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LaMar's Donuts
LaMar's Donuts serves famous, airy, delicious donuts. The company's slogan is "simply a better donut". Each day, LaMar's Donuts presents at least a couple of dozen varieties of fresh, unique donuts. The classic choice – the glazed donut – is critiqued as better than any competitors' glazed donuts and is just 220 calories. In addition to donuts, the menu features a sizeable beverage selection of coffees and teas.
Liquid capital required
Net worth required
Units in operation
All franchisees must operate their own business. Half of the franchisees own more than one LaMar's Donuts unit. Lamar's Donuts offers its franchisees ongoing support in the form of press releases, marketing support, and regular regional meetings. Though the company originated in Kansas City, LaMar's Donuts currently have locations in Colorado, Missouri, Kansas, Nebraska, Arizona, and Alabama.
LaMar's Donuts Franchise Opportunities - History
After about 30 years of experience manning the fryer at Jack Frost Donuts Company, Ray Lamar opened the first LaMar's Donuts in 1960. The Kansas City community fell in love with the donuts, and the store sold over 11,000 donuts each day. Due to its reputation, Lamar decided to franchise LaMar's Donuts in 1993.
LaMar's Donuts Franchise Cost / Initial Investment / LaMar's Donuts
Franchise Income
LaMar's Donuts has a franchise fee of $28,500 for a 10-year renewable agreement, although the total initial investment ranges from $300,000 to $500,000. Franchisees must also pay the company an ongoing royalty fee of 5% on all sales. To be considered for a franchise, interested parties must have a net worth that exceeds $350,000, with at least $150,000 available in liquid assets.
LaMar's Donuts Business Opportunities: Other Information
The donut industry is huge, earning about $10 billion annually. LaMar's Donuts is a standout amongst a crowded market, having been featured in magazines like The New Yorker, Gourmet, Better Homes and Gardens, Town and Country. Even the famous Zagat survey named LaMar's Donuts the spot that "perfected the art of the doughnut".
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LaMar's Donuts is listed in the Franchise Directory under the Food category. It's also listed in the section for Franchises Under $200,000.
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Healthy YOU Vending
You can join the wellness revolution by starting a business with Healthy YOU Vending – the nation's largest supplier of healthy vending machines. The proven Healthy YOU model requires no employees and allows you to monitor your machines from home or from mobile devices. There are no franchise fees, royalties, marketing fees or post-investment required purchases. Franchise level support but work with a home office that coaches but does not dictate.
Jon Smith Subs
Jon Smith Subs is a FRESH new opportunity with a track record for success, serving high quality subs, right off the grill, overstuffed and piled high with the freshest ingredients, since 1988.
The Great Greek Mediterranean Grill
The Great Greek Mediterranean Grill is a Fast-Casual franchise offering delicious, healthy, sought-after Greek food options with our authentic third generation recipes. We stand apart for our high quality ingredients, never-processed produce or meat, and outstanding hospitality.
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Ukraine fails to prevent numerous violations in 2018, - Amnesty International
The report states that human rights activists in this country suffer from attacks, and the authorities need more decisive steps to prevent the crimes and duly investigate the ones that already took place
Amnesty International logo
Amnesty International released a report on the current state of affairs in the human rights area in Ukraine.
'The Ukrainian authorities have failed to prevent or investigate numerous human rights violations committed by violent groups in 2018, perpetuating a feeling of impunity among their members which risks encouraging further attacks against human rights activists, political opponents and ethnic minorities,' reads the report.
Related: Ukraine's far-right C14 conducts anti-Roma cleansing operation at Kyiv train station
Related: Amnesty International considers forced feeding of Sentsov 'torture'
'Independent journalists and media companies, especially those who are accused of disseminating "pro-Russian" views, have increasingly come under pressure by both the authorities and members of violent groups. Ukrainian authorities should take decisive steps to stop the surge of violent attacks by ensuring effective, impartial and transparent investigations of all cases. Those who are suspected of being involved in violence should be brought to justice in fair trial proceedings', the document says.
Related: Human Rights Watch notices curbing of freedom of speech in Ukraine
'The authorities should also unequivocally recognize the important role played by civil society activists, and fully respect the right to freedom of expression, including media freedom, and victims of violence should be provided with access to adequate remedy', Amnesty International wrote.
human rights Amnesty international

redpajama_set_name: RedPajamaCommonCrawl


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'Agro-forestry will create jobs for youths, rid cocoa, cashew-producing states of criminals'
By Femi Ibirogba
22 February 2021 | 3:44 am
First, most of the cocoa trees in Nigeria have been existing from about 40 to 60 years and even more. Therefore, those plantations have declined in productivity and there is a need to either plant..
Dr Patrick Adebola is the newly appointed Executive Director of the Cocoa Research Institute of Nigeria (CRIN), Ibadan, Oyo State. He explains how most forests can be developed for agro-forestry farms to engage youths in cash crop production; the challenges confronting cocoa and cashew production, and availability of improved planting materials and distribution impediments, among others. FEMI IBIROGBA, Head, Agro-Economy, reports.
Nigeria produces fewer tonnes of cocoa beans yearly than Ghana and Cote D'Ivoire. What are the factors responsible for the poor productivity in Nigeria and how can they be resolved?
First, most of the cocoa trees in Nigeria have been existing from about 40 to 60 years and even more. Therefore, those plantations have declined in productivity and there is a need to either plant new ones or that the old plantation undergoes thorough rehabilitation. This is one of the reasons the productivity is low.
Secondly, there is the issue of the quality of planting materials. Generally, farmers tend to harvest from their farms and produce the seedlings themselves so that they can plant and they may not be able to get the maximum yields. The Cocoa Research Institute (CRIN) implores farmers to come for improved planting materials — seedlings that are high-yielding and will produce fruits early in about two years.
There was a national programme between 2013 and 2014 that was to mass-produce improved seedlings for free distribution to farmers. Why is the programme unsuccessful and why is the current government hesitant in this regard?
The government has been doing their part. From my understanding, every year, the government, through the Federal Ministry of Agriculture, in conjunction with CRIN, produces thousands of improved seedlings, which we distribute to farmers. The number may need to increase so that the impact would be better.
Can you give an estimate of the quantity of seedlings produced and distributed to farmers?
Last year, we produced almost a thousand improved seedlings.
Are these farmers, the old plantation farmers, rehabilitating their existing plantations?
The majority of the farmers are to establish new plantations. For this year, we have planned to produce about 500,000 cocoa seedlings of improved varieties for distribution to the farmers.
Has this plan been incorporated in the budget?
Yes, it is already in the budget. The budget has been appropriated…
Is CRIN in charge of the seedling production or outsourced in different region?
The institute has six sub-stations which are situated in the six geo-political zones in the country. We produce seedlings at the headquarters here, as well as in our sub-stations, where these mandate crops are being propagated. There is a station in Cross River State, in Abia State, in Ondo and Edo State each. The production of seedlings is spread across all our sub-stations so that farmers who live close to the sub-stations can get easy access to the seedlings.
What do you need as an institution to mass-propagate the improved varieties of cocoa seedlings that can close the production gap between Nigeria and Cote D'Ivoire?
A lot of technologies have emerged to help mass-propagate genetic materials that are of very high quality and are of high demand. We do the manual production, using the seeds to propagate those seedlings. But there are other technologies like the semi-autotrophic hydroponics (SAH), which can be used to mass-propagate improved planting materials. We are trying to see how this can be introduced to cocoa and although we have attempted that in an experimental trial.
How is the hydroponic production carried out?
The process is easy, having the substrates and parts of the planting materials, a part of the leaf of stem, which will be propagated using a special medium. We root and harden them and make them available for farmers. Seedlings can be produced in millions using the hydroponic technology.
The process does not begin in the laboratory and it is not solely produced in the lab but we keep them in an environment, conducive for them to root easily and for them to acclimatise.
Why is there low cashew production and what factors have led to this?
Cashew is another cash crop that Nigeria is blessed with. Cashew can practically grow anywhere in Nigeria. The reason for the low production is probably because people are not aware that it is a money-spinning kind of business. The institute is ready and have been supplying planting materials of Jumbo nuts, Indian dwarf and the likes that are given to farmers. I think that we are capable of supplying planting materials upon request by farmers.
Will you suggest plantation expansion or intensive production and increased productivity of available hectares?
It is a combination of several factors. One, most of the trees are not as productive as we would expect them. So, it is good to go for high-yielding materials to establish new plantations. It is also possible for old plantations to be rejuvenated. These are the areas to be considered to increase production.
Most of the cocoa processing factories in Nigeria are gone and there are no standardised cashew processing factories. Is there a correlation between shutting down of the factories and production of these cash crops?
I wouldn't say that there is a correlation between the two. The issue remains that the agricultural sector was and has been neglected because of the oil boom. Therefore, the farms are abandoned and they become moribund and there is little or no interest in farming. Thankfully, the government is bringing us back into agriculture. The plantations were neglected and this is why most of the plantations do not produce good yields.
Going to industries, it is the neglect of a particular sector that affects industrialisation. If the primary source (raw materials) are not available due to the neglect and abandonment, the industry will not be productive. However, things are changing. Industries are springing up but there are a few processors of cocoa and cashew. I am positive that the industries will continue to increase in the entire value chain of cashew.
You mentioned that farming has been abandoned for so long. Regardless of this, there are no jobs for the youths and over 14 million youths are said to be unemployed. How can the younger generations be attracted into the agricultural sector, especially in cash crop production?
When a plantation is established in Nigeria, it takes only years before it begins to yield, and the farmer can begin to harvest, under favourable conditions. The plantation can exist for more than 50 years and for this reason, we are appealing to the youths to come into tree crop farming, because it is highly lucrative. These generations of youths rely on white-collar jobs, which happen to be scarce these days. But with the support of the government, I think this is an opportunity for youths to come into agriculture.
What advice will you give to state governments that are known for the production of cocoa and cashew to empower interested youths?
I will recommend the grouping of the youths in those states by the state government, providing them with land, incentives in terms of inputs, while partnering with CRIN to also provide the technical know-how for planting and harvesting and marketing.
Are you suggesting that the forests occupied by criminals can be transformed into cocoa and cashew production zones?
Yes, I suggest this because forests are not to be left fallow and unutilized, and this is something the state government can look into.
Let them group the youths and allocate hectares of land to them for tree crop farming and provide them with planting materials and the essential training for production, harvest and marketing of the crops.
What do you suggest as panaceas for the herder/farmer crisis, engulfing the country at the moment?
This issue is like a hot potato and it's been ongoing for years. Although it is nothing new, the casualties in these clashes are alarming and the government should pay reasonable attention to it. There has been a lot of suggestions pertaining to ranching, cattle colonies, and I think that these solutions are workable if they are applied genuinely. We also need to understand that both farmers and herders are citizens of the country, hence protecting their interest is key in that, this crisis can be resolved amicably. I believe that it can be a win-win situation if the place of dialogue is prioritised.
As the newly appointed Executive Director of CRIN, how do you intend to help scale up production of cocoa, cashew, kola nut, tea and other cash crops to meet up with the demands yearly?
CRIN is an institute saddled with the mandate of making sure that the genetic potential of the five mandate crops — cocoa, cashew, coffee, kola and tea — are improved. We will play our part.
Yes. We have been undergoing this research for over 50 years and it has yielded a lot of result. We have been able to get improved materials for the five mandate crops that are being propagated yearly to farmers. Going by your question about our vision, I will say that our vision is to continue to carry out quality research so as to come out with highly productive varieties that will be distributed evenly to farmers to help improve their production.
Secondly, we are saddled with the responsibility of transforming some of the mandate crops into by-products to add value. We have a section in the institute which is responsible for value addition. So, our intention is also to continue along that line. The technology is readily available and we are anticipating the investors to take them up. We are also ready to train and deliver those technologies to whoever can take them up or those who we can partner with.
Do you imply that the technologies can be imparted into youths?
Certainly. Our institute is funded by the public sector and we are capable of making the knowledge available to youths, to women and would-be entrepreneurs, who are interested in taking up those technologies. With our institute being funded by the public resources, I believe that the transfer of this knowledge will be without service charge.
Patrick Adebola
'Seedlings, production techs impeding Nigeria's cocoa industry'
Apart from neglect of agricultural infrastructure by the government, non-availability of improved high-yielding cocoa seedlings, scarcity of other inputs such as agro-chemicals, old production technologies and climate change ...
Combating fire outbreaks, heat stress on poultry, other farms
Apart from the general stifling economic environment and rising cost of feed ingredients, factors that make poultry production unsustainable are thermal heat stress, Avian Influenza (bird flu) infestation and fire disasters. Hence, poultry farm owners, especially egg producers, have been advised to take proactive measures such as heat mitigation palliatives, fire and bird flu outbreak…
Maximising public-private partnership potential for food tech adoption
To avoid the frustration of scientists and technologists working on agricultural research and development in Nigeria, the government has been urged to deepen the town-and-gown relationship
Nigeria struggles with cocoa production as China breaks through
With rugged determination and twist of fortune for China, it now successfully grows and exports cocoa beans to Europe despite difficult production conditions.
Battle intensifies against food contaminants, export rejection
Some agencies and institutes of the Federal Government in food research and development have moved to stem rejections of exports of food and industrial crops of Nigerian origin by training farmers...


