received G-CSF injection  without BMMCs treatment. Group #4 was treated with 0.3 ml of saline  as vehicle control. For group #1, daily G-CSF injection  started after BMMC transplantation and continued once a day for four successive weeks. The same G-CSF administration protocol was used for group #3  to provide a cell density > 5 × 108 cells/L. Then BMMCs were pre-labeled by PKH26 and DAPI according to the manufacturer's instruction [7 cells/ml. The viability of BMMCs was greater than 95% measured by trypan blue exclusion. For a given recipient mouse 1 × 107 double-labeled BMMCs were injected via the tail vein.As shown previously, BMMCs can be isolated and labeled in vitro prior to transplantation ,21. Brietruction . The fin6 cells/ml) were incubated in 2% fetal bovine serum in PBS at 4°C for 20 minutes with 1 μl of mouse monoclonal antibodies specific for CD34, CD44, CD45, Sca-1 or treated with normal mouse serum as a control. Samples were then analyzed by FACS Calibur with CellQuest software .Putative BMMCs double labeled by PKH26 and DAPI  of adhesive-backed paper were used as bilateral tactile stimuli by placing on the distal-radial area of the wrist of each forelimb. Mice were pre-trained for 1 week before tests and assessed basal level of time scores for adhesive removal before MCA occlusion. Mice were tested in 3 trials to measure the mean time to remove the adhesive papers, with a cutoff time of 300 seconds.For adhesive removal tests, square-shaped pieces  were modified and calculated following the method of Lu D et al . The modMice were sacrificed and perfused transcardially for 2 minutes at 120 mmHg with 4% paraformaldehyde in 0.01 M phosphate-buffered saline  via a left ventricular stab under overdose pentobarbital anesthesia . The brains were dissected out and cryoprotected in 30% sucrose at 4°C overnight, embedded in optimal cutting temperature compound, frozen, and prepared into 8-μm-thick frozen sections using a cryostat.2O2 in PBS for 30 minutes, and pre-incubated in 5% normal rabbit serum in PBS with 0.3% Triton X-100 for 30 minutes. Sections were then incubated with PCNA (1:200) or DCX (1:100) antibody. Sections were further reacted with biotinylated rabbit anti-mouse (PCNA) or goat (DCX) antibody at 1:400 concentration  for 2 hours, and subsequently with the ABC reagent (1:400)  for another 1 hour. The immunoreactivity was then visualized by using DAB (0.05%) and H2O2 (0.003%). Slides were then dipped in hematoxylin, dehydrated and coverslippered.To assess the rate of proliferation and survival of cells after BMMC transplantation, a one in eight series of sections from each brain was collected randomly to identify PKH26 and DAPI double positive cells. Additional sections were processed for immunolabeling of PCNA and DCX using microwave antigen retrieval as previously described ,21. Brie® 488 conjugated donkey anti-mouse or anti-goat IgGs . Sections were then washed and mounted with anti-fading medium before examination on a laser confocal scanning microscope . Sections from every mouse in the recipient group were processed in parallel under identical conditions to minimize variations in staining intensity.For immunofluorescence, sections were incubated with PCNA or DCX antibody overnight, washed extensively and followed by a 2 hours reaction with Alexa FluorAll data are presented as mean ± standard deviation. One set of sections from each animal was selected randomly. In each frozen section, 10 microscopic fields were captured randomly at a magnification of 200X. All analyses were processed using the statistical software GraphPad Prism 4. Means were compared using one-way ANOVA with Bonferroni's multiple comparison tests. Statistical significance was set at a p value < 0.05.The authors declare that they have no competing interests.XMZ designed the experiments, established the animal model, performed the histological and morphological evaluations and drafted the manuscript. FD carried out bone marrow mononuclear cell transfusion. XNH and WL performed tissue processing and data analysis. DY and RW participated in the fluorescence-activated cell sorting analysis. CJY and HYH performed the statistical analysis. JF supervised and coordinated the study, and finalized the manuscript. All contributors read and approved the final manuscript.