The decay of Jo'burg's centre can be ascribed to many factors but perhaps none more so than the absence of Body Corporates. These had become relics of a more genteel era; the communal responsibilities that are contentious in even the most well-heeled blocks were not markedout. Windows were broken and not repaired. Lifts froze and their shafts became tips.
The relationship between tenants and owners or their agents deteriorated with disputes over the state of the buildings, and in some cases resulted in unpaid rents and dues. The buildings started looking like fire hazards, and the City Council began closing on them for unpaid utilities. In between the needs of City Council and the aspirations of developers anticipating the bloom of an African city lies the fate of Jo'burg's residents. The outcome will decide whether or not Johannesburg becomes, again, a city of exclusion.


A new selection of our best liquids EACH WEEK!
This is the perfect way for you to find your next favorite vape! We select 5 of our premuim liquids and pack them up for you to try!
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I have a confession to make. I woke up one morning and discovered that my clothes were snug. More than I was comfortable with. I had been doing a lot of traveling, and meeting a lot of people. A sports injury sidelined me from running, but my schedule was so packed that I quit exercising altogether. As I was growing my client base, I was also growing another base: my waist. I was in a constant state of anxiety, and racing from work to church to home. Yes, I was successful, but at what cost?
How many of us pursue success at the cost of our personal well-being? Far too many of us, I'd venture to say. Yet our success will be short lived unless we make time to take care of ourselves. When I was lying on a gurney in the emergency room, staring at the bright lights in the trauma center, I realized how quickly things can change when my body is not taken care of. So I'm offering these suggestions so that as you succeed, you will be able to enjoy your success with good health.
Studies show that dehydration affects our performance. Be sure to drink at last 8 glasses of water a day to stay on track with your sales and business goals.
Sleep deprivation affects our thinking and our performance. Getting enough sleep is essential to business success.
Fruits and vegetable have vitamins you need to feel your best. The right fuel for your body energizes you to tackle your biggest challenges with power.
Exercise releases dopamine, the feel good hormone, clears you head. Just walking 30 minutes a day 5 days a week brings fresh perspective. When you think better, you are more successful.
Connecting with God connects you with wisdom to handle the challenges you face everyday.
Moderation is the key. Relying on drugs to get through the day or relax works against you. The less dependence you have on caffeine and alcohol, the better you will feel.
Good health starts with your mouth. Research shows that heart health is affected by oral hygiene. Healthy mouth, healthy body.
Staying healthy is the foundation of your success. Without a strong body, your success is like a house built on sand. When storms come, they erode the foundation and the house eventually collapses. Likewise, without good health, your success will be built on shifting sand, and will be short lived. Make the time to eat well, get enough sleep, exercise pray, and take care of your body. Then you can enjoy the success you build!


A frequently overlooked detail in weddings is the Bride's Room. What is often forgotten is how many photos are taken of the bride and her bridesmaids as they hang out and get ready for the day. Just taking a little extra time and energy to create a really beautiful environment can make such a difference for documenting this special day.
Kelsey and Travis's venue was the home and property of some family friends. We were fortunate enough to have the master suite as our canvas (as well as some very gracious home owners). Removing and rearranging furniture was step one. Bringing in some beautiful and functional pieces for the day was step two. Having the fabulous creative team of Adrianne and Cheri add their magic made everything come together for the perfect Bride's Room.
Something as simple as covered dining room chairs add a special touch to the seating areas.
Candles, flowers and chocolates–always a special touch.
Hobnob has some unique pieces for rental that Kelsey was able to use.
One of those is the antique vanity. It provides a beautiful backdrop for photos of the bride.
Wherever girls are getting ready, an abundance of mirrors is always necessary. So, we brought in a few extras and made use of all that were available in the home.
Simply adding flowers makes an ordinary space special.
Every little detail helped to create the perfect space for making memories of a lifetime.
It's not just about the ceremony and the reception. The festivities begin when the day begins, so it's important to think through every part of the day and sprinkle details and special touches throughout.