A 30-year-old man, who had been treated with craniospinal irradiation, total-body irradiation, and bone marrow transplantation for acute lymphoblastic leukemia at 20 years of age, complained of dysphagia. The patient had spike fever with leukocytosis . Serum granulocyte colony-stimulating factor (G-CSF) level was also increased (53.7 pg/ml). Immunohistochemistry revealed positive staining for anti-G-CSF antibody in carcinoma cells obtained by endoscopic biopsy. The patient was diagnosed with G-CSF-producing locally advanced esophageal squamous cell carcinoma. The clinical diagnosis was T4; tumor invaded aorta, with regional lymph node metastases (N1). The patient underwent transthoracic esophagectomy with three-field lymph node dissection and gastric tube reconstruction following a radiation dose of 41.4 Gy with 5-fluorouracil continuous infusion as neoadjuvant therapy. There were no viable cancer cells in the resected esophageal specimen and lymph nodes. The patient had no evidence for typical risk factors for developing esophageal cancer. After the operation, neutrophils and G-CSF decreased to normal levels. The patient had recurrence of regional and distant multiple lymph node metastases at 3 months after operation. New treatments for childhood cancer have decreased the number of deaths from the primary cancer. However, the treatment of cancer—including chemotherapy, radiation therapy or stem cell transplantation—may cause various late effects for childhood cancer survivors months or years after successful treatment has ended. Large cohort studies demonstrated increasing risk of second and subsequent primary cancer –3. One oMeanwhile, granulocyte colony-stimulating factor (G-CSF)-producing malignant tumor with leukocytosis has been reported to occur in various organs. G-CSF-producing tumor was first described by Robinson , and hasWe report herein a rare case of esophageal squamous cell carcinoma following therapy for acute lymphoblastic leukemia that showed increased leukocyte and serum G-CSF levels. Expression of G-CSF in the cytoplasm of cancer cells was found by immunohistochemical staining.l-asparaginase, vincristine, and steroid, and whole-brain radiotherapy of 18 Gy at age 11 years. At age 14 years, radiation to the whole brain of 24 Gy and the whole spine of 18 Gy were performed for central nervous system recurrence. At age 20 years, because of bone marrow relapse, he was treated with remission reinduction chemotherapy, bone marrow transplantation, and total-body irradiation of 12 Gy. Thus, the esophagus might have been exposed to radiation of 30 Gy during the overall treatment for acute lymphoblastic leukemia. Esophagogastroduodenoscopy showed a type 2 advanced cancer in the upper thoracic esophagus  and localized radiation as neoadjuvant therapy were designed because of transplantation-induced chronic renal failure, estimated creatinine clearance rate using Cockcroft–Gault formula of 39.1 ml/min, and irradiation history for leukemia. A total dose of 41.4 Gy was given in 23 fractions of 1.8 Gy, with 5 fractions per week starting on the first day of the first cycle of chemotherapy. The target volume comprised the primary tumor and enlarged regional lymph nodes. The spinal cord was excluded from the target area as much as possible, so as to not extend beyond 15 Gy. The patient completed the neoadjuvant therapy without severe adverse events, and the preoperative therapy evaluation was partial response (PR) with downstaging from T4 to T3  19,020/μl]. Serum G-CSF level was also increased . Immunohistochemistry revealed positive staining for anti-G-CSF antibody in the cytoplasm of cancer cells  marked leukocytosis, 2) elevation of serum G-CSF concentration, (3) decrease in leukocyte count after removal of the tumor, and (4) positive staining for G-CSF in tumor cells. There have been several case reports on G-CSF-producing esophageal squamous cell carcinoma –31, but  elevatioAlthough there have been a number of reports on incidence of therapy-induced second solid tumors, few have focused on treatment, and prognosis is considered to be poor , 33. ImpIn conclusion, G-CSF-producing esophageal squamous cell carcinoma is relatively rare and denotes a dismal prognosis. In the case of a patient with leukocytosis, the possibility of G-CSF-producing cancer should be considered. It is important for patients who have history of treatment for cancer by radiotherapy, chemotherapy, and allogeneic hematopoietic cell transplantation to be checked for second cancer occurrence, particularly within the irradiated area.