Nestled in the heart of Glasgow, The Anchor Line is a fabulous, multi award-winning restaurant situated on St Vincent Place. From the group that brought us some of Glasgow's favourite dining spots including little sister, Atlantic Bar & Brasserie, Cafe Andaluz, Amarone and more, The Anchor Line is known for its incredible menu and buzzing ambience. Adorned with decorative tales of a bygone era, the restaurant celebrates the grand ocean voyages of the 1920s and 30s from Glasgow to New York, complete with appropriate decadence including a theatrical open bar, grand marble pillars and luxurious furnishings.
"Great food ahoy! I visited the Anchor Line with a group of colleagues while at a conference. What a lovely place! The menu is excellent and the food is fresh and marvellously prepared. Can't speak highly enough of this restaurant. It's well worth a visit."
"I visited for the first time after my friend recommended it. The ambience is lovely – it almost feels like being on a cruise! They have an extensive wine menu and the waitress was lovely and attentive without being invasive. I really enjoyed this visit and looking forward to coming again."
Voucher is valid from Thu 3 Jan 2019 until Fri 22 Mar 2019, Sun – Thu 12noon – 9pm and Fri 12noon – 5pm; subject to availability.
Includes a choice of cocktail each from Peach Ices Tea, raspberry Mule and Peely Wally Weegie.
Alcohol can be substituted for a draught soft drink.


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Kiss Árpád, Kiss Árpád Lajos Elek (Németbogsán, 1878. december 27. – Budapest, 1934. január 2.) színész, színigazgató.

Életútja

Apja, dr. Kiss Mihály/Miksa járásorvos, anyja Asbóth Ilona. A Ludovika Akadémia elvégzése után öt évig katonatiszt volt, majd rövid ideig Hegyi Aranka növendéke. 1903–04-ben Mezei Kálmánnál volt gyakorlaton. A pályát Újpesten kezdte, a Vasgyáros Bligny hercege volt az első szerepe. 1908–09-ben Kunhegyi Miklósnál lépett színpadra. Vidéki működése alatt egyben titkárként is működött, majd 1909. április hó 14-én színigazgatásba fogott és mint ilyen 1914–1918 között Szatmárt, 1914–15-ben, illetve 1922-től 1930-ig Békéscsabán, 1918–1920 között Nyíregyházán, 1918–1922 között Ungváron és Munkácson, 1923–1928 között, valamint 1930–31-ben Makón, 1928 és 1932 között Sopronban, 1930–32-ben Székesfehérvárott és Veszprémben, 1931–32-ben Szombathelyen működött. 1932-ben csődbe ment. Az Országos Színészegyesület tanácsosa volt. Halálát szervi agybaj okozta.

Neje Kövi (Kövy) Kornélia színésznő, aki született 1881. július 9-én, Keszthelyen, 1909. április 14-én kötöttek házasságot Budapesten. Színipályára lépett 1896. augusztus 23-án Leszkay Andrásnál, 1928-ban nyugdíjba ment.

Jegyzetek

Források

 Magyar színházművészeti lexikon
 Magyar Színművészeti Lexikon (1929-1931, szerk. Schöpflin Aladár)

Magyar színészek
Színigazgatók
1878-ban született személyek
1934-ben elhunyt személyek

redpajama_set_name: RedPajamaWikipedia


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Gorka: Sarah Palin and I Support 'Anti-Establishment, Anti-RINO' Roy Moore over Mitch McConnell's Candidate
AP/Getty
John Hayward
Dr. Sebastian Gorka, former deputy adviser to President Trump and Breitbart News National Security editor, praised President Trump on Wednesday's Breitbart News Daily for going into "the belly of the beast" to deliver a sensational address to the United Nations. He also explained why he supports Judge Roy Moore for Senate in the Alabama GOP primary over the candidate President Trump endorsed, Luther Strange.
The U.N., which "was supposed to create global peace and stability after 60 million people were killed in World War 2," has become a "hive of duplicitous globalist mantras" in Gorka's estimation. He denounced absurd U.N. spectacles such as nations like Venezuela and Cuba's attacks on the United States and Israel over human rights.
"He just spoke truth to power," Gorka said of Trump's speech. "He called out our enemies. He attacked socialism and communism. He even went on record on key policy things such as the disastrous JCPOA Iran deal we inherited from the Obama administration and called it an embarrassment."
"I know the guy who wrote the speech. I'm glad he managed to smuggle it past all the anti-MAGA forces that are in the administration," said Gorka, tongue not entirely planted in cheek. "I'm glad it was a home run."
Kassam noted that Gorka remained staunchly and outspokenly loyal to President Trump after leaving the White House, but now, he has joined a coalition that seeks to help Roy Moore win the Alabama Senate primary over the candidate Trump endorsed, Luther Strange.
"I've said again and again and again, I said in my resignation letter that is up at Breitbart, the president is being served by people who are giving him the wrong advice," Gorka said.
"That's why Steve and myself left the building, so we can get the agenda back on track," he said, referring to former White House strategist and fellow Breitbart alumnus Steve Bannon. "I joined the Make America Great Again Coalition, that's MAGACoalition.com, to assist him as I promised, from the outside."
"That's why we're going to Montgomery tomorrow, to Alabama," he explained. "I'm going to be on the stage with Gov. Sarah Palin, and we're going to support the candidate who isn't being supported by Mitch McConnell. We're going to support the candidate who is anti-Establishment, anti-RINO, just as the president truly is. We've got to put the pressure on, even when the president is getting bad advice, because the stakes are just too high."
Gorka said those stakes include nothing less than "the founding principles of this republic being systematically undermined and the perpetuation of a permanent political class."
"It's like the bureaucrats in Brussels that were roundly rejected by the British people in the Brexit vote. That's what MAGA is about. It's about getting rid of people who think they know better than the people who elected them, and think they're smarter, and create these sinecures for life as professional politicians. That's what MAGA is about," he said.
"I reject the word 'populism.' I think it's become pejorative," Gorka argued. "It's about the reassertion of sovereignty. That's what the president's speech was about yesterday: the reassertion of sovereignty as a nation-state and true democracy and republican federalism in America."
Speaking as a Briton, Kassam observed that too many Americans do not realize just how unique their national commitment to liberty is, even when measured against other Western countries.
"What does the Bible say? A man can never be a prophet in his own country," he reflected. "I chose this nation. You have the outside perspective as well. When I talk about founding principles and threats to the Republic, that's not pablum. That's not rhetoric. I truly mean it."
"I chose this nation. I'm a legal immigrant. That's why I joined the MAGA Coalition. Check it out. It's the super PAC for the forgotten men and women that voted for Donald Trump, the rank outsider, to become the most powerful man in the world," he said.
"Let's just cut to the chase here," Gorka suggested. "What's on the other side? This gels perfectly with the British experience in Brexit and everything else. We have people on the other side – Hillary actually said this in a speech she gave – who say borders should be dissolved."
"Can we just wrap our heads around the consequences of that? What does British mean? What does American mean? What do the founding documents of this nation mean, if there are no borders in our hemisphere, as Hillary Clinton told a group of bankers should be the reality?" he asked.
"The stakes could not be higher than they are today. That's why we have to help the president deconstruct this bureaucratic administrative state; drain the Swamp – because otherwise, I'm sorry, democracy is dead," Gorka warned.
Breitbart News Daily airs on SiriusXM Patriot 125 weekdays from 6 a.m. to 9 a.m. Eastern.
PoliticsRadioAlabama Senate primaryBreitbart News DailyDonald TrumpDr. Sebastian GorkaLuther StrangeMAGARoy MooreUnited Nations


Searching for news... If nothing shows up chances are not too much is happening in Elk County, KS these days.
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About IAI
Host Response and Inflammation
Regulatory Roles of CD1d-Restricted NKT Cells in the Induction of Toxic Shock-Like Syndrome in an Animal Model of Fatal Ehrlichiosis
H. L. Stevenson, E. C. Crossley, N. Thirumalapura, D. H. Walker, N. Ismail
H. L. Stevenson
Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch at Galveston, 301 University Boulevard, Galveston, Texas 77555-0609
E. C. Crossley
N. Thirumalapura
D. H. Walker
N. Ismail
For correspondence: naismail@utmb.edu
DOI: 10.1128/IAI.01242-07
CD1d-restricted NKT cells are key players in host defense against various microbial infections. Using a murine model of fatal ehrlichiosis, we investigated the role of CD1d-restricted NKT cells in induction of toxic shock-like syndrome caused by gram-negative, lipopolysaccharide-lacking, monocytotropic Ehrlichia. Our previous studies showed that intraperitoneal infection of wild-type (WT) mice with virulent Ehrlichia (Ixodes ovatus Ehrlichia [IOE]) results in CD8+ T-cell-mediated fatal toxic shock-like syndrome marked by apoptosis of CD4+ T cells, a weak CD4+ Th1 response, overproduction of tumor necrosis factor alpha and interleukin-10, and severe liver injury. Although CD1d−/− mice succumbed to high-dose IOE infection similar to WT mice, they did not develop signs of toxic shock, as shown by elevated bacterial burdens, low serum levels of tumor necrosis factor, normal serum levels of liver enzymes, and the presence of few apoptotic hepatic cells. An absence of NKT cells restored the percentages and absolute numbers of CD4+ and CD8+ T cells and CD11b+ cells in the spleen compared to WT mice and was also associated with decreased expression of Fas on splenic CD4+ lymphocytes and granzyme B in hepatic CD8+ lymphocytes. Furthermore, our data show that NKT cells promote apoptosis of macrophages and up-regulation of the costimulatory molecule CD40 on antigen-presenting cells, including dendritic cells, B cells, and macrophages, which may contribute to the induction of pathogenic T-cell responses. In conclusion, our data suggest that NKT cells mediate Ehrlichia-induced T-cell-mediated toxic shock-like syndrome, most likely via cognate and noncognate interactions with antigen-presenting cells.
Patients with severe human monocytotropic ehrlichiosis (HME) may develop sepsis or toxic shock-like syndrome (9), including elevated liver enzyme levels, hepatic injury and inflammation (34), and lymphopenia (33). HME is caused by Ehrlichia chaffeenesis, an obligately intracellular gram-negative bacterium that lacks lipopolysaccharide (LPS) and peptidoglycan (24). The clinical and pathological manifestations of the disease observed after intraperitoneal (i.p.) inoculation of mice with a virulent Ehrlichia species isolated from Ixodes ovatus ticks and referred to as I. ovatus Ehrlichia (IOE) reproduces the manifestations reported for severe and fatal human disease. This animal model of fatal HME has enabled us to further characterize the host immune response. Studies using this model have determined that decreased interleukin-12 (IL-12) production, a weak CD4+ Th1 response, expansion of pathogenic tumor necrosis factor alpha (TNF-α)-producing CD8+ T cells (19), elevated serum TNF-α and IL-10 levels (20, 37), and development of hepatic apoptosis and necrosis are associated with disease progression and development of fatal toxic shock-like syndrome (36). Recently, we determined the roles of CD8+ T cells in the pathogenesis of fatal ehrlichiosis by using two different types of CD8+ T-cell-deficient mice (β2m−/− and TAP−/−). Interestingly, we observed that β2m−/− mice that are deficient in both CD8+ and CD1d-restricted natural killer T (i.e., invariant NKT [iNKT] and variant NKT) cells are more resistant to fatal disease than TAP−/− mice that are deficient only in CD8+ T cells and that 90% of β2m−/− mice survived past day 30 postinfection (18). These observations, as well as the report by Mattner et al. (26) that NKT cells are directly activated by Ehrlichia muris, a species closely related both antigenically and phylogenetically to E. chaffeensis and IOE, persuaded us to examine more closely the role of NKT cells in severe ehrlichiosis.
NKT cells are a unique subset of T lymphocytes that express a semi-invariant T-cell receptor (TCR) and markers of natural killer (NK) cells. These cells have been identified as a novel lymphocyte population that acts in innate immune responses and also influences the acquired immune response (40). Unlike conventional antigen-specific T lymphocytes, NKT cells recognize glycolipids and phospholipids, rather than peptide antigens, presented by the nonclassical major histocompatibility complex (MHC) class I molecule CD1; however, the only CD1 receptor present in mice is CD1d. CD1d-restricted NKT cells comprise two major subsets, iNKT cells, which express exclusively an invariant TCR α chain (Vα14Jα18 in mice) and are activated by α-galactosylceramide, and variant NKT cells, which express more diverse TCRs. CD1d is an MHC class I-like molecule expressed on the surface of many cells, including hepatocytes (41) and professional antigen-presenting cells (APCs), including splenic dendritic cells (DCs), macrophages, and B cells (8). Expression of CD1d in the thymus is essential for positive selection of iNKT cells in the periphery.
NKT cells play a critical role in linking innate immunity and adaptive immunity by influencing the activation and effector functions of several hematopoietic cells, including DCs, macrophages/Kupffer cells, NK cells, and T and B lymphocytes (5). Fujii et al. (11) have shown that activation of iNKT cells by α-galactosylceramide results in up-regulation of CD40, CD80, CD86, and MHC class II on APCs similar to the up-regulation induced by Toll-like receptor ligation. This up-regulation may be important for the host's recognition of pathogens that lack LPS and peptidoglycan, such as ehrlichiae. Previously, we have shown that antigen-specific activation of NKT cells by Alphaproteobacteria lacking LPS, such as E. muris and Sphingomonas capsulata, is CD1d dependent and Toll-like receptor independent (26). This study further demonstrated that glycosylceramides are alternative ligands to LPS for innate recognition of the gram-negative, LPS-negative bacterial cell walls by NKT cells. In addition, activated NKT cells influence the Th1/Th2 phenotype of CD4+ T cells through their early and rapid production of large amounts of Th1 and Th2 cytokines, such as gamma interferon (IFN-γ) and IL-4, respectively. NKT cells also promote the induction of effector CD8+ T cells by enhancing the ability of APCs, mainly DCs, to activate naïve CD8+ T cells (38). Probably even more relevant is the capacity to activate cytotoxic T-lymphocyte (CTL) responses in the absence of CD4+ T-cell help. This phenomenon may be an important mechanism in severe ehrlichiosis, in which a significant decrease in the number of CD4+ Th1 cells has been observed (19).
We hypothesized that NKT cells may mediate tissue damage and toxic shock-like syndrome in the animal model of severe and fatal ehrlichiosis directly or indirectly by activating other cells of the immune system, including pathogenic CD8+ T cells. We investigated our hypothesis by studying lethal ehrlichial infection (i.e., i.p. inoculation of IOE) in CD1d−/− mice and identified a critical requirement for both CD1d expression and NKT cells in immunopathology and toxic shock. Our results indicate that NKT cells play both beneficial and detrimental roles during severe ehrlichial infection. On the one hand, NKT cells are critical in controlling the early bacterial burden and increase the number of costimulatory molecules on APCs. In contrast, they also promote Ehrlichia-mediated toxic shock-like syndrome. Understanding the immunopathology mediating fatal ehrlichiosis is critical for designing effective immune-based therapy not only against ehrlichiosis but also against toxic shock-like syndrome caused by Ehrlichia and other pathogens.
Mice.BALB/c wild-type (WT) or CD1d−/− (C.129S2-Cd1tm1Gru/J; stock number 003814) mice were used for all experiments (Jackson Laboratories, Bar Harbor, ME) except those whose results are shown in Fig. 1, in which we compared the disease characteristics in WT BALB/c mice to those in WT C57BL/6J mice. The mice were gender matched for each experiment and were 6 to 12 weeks old. Mouse handling and experimental procedures were conducted in accordance with the recommendations of the University of Texas Medical Branch Institutional Animal Care and Use Committee.
Survival, organ bacterial burdens, serum TNF-α and ALT concentrations, and liver lesions were similar in C57BL/6 and BALB/c WT mouse strains. Both WT BALB/c and C57BL/6 mice (nine mice per group) were inoculated with a high dose of IOE (1 × 105 genome copies) and monitored daily for signs of illness. The data represent the results of three independent experiments with three mice per group. (A) Survival rate of both WT C57BL/6 and BALB/c mice. (B) Bacterial burdens in the liver, lung, spleen, and kidney were not significantly (P > 0.05) different for the two mouse strains on day 7 postinfection. The bars indicate the averages and the error bars indicate the standard deviations for triplicate amplifications with three mice per group. (C and D) Serum levels of TNF-α (C) and ALT (D) were not significantly (P > 0.05) different in IOE-infected C57BL/6 and BALB/c WT mice on day 5 postinfection. The bars indicate the means and the error bars indicate the standard deviations for three mice per group. Similar results were obtained in three independent experiments.
Experimental design.For all experiments a highly virulent Ehrlichia sp. (IOE), originally isolated from I. ovatus ticks in Japan, was used (17, 35). The genome copy numbers of IOE in our inocula were determined as previously reported (37). Mice were inoculated i.p. with a high dose of IOE (∼1 × 105 genome copies), and three mice per group were sacrificed on days 5 and 7 postinfection to determine the bacterial burden and the levels of cytokines and liver enzymes in the serum, as well as organ pathology. Mice were monitored daily for signs of illness, including the presence of distress, loss of appetite, roughened fur, and decreased activity. On days 0, 5, and 7 individually tagged mice were monitored for changes in temperature and weight as previously described (19).
Determination of the ehrlichial load in collected organs by quantitative real-time PCR.To determine the relative differences in the ehrlichial burden between the different groups of mice, approximately 10 mg each of liver, lung, spleen, and kidney were collected at different time points postinfection and homogenized. DNA was extracted using a DNeasy tissue kit (Qiagen, Valencia, CA) with an elution volume of deionized water of 100 μl, and ehrlichial burdens were determined using an iCycler IQ multicolor real-time detection system (Bio-Rad, Hercules, CA). The following primers (Sigma-Genosys, St. Louis, MO) and probes (Biosearch Technologies, Novato, CA) targeting both the IOE and E. muris dsb (which encodes a thio-disulfide oxidoreductase) and host glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used: EM/IOE dsb forward primer (5′ CAGGATGGTAAAGTACGTGTGA 3′), EM/IOE dsb reverse primer (5′ TAGCTAAYGCTGCCTGGACA 3′), EM/IOE probe (5′ 6-carboxyfluorescein-AGGGATTTCCCTATACTCGGTGAGGC-BHQ1 3′), GAPDH forward primer (5′ CAACTACATGGTCTACATGTTC 3′), GAPDH reverse primer (5′ CTCGCTCCTGGAAGATG 3′), and GAPDH probe (5′ Cy5-CGGCACAGTCAAGGCCGAGAATGGGAAGC-BHQ2 3′). The comparative cycle threshold method (4) was used to determine the ehrlichial burdens in the harvested organs.