Our aim was to characterize clinical properties and laboratory parameters in patients with or without cerebrospinal fluid (CSF) findings suggestive of central nervous system (CNS) involvement, and especially those who developed serious CNS complications during acute nephropathia epidemica (NE) caused by Puumala hantavirus (PUUV) infection.A prospective cohort of 40 patients with acute NE and no signs of major CNS complications was analyzed. In addition, 8 patients with major CNS complications associated with NE were characterized. We collected data of CNS symptoms, CSF analysis, brain magnetic resonance imaging (MRI) results, electroencephalography (EEG) recordings, kidney function, and a number of laboratory parameters. Selected patients were evaluated by an ophthalmologist.Patients with a positive CSF PUUV IgM finding or major CNS complications were more often males (p < 0.05) and they had higher plasma creatinine values (p < 0.001) compared to those with negative CSF PUUV IgM. The degree of tissue edema did not explain the CSF findings. Patients with major CNS complications were younger than those with negative CSF PUUV IgM finding . Some patients developed permanent neurological and ophthalmological impairments.CNS and ocular involvement during and after acute NE can cause permanent damage and these symptoms seem to be attributable to true infection of the CNS rather than increased tissue permeability. The possibility of this condition should be borne in mind especially in young male patients. Myodes glareolus); human infection is acquired by inhalation of the virus from aerosolized excreta [Puumala hantavirus (PUUV) causes a hemorrhagic fever with renal syndrome (HFRS) also called Nephropathia epidemica (NE). Although the condition is endemic in Northern Europe, an increasing number of cases has been reported throughout Europe, possibly due to climate change -3. The v excreta . After a excreta .Many patients with acute NE display signs of central nervous system (CNS) involvement -7 and evIn this study, we characterized the clinical properties of patients with encephalitis or significant CNS injury caused by acute PUUV infection. We also compared the clinical properties of NE patients with positive CSF PUUV IgM finding to those negative for CSF PUUV IgM. Our intention was to identify patient populations that might be at elevated risk of developing significant CNS illness associated with acute NE. In addition, our aim was to analyze the clinical findings in order to search for an explanation for the CNS involvement.The patients of this study were partly recruited from those hospitalized for acute NE in the Oulu University Hospital in Northern Finland during September 2005 to February 2008 and the patient population in full has been described in detail elsewhere . For theWe compared the clinical presentation of acute NE without major CNS complications between the patients with CSF PUUV IgM-negative cases (21 patients) to those with CSF PUUV IgM-positive findings (19 patients) as well as with the patients who suffered major CNS complications (8 patients). The study protocol was explained to each patient and they had the right to refuse or withdraw from the study according to the Helsinki declaration. The study was approved by the Ethics Committee of the Oulu University Hospital and all participants signed an informed consent form.Eight patients were identified to have experienced major CNS complications associated with acute NE Table . These pPatients were examined as previously described in detail . BrieflyThe laboratory diagnosis of NE was based on PUUV serology that was initially analyzed using a commercial enzyme-linked immunosorbent assay of IgM antibodies . In some cases, the samples were also analyzed with an indirect immunofluorescence test for PUUV-IgG which displayed a granular staining pattern in cases of typical acute infection . The CSFThe patients were divided into three categories: those without major CNS complications and negative CSF PUUV-IgM (21 patients), patients without CNS complications and a positive for CSF PUUV-IgM finding (19 patients), and those with major CNS complications caused by acute NE (8 patients). In addition, we explored the clinical data of the patients positive for CSF PUUV-IgM and elevated CSF white cell count (data not shown in the tables). The results were analyzed with Chi-square test, Pearson bivariate correlation test, or t-test  for statistical significance.Our first task was to analyze if the laboratory findings differed between the patients with or without symptoms associated with affected CNS during acute NE. We found that all 21 patients with negative CSF PUUV IgM as well as nearly all