Determination of serum cytokine and ALT concentrations.At different time points after infection, serum samples were collected from infected mice. IFN-γ, TNF-α, and IL-10 levels were determined by using a Quantikine enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN) according to the manufacturer's recommendations. The serum concentrations of the liver enzyme alanine transaminase (ALT) (an enzyme that is released from injured hepatocytes) were determined by the Clinical Chemistry Laboratory, University of Texas Medical Branch.
Histology and ultrastructural analysis.Formalin-fixed, paraffin-embedded samples from the liver, lung, and spleen were sectioned and stained with hematoxylin and eosin (H&E). Histological sections were evaluated qualitatively, and liver lesions were assessed by using four parameters, hepatocyte damage, frequency of lesions, size of inflammatory lesions, and extent of lobular and perivascular inflammation, as described previously (30).
Isolation of splenic and hepatic mononuclear cells.Spleens and livers were harvested at different time points postinfection, and mononuclear cells were purified for flow cytometric analysis. Splenocyte single-cell suspensions were prepared in RPMI 1640 medium (500 ml) supplemented with 10% heat-inactivated fetal bovine serum, 1% HEPES buffer, and 1% penicillin G (50,000 U)-streptomycin sulfate (50,000 μg). Hepatic mononuclear cells were isolated using a modified enzymatic dispersal protocol as described previously (28), which allows the maximum yield and viability of hepatic mononuclear cells. Briefly, mouse livers were perfused with cold phosphate-buffered saline through the portal vein, and liver tissues were collected in Hanks' balanced salt solution (HBSS) without Ca2+ and Mg2+ and washed. Each liver was then perfused with warm HBSS (37°C) with Ca2+- and Mg2+-containing collagenase IV (500 mg/liter; 312 U/mg), DNase I (50 mg/liter), fetal calf serum (FCS) (2%), and bovine serum albumin (0.6%). Tissue was teased apart into ∼1- to 3-mm fragments while it was submersed in the warm HBSS and then incubated at 37°C with frequent shaking. After 15 min cold HBSS without Ca2+ and Mg2+ was added to stop the enzymatic digestion. The tissue was then passed through a 30-μm cell strainer to remove any large clumps or undissociated tissue. The filtered suspension was then centrifuged (500 × g) for 10 min at 4°C, and the cell pellet was washed twice in cold HBSS. Hepatocytes were removed by differential centrifugation (36 × g) for 1 min at 4°C, and the final pellet was resuspended in RPMI 1640 medium containing 10% FCS and 1% HEPES. Hepatic mononuclear cells were further purified for intracellular cytokine staining by overlaying cells obtained from each mouse liver carefully onto 5 ml of Lympholyte M (Cedarlane Laboratories, Burlington, NC), followed by centrifugation and collection of the upper two-thirds of the resulting density gradient. Trypan blue dye exclusion was used to determine cell viability.
Flow cytometry and intracellular cytokine staining.Splenocytes and/or hepatic mononuclear cells were harvested, counted, resuspended in fluorescence-activated cell sorter staining buffer (Dulbecco's phosphate-buffered saline without Mg2+ or Ca2+ containing 1% heat-inactivated FCS and 0.09% sodium azide), and filtered (0.2-μm-pore-size membrane), the pH was adjusted to 7.4 to 7.6, and aliquots were placed into a 96-well V-bottom plate (Costar, Corning, NY) at a concentration of 106 cells per well. Fc receptors were blocked with a monoclonal antibody (clone 2.4G2) against mouse CD16 and CD32 cell surface antigens for 15 min. The cells were then washed and pelleted. Optimal concentrations were determined for the following monoclonal antibodies: allophyocyanin-Cy5-conjugated CD3 (clone 145-2C11) and CD11c (clone HL3); fluorescein isothiocyanate-conjugated CD4 (clone GK1.5), B220 (clone RAB-632), and Fas (clone FAS); phycoerythrin-conjugated CD8a (clone 53-6.7), CD40 (clone 3/23), and granzyme B (eBioscience, San Diego, CA) (clone 16G6); and peridin-chlorophyll protein-conjugated CD11b (clone M1/70), CD4 (clone RM4-5), and CD8 (clone 53-6.7). Corresponding isotype controls were used. All antibodies were obtained from BD Pharmingen (San Diego, CA) unless otherwise indicated. For ex vivo intracellular granzyme B detection, splenocytes or hepatic mononuclear cells were incubated at 37°C for 4 h in complete medium supplemented with BD Golgi plug (BD Pharmingen) according to the manufacturer's recommendations. Lymphocyte populations were gated based on forward and side scatter parameters, 20,000 to 50,000 events were collected using a BD FACSCalibur (BD Immunocytometry Systems, San Jose, CA) flow cytometer with CellQuest software (Immunocytometry Systems), and data were analyzed using FlowJo (Tree Star Inc., Ashland, OR) flow cytometric analysis software.
Statistical analysis.Data were analyzed using SigmaPlot software (SPSS, Chicago, IL), and P values were determined using the Student two-tailed t test. P values less than 0.01 were considered highly significant, and P values less than 0.05 were considered significant. Mouse groups contained three mice unless otherwise indicated, and the standard deviation was determined for each group. The standard error of the mean was used for analysis of combined data from more than one experiment.
Susceptibility, in vivo bacterial burdens, TNF-α production, and pathological changes in the liver of WT BALB/c mice following lethal ehrlichial infection are similar to those of WT C57BL/6 mice.To determine whether the outcome of ehrlichial infection is influenced by the genetic background of BALB/c mice, we compared the host defenses and immune responses to Ehrlichia of these mice to those of C57BL/6 WT mice. Both groups of WT mice succumbed to i.p. inoculation with a high dose of IOE by day 8 postinfection (Fig. 1A). No significant differences in organ bacterial burdens were observed on day 5 (data not shown) or day 7 postinfection (Fig. 1B). We next examined whether BALB/c mice developed toxic shock-like syndrome following lethal ehrlichial infection like C57BL/6 mice (19). To this end, we characterized some of the main features of Ehrlichia-induced toxic shock, including high serum levels of TNF-α and hepatic injury. Similar to infected C57BL/6 mice, WT BALB/c mice infected i.p. with a high dose (∼1 × 105 genome copies) of IOE had high concentrations of serum TNF-α (Fig. 1C) and serum ALT (Fig. 1D) on day 5 postinfection. Similar to analyses of IOE-infected C57BL/6 mice (19, 20, 36, 37), hepatic histopathology analysis of WT BALB/c mice showed apoptosis of both Kupffer cells and hepatocytes on day 5 postinfection and severe multifocal hepatic necrosis on day 7 postinfection (data not shown). These data suggest that lethal ehrlichial infection causes pathology and fatal disease in both WT strains of mice, indicating that IOE induction of toxic shock-like syndrome occurs independent of the genetic background of the mice.
Resistance to Ehrlichia-induced toxic shock in the absence of CD1d and CD1d-reactive NKT cells.To identify a role for CD1d-restricted NKT cells in Ehrlichia-induced fatal toxic shock syndrome, we examined the effect of ehrlichial infection on the host defenses and the pathogenesis of ehrlichiosis in WT BALB/c and NKT cell-deficient CD1d−/− mice with a BALB/c background. Because CD1d is required for positive selection of NKT cell precursors in the thymus (in addition to stimulation of variant NKT and iNKT cells in the periphery), CD1d−/− mice lack the CD1d-restricted iNKT cell population (27). Briefly, WT and CD1d−/− mice were infected i.p. with a high dose of IOE (∼1 × 105 genomes copies), their survival was monitored, and the host immune response was evaluated on days 5 and 7 postinfection. BALB/c CD1d−/− mice succumbed to high-dose IOE infection on day 8 postinfection, 1 day after the death of WT BALB/c mice (Fig. 2A). However, unlike WT BALB/c mice, CD1d−/− mice did not develop signs of toxic shock-like syndrome. Compared to WT mice on day 5 postinfection, fatal ehrlichial infection did not result in weight loss or hypothermia (data not shown) in CD1d−/− mice. In addition, the IOE-infected CD1d−/− mice had significantly lower serum concentrations of the liver enzyme ALT on day 5 postinfection (Fig. 2B).
Survival, serum levels of liver enzyme and cytokines, and organ bacterial burdens in IOE-infected CD1d−/− mice and BALB/c WT controls. (A) Survival rates of BALB/c CD1d+/+ and CD1d−/− mice following i.p. inoculation of a high dose of IOE (1 × 105 genome copies). There was no significant difference in survival between the two groups of mice (nine mice per group). The data represent the results of one of three independent experiments with a total of nine mice per group. (B to E) Serum levels of ALT, TNF-α, IFN-γ, and IL-10 on day 5 following i.p. infection of knockout and WT BALB/c mice with a high dose of IOE. CD1d−/− mice had significantly lower (P < 0.01) serum levels of ALT (B) and TNF-α (C) than infected WT mice. The serum levels of IFN-γ (D) and IL-10 (E) were not significantly different for the two groups. (F and G) Ehrlichial burdens in different organs of CD1d−/− and WT mice on days 5 (F) and 7 (G) postinfection. CD1d−/− mice had significantly (P < 0.01) greater bacterial burdens in all analyzed organs on day 5 postinfection and in only the liver on day 7 postinfection. The bars indicate the means and the error bars indicate the standard deviations for three mice per group. Similar results were obtained in three independent experiments. P values less than 0.01 were considered highly significant (filled diamond), and P values less than 0.05 were considered significant (asterisk). KO, knockout.
We next examined the effects of the CD1d molecule and NKT cells on the systemic levels of cytokines known to be associated with Ehrlichia-induced toxic shock. Similar to the results of our previous studies (37) using WT C57BL/6 mice, fatal disease in IOE-infected BALB/c mice was associated with significant high serum levels of TNF-α, IFN-γ, and IL-10 (Fig. 2 C to E). In contrast, CD1d−/− mice had lower concentrations of TNF-α (Fig. 2C) but not of IFN-γ (Fig. 2D) or IL-10 (Fig. 2E) on day 5 postinfection.
Our previous studies demonstrated that IOE-induced tissue damage and toxic shock occur in WT C57BL/6 mice in the absence of overwhelming infection. Therefore, we first compared the differences in bacterial burden between the WT BALB/c and knockout mice using real-time PCR. The CD1d−/− mice had significantly higher ehrlichial burdens in the liver, lung, spleen, and kidney on day 5 postinfection and in the liver on day 7 postinfection (Fig. 2F and 2G). The difference in bacterial burden was most striking in the liver on day 5 postinfection, in which the CD1d−/− mice had a fourfold-greater ehrlichial burden than the WT mice. Examination of liver histopathology in infected CD1d−/− mice on day 5 postinfection revealed less inflammation and very few apoptotic cells (Fig. 3A and 3C) compared to WT BALB/c mice, in which acute inflammation and a significantly higher number of apoptotic liver cells, including both hepatocytes and cells lining the sinusoidal spaces and vascular lumens (i.e., Kupffer cells, monocytes, and/or endothelial cells) (Fig. 3B and 3C), were observed. No evidence of necrosis was detected in BALB/c WT or CD1d−/− mice on day 5 postinfection. Electron microscopic analysis of livers from infected WT and CD1d−/− mice further confirmed the difference in apoptosis between the two groups and also demonstrated that most apoptotic cells were indeed Kupffer cells/macrophages (data not shown). Interestingly, on day 7 postinfection, WT and CD1d−/− mice both developed multifocal hepatocellular necrosis similar to that previously reported for WT C57BL/6 mice (37). Thus, although the absence of CD1d and iNKT cells did not prevent Ehrlichia-induced mortality, the CD1d molecule and/or CD1d-restricted NKT cells mediated the early systemic overproduction of proinflammatory TNF-α, apoptotic death of host cells, liver injury, and hypothermia that collectively comprise the major manifestations of Ehrlichia-induced toxic shock syndrome.
Differences in hepatic histopathology between WT BALB/c and CD1d−/− mice. CD1d−/− mice (A) had fewer apoptotic cells (arrows) in the liver than CD1d+/+ WT BALB/c mice (B) on day 5 postinfection. H&E-stained liver sections showed similar results for three mice per group. (C) Numbers of apoptotic cells or nuclei and apoptotic bodies present in H&E-stained liver sections from each mouse in 10 medium-power fields (magnification, ×200). The bars indicate the means and the error bars indicate the standard deviations for three mice per group. Similar results were obtained in two independent experiments. P values less than 0.01 were considered highly significant (filled diamond). The cells undergoing apoptosis were similar to those reported previously (36) and included sinusoidal lining cells, as well as hepatocytes. KO, knockout.
Absence of CD1d and CD1d-reactive NKT cells restores the populations of splenic CD4+ and CD8+ T cells.We demonstrated previously (18, 19, 37) that fatal Ehrlichia-induced toxic shock-like syndrome is associated with significant apoptosis of splenic CD4+ T cells and decreases in the percentages and absolute numbers of CD4+ and CD8+ T cells in the spleen. Substantial lymphopenia has also been reported in patients with severe disease. To determine the role of CD1d and CD1d-restricted NKT cells in T-cell apoptosis, we examined the differences in the percentages of CD4+ and CD8+ lymphocytes in the WT and CD1d−/− mice by flow cytometry. The IOE-infected CD1d−/− mice had higher percentages (Fig. 4A) and absolute numbers of splenic CD4+ and CD8+ T cells than the WT mice on day 7 postinfection (the splenocyte mononuclear cell yields determined by trypan blue exclusion were similar in the two groups of mice [data not shown]). These data suggest that CD1d-restricted NKT cells mediate the significant reduction in the total number of CD4+ and CD8+ T cells.
Absence of NKT cells in IOE-infected CD1d−/− mice restored the percentages of both CD4+ and CD8+ T cells in the spleen and decreased Fas expression on CD4+ T cells and granzyme B expression in CD8+ T cells. BALB/c WT and CD1d−/− mice were inoculated with a high dose of IOE. The percentages and absolute numbers of CD4+ and CD8+ T cells, as well as the expression of Fas and granzyme on splenic CD4+ and hepatic CD8+ T cells, were determined by flow cytometry on day 7 postinfection. Lymphocytes were gated based on size and granularity, and 50,000 events were studied. (A) CD1d−/− mice had significantly higher percentages and absolute numbers of CD4+ and CD8+ T cells (splenocyte yields were not significantly different as determined by trypan blue exclusion) than CD1d+/+ BALB/c mice on day 7 postinfection. The bars indicate the means and the error bars indicate the standard deviations for three mice per group. Similar results were obtained in two independent experiments. (B and C) CD1d−/− mice had lower expression of Fas on splenic CD3+ CD4+ T cells and of granzyme B in hepatic CD3+ CD8+ T cells on day 7 postinfection. The dot plots shown are similar to those obtained for all three mice per group, and the data are representative of two independent experiments. The percentages below the plots are the percentages of Fas+ CD4+ T cells (upper values) and granzyme-positive CD8+ T cells (lower values) shown in the right upper quadrants of each dot plot. (D and E) Percentages of splenic CD3+ CD4+ T cells and hepatic CD3+ CD8+ T cells expressing Fas and granzyme B, respectively, shown graphically for statistical comparison. The bars indicate the means and the error bars indicate the standard deviations for three mice per group. P values less than 0.01 were considered highly significant (filled diamond), and P values less than 0.05 were considered significant (asterisk). KO, knockout.
Fas/FasL interaction plays a role in CD1-restricted-NKT-mediated apoptosis of CD4+ T cells.In several infectious and noninfectious disease models, it has been demonstrated that the induction of apoptosis in CD4+ T cells, as well as cytotoxic functions acquired by NKT cells, involves the Fas/FasL pathway (2, 39). We hypothesized that NKT cells mediate apoptosis of Ehrlichia-specific CD4+ effector cells via this mechanism. Consistent with this hypothesis, we observed that lethal ehrlichial infection resulted in up-regulation of Fas expression on splenic CD4+ T cells from WT and CD1d−/− mice (Fig. 4B and 4D) on day 7 postinfection. However, the absence of CD1d-restricted NKT cells in infected CD1d−/− mice significantly down-regulated the expression of Fas on splenic CD4+ T cells compared to the cells of infected WT mice (Fig. 4B and 4D).
CD1d-restricted NKT cell-mediated activation of cytotoxic CD8+ T cells involves granzyme B.As mentioned above, our previous studies demonstrated that there is a strong correlation between fatal disease and the expansion of TNF-α-producing CD8+ T cells. In addition, we recently reported that β2m−/− mice that lack both CD8+ T cells and CD1d-restricted NKT cells are more resistant to lethal ehrlichial infection than WT mice (18). To determine the contribution of CD1d-restricted NKT cells to the induction of pathogenic cytotoxic CD8+ T cells, we examined differences in intracellular expression of granzyme B (an important molecule involved in cytotoxic effector functions of CD8+ T cells) (32) in hepatic CD8+ T cells from CD1d−/− and WT IOE-inoculated mice. We chose the liver since it is the predominant organ that has marked ehrlichial tropism as well as extensive Ehrlichia-induced pathology. Ehrlichial infection of the CD1d−/− mice resulted in expression of intracellular granzyme B in CD3+/CD8+ T cells that was significantly lower than that observed in the WT mice (Fig. 4C and 4E). Taken together, our data suggest that CD1d-restricted NKT cells could potentially mediate apoptosis of CD4+ T cells via the Fas/FasL pathway and induction of cytotoxic CD8+ T cells in Ehrlichia-induced toxic shock.
IOE infection suppresses the expression of the costimulatory molecule CD40 on DCs, while NKT cells up-regulate CD40 expression on APCs.Based on the data described above, we hypothesized that NKT cells mediate induction of pathogenic CD8+ T cells and play a direct role in tissue injury and apoptosis of host cells during Ehrlichia-induced toxic shock via cognate interactions with APCs, including macrophages, DCs, and B cells. To test this hypothesis, we first quantified differences in CD11b+ cell frequency in harvested splenocytes by flow cytometry. Our data showed that CD1d−/− mice had significantly higher percentages and absolute numbers of CD11b+ cells in the spleen on day 5 postinfection (Fig. 5A). These observations correlate with the decreased number of apoptotic hepatic phagocytic cells and hepatocytes in the IOE-infected CD1d−/− mice compared to WT mice (Fig. 3A and B).
Absence of NKT cells resulted in significantly greater numbers of splenic CD11b+ cells but significantly decreased the expression of the costimulatory molecule CD40 on splenic CD11b+, CD11c+, and B220+ cells on day 5 postinfection. BALB/c mice were inoculated with IOE (1 × 105 IOE genome copies) and were sacrificed on day 5 postinfection. Splenocytes were isolated and stained with fluorescently labeled murine monoclonal antibodies, which was followed by flow cytometric analysis, and 20,000 events were collected. (A) CD1d−/− mice had significantly higher percentages of CD11b+ cells than WT mice. (B) CD11b+, CD11c+, and B220+ populations of cells were gated within their representative dot plots, and the mean fluorescence intensity of CD40 expression was determined using FlowJo. The results show that there was significant suppression of CD40 expression on CD11c+ cells in WT mice during IOE infection compared to uninfected WT mice. In addition, decreased expression of CD40 on CD11c+, CD11b+, and B220+ cells was detected in infected CD1d−/− mice compared to infected WT mice on day 5 after IOE infection. The data are representative data for two independent experiments. P values less that 0.01 were considered highly significant (filled diamond). KO, knockout.
We next determined the effect of IOE-activated NKT cells on the activation and/or maturation of APCs by measuring differences in the up-regulation of the costimulatory molecule CD40. CD40 was chosen since it is the main costimulatory molecule that provides strong DC maturation signals through binding of CD40L on the surface of activated conventional T cells or NKT cells (11). In addition, increasing CD40 expression on APCs is a mechanism by which a robust CTL response can be initiated in the absence of CD4 help (10), which is important during severe ehrlichiosis, in which a significant decrease in the number of CD4+ Th1 cells has been observed (19). Compared to uninfected controls, IOE infection of WT BALB/c mice resulted in down-regulation of CD40 expression on CD11c+ DCs but not on CD11b+ CD11c− macrophages or B220+ cells (Fig. 5B). Further down-regulation of CD40 expression on CD11c+ DCs was observed in the absence of CD1d-restricted NKT cells in infected CD1d−/− mice. Similarly, CD40 expression was significantly lower on CD11b+ CD11c− macrophages and B220+ cells in the CD1d−/− mice than in the WT mice. The difference was most significant (P < 0.001) for B220+ cells (Fig. 5B). These data suggest that unlike the maturation of other APCs, DC maturation is suppressed directly by IOE and that CD1d-restricted NKT cells promote up-regulation of costimulatory molecules during ehrlichial infection.
Previous studies in our laboratory have shown that mice lacking functional CD8+ T and NKT cells (β2m−/−) are more resistant to Ehrlichia-induced toxic shock than mice deficient in only CD8+ T cells (TAP−/−), suggesting not only that pathogenic CD8+ T cells mediate Ehrlichia-induced toxic shock-like syndrome but also that NKT cells are involved in ehrlichial pathogenesis (18). Using a mildly virulent E. muris strain, we have also shown that NKT cells are directly activated by E. muris in a CD1d-dependent manner and that NKT cells play a critical role in protection against in vivo infection by E. muris (26). Because of these observations and the fact that NKT cells are abundant in the liver, the main organ with marked ehrlichial tropism, we examined the role of CD1d and CD1d-restricted NKT cells in fatal ehrlichial infection caused by the highly virulent organism IOE. In this study, we showed that CD1d-restricted NKT cells mediate toxic shock-like syndrome. Similar to IOE-infected C57BL/6 mice, WT BALB/c mice infected with a high dose of IOE develop toxic shock-like syndrome that closely mimics severe HME in immunocompetent patients and is characterized by lymphopenia, hepatic apoptosis, lymphohistiocytic infiltrates in the liver, and elevation of the levels of hepatic enzymes (34, 36). These observations were similar to those previously reported by Bitsaktsis et al., who compared IOE infection of WT BALB/c mice and IOE infection of C57BL/6 mice (7). These data allowed us to further examine the role of NKT cells using CD1d−/− mice with the BALB/c background.