patients  with positive CSF PUUV IgM complained of symptoms suggestive of CNS involvement . Since such a high proportion of the patients did experience CNS-related symptoms, no statistically significant differences in age, gender, any CSF finding, or other clinical or laboratory parameters based on these symptoms could be detected.The CSF PUUV IgM-positive finding may be considered to indicate intrathecal antibody production and the possibility of a true CNS infection. Based on this hypothesis, we compared the properties of IgM-positive and -negative patients. Selected clinical and laboratory findings of the study patients without major CNS complications divided according to CSF PUUV IgM-negative or -positive finding are summarized in Table It is possible that the CSF protein concentration and white cell count can be elevated either due to a true PUUV infection in the CNS or because of increased tissue permeability caused by the acute NE. We explored the possibility that clinical and laboratory parameters should possibly explain the mechanisms of the CSF findings. There was a statistically significant correlation between the CSF protein concentration and the severity of thrombocytopenia . The patients with elevated CSF protein concentration had a high blood white cell count  and there was a correlation between the CSF protein concentration and the plasma creatinine level . In contrast, the degree of tissue edema or urine output displayed no association with the CSF protein concentration or white cell count. We were able to analyze albumin concentrations from 26 CSF samples in order to investigate possible blood-brain barrier injury. We found that CSF albumin concentration was higher in patients with positive PUUV IgM CSF finding compared to PUUV IgM negative cases . The CSF/serum albumin ratio, however, was not different between the CSF PUUV IgM positive and negative cases. In addition, the CSF albumin concentrations were similar in the CSF samples with elevated white cell count compared to those with low CSF white cell count.The patients with high plasma creatinine levels also exhibited the most significant tissue edema as demonstrated by the correlation between with the weight change during hospitalization and plasma creatinine concentration . These patients also had the lowest urine output during the first day of treatment . As mentioned above, the patients with high plasma creatinine concentration had high CSF protein concentrations  and they also had a high blood white cell count .Selected clinical, imaging, and laboratory findings of patients with major CNS involvement are summarized in Tables The imaging findings of the patients with major CNS complications caused by NE are summarized in Table Three male patients with major CNS findings experienced a sudden, transient, and complete loss of vision for less than 5 minutes associated with severe headache, vomiting, and dizziness immediately before or during their hospitalization . One patient (patient 2) experienced a significant reduction of visual acuity to hand movement level, light sensitivity, and ocular pain during the first days of hospitalization. Another patient (patient 4) suffered from intermittent blurring of vision, diplopia, sense of defects in visual field, and nystagmus for at least 18 months after the acute NE. His visual field appeared normal in both Goldmann and Humphrey 24-2 perimetry and no abnormalities were noted in diplopia test.A tendency towards myopia in the acute phase compared to control examination was noted in the three patients -DQ6 haplotype . In thatOur current report demonstrates that the brain MRI and hormonal studies should be considered at least whenever severe NE is diagnosed in a young male patient. It should be appreciated that the illness may lead to long term neurological and ophthalmological impairments which may be caused by a true CNS infection. At present, little is known about the biological mechanisms leading to injury and further studies should be conducted.Acute PUUV hantavirus infection can cause permanent CNS and ocular damage. In our patient population the CNS injury was observed in young adult male patients. It seems possible that these symptoms are attributable to true infection of the CNS rather than increased tissue permeability.The authors declare that they have no competing interests.TH, NH, and HK examined the patients. SMM, TS, OV, AP, AV, and OV have participated to the laboratory analysis. EP and AK analyzed the brain MRI studies. PS is responsible for the endocrinological evaluation. SR analyzed the EEG examinations. All authors have participated to the preparation of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/217/prepub