Although both the WT and knockout mice succumbed to disease and all mice became terminally ill by days 7 and 8 postinfection (Fig. 2A), respectively, the pathogenesis of fatal disease in NKT cell-competent mice and the pathogenesis in NKT cell-deficient mice appear to be mediated by different mechanisms. Increases in the levels of the serum cytokines TNF-α, IL-10, and IFN-γ have been observed during Ehrlichia-induced toxic shock (37). Significantly lower levels of serum TNF-α (Fig. 2B) were observed in the knockout mice than in the WT mice, indicating that NKT cells are directly or indirectly responsible for the production of this proinflammatory cytokine during Ehrlichia-induced toxic shock-like syndrome. The observation that abrogation of systemic TNF-α production does not protect IOE-infected CD1d−/− mice from lethal disease is consistent with our previous data showing that TNF neutralization fails to protect IOE-infected WT mice from lethal infection and results in less apoptosis in the liver. On the other hand, TNF receptor-deficient mice are partially protected (20), suggesting that TNF is not solely responsible for Ehrlichia-induced fatal disease and that other mechanisms mediate this fatal disease. An interesting finding is that despite the normal levels of serum TNF-α, the absence of NKT cells did not influence the high serum levels of IFN-γ or IL-10 (Fig. 2C and 2D), which were also observed in infected WT mice. The presence of high levels of IL-10 in CD1d−/− mice is also consistent with our previous data suggesting that IL-10 is linked to fatal disease. However, the source of IL-10 appears to be independent of NKT cell activation.
As mentioned above, the liver is one of the main sites of pathology during severe ehrlichiosis, and relatively high concentrations of NKT cells are found in this organ. We focused our investigations on several different characteristics of liver involvement, including the serum levels of ALT, the hepatic bacterial burdens, and liver histopathology. The differences in these parameters between the WT and CD1d−/− mice showed that CD1d-restricted NKT cells mediate early liver damage following lethal ehrlichial infection. The infected CD1d−/− mice had significantly lower levels of serum ALT (Fig. 2B), higher bacterial burdens in the liver on days 5 and 7 postinfection (Fig. 2F and 2G), much less apoptosis of both hepatocytes and Kupffer cells, and less inflammation in the liver on day 5 postinfection (Fig. 3). The bacterial burdens in the lung, spleen, and kidney were also significantly greater in the absence of NKT cells on day 5 postinfection but not on day 7 postinfection, suggesting that NKT cells assist in controlling the bacterial burden in these organs only at early stages of ehrlichial infection (Fig. 2F). One possible explanation accounting for the differential effects of NKT cells on the bacterial burdens in different organs could be the abundance of NKT cells in the liver and the ability of these cells to eliminate infected target cells via engagement of death receptors. In support of this conclusion, we also observed that mice lacking NKT cells had significantly higher percentages (Fig. 4A) and absolute numbers (data not shown) of CD11b+ phagocytic cells than the WT controls. Numerous hepatocytes were apoptotic in the WT mice, and as previously reported by Sotomayor et al. (36), most of these dead hepatocytes did not contain ehrlichial antigen as determined by immunohistochemical staining. Several studies have suggested that apoptosis leads to secondary necrosis and activation of CD8+ T cells via cross-priming pathways (31). Thus, cross-presentation of apoptotic cells that contain ehrlichial antigens by DCs may assist in the activation of pathogenic cytotoxic CD8+ T cells.
Next, we examined the role of CD1d-restricted NKT cells and CD1d molecules in impeding induction of the specific acquired immune response against Ehrlichia for two reasons. First, lymphopenia occurs during severe HME (15, 33) and in animal models of fatal ehrlichiosis (7). Second, recently, CD4+ T-cell apoptosis was reported to occur during severe ehrlichiosis (18) and occurs in other septic conditions secondary to activation-induced cell death, which has also been linked with IL-10-mediated up-regulation of Fas (1). The absence of NKT cells resulted in significantly decreased expression of Fas death receptors on splenic CD3+ CD4+ T cells (Fig. 4B and 4D). In addition, the absence of NKT cells resulted in decreased activation of cytotoxic CD8+ T cells, as shown by decreased intracellular granzyme B expression in hepatic CD3+ CD8+ T cells (Fig. 4C and 4E) from IOE-infected CD1d−/− mice compared to IOE-infected WT controls on day 7 postinfection. These observations correlated with the observed differences in the percentages and absolute numbers of CD4+ and CD8+ T cells in the spleen (Fig. 4A). We hypothesize that splenic CD4+ T cells die prematurely during ehrlichial infection due to Fas-mediated activation-induced cell death and that NKT cells mediate the induction of pathogenic cytotoxic CD8+ T cells, followed by their migration to the main sites of infection, such as the liver. A recent study in our laboratory supported this concept as only splenic CD4+ T cells, and not splenic CD8+ T cells, concurrently express the early apoptotic marker annexin V (18). IL-10 may also be involved in the early induction of apoptosis of CD4+ T cells, as this is a known mechanism by which immune homeostasis is reestablished postinfection (3) and systemically IL-10 is present at high concentrations late during severe ehrlichiosis. In addition, IFN-γ has been shown to promote FasL- and perforin-mediated hepatocellular destruction by CTLs (32). Although our previous study showed that mice lacking perforin did not survive ehrlichial infection, granzyme B may still be important in IOE-induced toxic shock-like syndrome for at least two reasons. First, severe ehrlichiosis, like other septic conditions, appears to be a multifactorial process in which elimination of one factor or cell type is not sufficient to protect mice from fatal disease. Second, recent reports have identified a perforin-independent pathway for target cell killing by granzyme B (1, 13).
NKT cells can bias the immune response toward a Th1 or Th2 phenotype by producing high concentrations of particular cytokines early during the immune response; additionally, they also express CD40L on the cell surface, which has been reported to increase the expression of the costimulatory molecule, CD40, on APCs. Up-regulation of CD40 has been described as "empowering" APCs to be more efficient in presenting antigen and in producing cytokines and thus be more robust activators of the adaptive immune response, including CTLs (10, 25). Therefore, we examined whether NKT cells mediate toxic shock and liver injury via their cognate interaction with APCs. CD1d−/− mice that lack iNKT cells had significantly lower expression of CD40 on APCs, including DCs, phagocytic cells, and B cells, suggesting that NKT cells up-regulated CD40 expression on these cells. Interestingly, IOE infection decreased CD40 expression on CD11c+ cells, as indicated by the reduction in the level of this costimulatory molecule in both WT and CD1d−/− Ehrlichia-infected mice compared to uninfected control mice. Increasing the level of CD40 on APCs can mediate either protective or pathogenic immune responses depending on the cytokine environment and type of APCs. For example, an increased level of CD40 results in increased production of reactive oxygen species and TNF-α by macrophages (16, 22), and CD40-CD154 (CD40L) ligation is required for IL-12 production and induction of Th1 immunity by CD8α+ DCs in vivo (43). Alternatively, agonists of either CD40 on DCs (14) or CD40L on CD8+ T cells (10) can initiate a CTL response independent of CD4+ T-cell help. This situation is particularly important during severe ehrlichiosis, in which a decrease in the number of protective IFN-γ-producing CD4+ T cells and an increase in the number of pathogenic TNF-α-producing CD8+ T cells have been observed (19). The observed differences in the expression of CD40 on APCs may have resulted in differences in CD4+ and/or CD8+ T-cell activation between the WT and CD1d−/− mice. Additionally, CD40-CD154 ligation also contributes to lethality in cecal ligation- and puncture-induced sepsis (12).
In a recent review Tupin et al. (42) thoroughly analyzed the different roles of invariant NKT cells in response to a variety of infectious agents, including bacteria, viruses, and parasites. These authors reported the roles of Vα14 iNKT cells in bacterial infections, and in many infections, such as infections with Streptococcus pneumoniae (21), Pseudomonas aeruginosa (29), and Borrelia burgdorferi (23), these cells played protective roles by prolonging survival or decreasing the bacterial burden. In contrast, in infections with other disease-causing bacteria, such as Chlamydia trachomatis, NKT cells induced a pathogenic response (6). To our knowledge, fatal ehrlichiosis is the first bacterium-mediated disease for which both beneficial and detrimental roles have been observed for NKT cells. These cells are protective because they help control the initial bacterial burden; however, they also play a role in inducing toxic shock-like syndrome by playing a role in increasing the serum TNF-α concentrations and liver injury, promoting Fas-induced apoptosis of protective CD4+ T cells, and inducing pathogenic granzyme B-producing CD8+ T cells. The dual roles played by NKT cells during fatal ehrlichiosis need to be considered when vaccines or other immunotherapeutics targeting these and other cells capable of affecting multiple downstream events are designed.
We thank Sherrill Hebert and Doris Baker for their excellent secretarial assistance, Colette Keng and Jennifer Buezo for their technical assistance, and Linda Muehlberger for expert histological preparations.
This work was funded by grant AI31431 to D.H.W. and Emerging and Tropical Infectious Diseases training grant T32 AI7526 to H.L.S. from the National Institute of Allergy and Infectious Diseases.
Received 10 September 2007.
Returned for modification 22 October 2007.
Accepted 15 January 2008.
↵▿ Published ahead of print on 22 January 2008.
Editor: R. P. Morrison
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Infection and Immunity Mar 2008, 76 (4) 1434-1444; DOI: 10.1128/IAI.01242-07
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Nuclear medicine is considered mainly as one of the outpatient facilities that provide patients diagnostic services except for the therapy patients who may require isolation for a period away from other patients. For that reason, the patient's services within these units are varied widely and require more patient care techniques. The aim of the present study is to assess the patient satisfaction for the Nuclear Medicine Services in Riyadh region, Kingdom of Saudi Arabia. The study used the survey method for assessment of the patient satisfaction. The purpose of this survey was to gather meaningful opinions, comments, and feedback from patients visiting the nuclear medicine department in different hospitals. Statistical analysis was done for the survey output results to identify the area of strengths and the areas need more improvements. It was concluded that the politeness of the department employees, the privacy of the examination and guaranteed confidentiality of the treatment scored the highest satisfaction mean scores recorded by the patients. On the other hand, multiple appointment options, number of seats available in the waiting room, and cleanliness of the bathrooms had the lowest mean scores.


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Q: Write CSV file inside zip file using FileSystem I want read the CSV files inside the zip file and edit the record based on some condition. I am using FileSystem class to create zip file system and then edit csv files. I used the below code read csv files
public static List<String> getFileList() {
        List<String> fileList = new ArrayList<>();
        ZipFile zip = null;
        try {
            zip = new ZipFile(zipFile);
            Enumeration<? extends ZipEntry> entries = zip.entries();
            while (entries.hasMoreElements()) {
                ZipEntry entry = entries.nextElement();
                if (entry.getName().contains("**") && !entry.getName().contains("***")) {
                    fileList.add(entry.getName());
                }
            }
        } catch (IOException e) {
            e.printStackTrace();
        }
        return fileList;
    }

    public static void modifyTextFileInZip() throws IOException {
        Path zipfilePath = Paths.get(zipFile);

        try (FileSystem fs = FileSystems.newFileSystem(zipfilePath, null)) {
            for (String file : getFileList()) {
                Path source = fs.getPath(file);
                String tempFileDest = source.getParent().toString() + "/" + "temp_" + source.getFileName();
                tempFileDest = tempFileDest.replace(".red", "");
                Path temp = fs.getPath(tempFileDest);
                editFiles(source, temp);
            }
        }
    }

    public static void editFiles(Path source, Path dest) {
        CSVReader reader;
        try {
            reader = new CSVReader(new InputStreamReader(Files.newInputStream(source)));
            List<String[]> records = reader.readAll();
            Iterator<String[]> iterator = records.iterator();

            Optional<String> fileName =
            Files.list(source.getParent())
                    .filter(file -> file.getFileName().toString().endsWith("***") &&
                            !file.getFileName().toString().endsWith("****"))
                    .map(file -> file.toString())
                    .findFirst();
            //System.out.println(fileName);

            while (iterator.hasNext()) {
                String[] record = iterator.next();
                String val = null;
                if("****".equals(record[5])){
                    try {
                        val = fileName.orElseThrow(() -> new RuntimeException("File Not found"));
                        val = "**" + val;
                        //System.out.println(val);
                    } catch(Exception e){
                        System.out.println("File not Found");
                    }
                    record[5] = val;
                }

                for(String cell: record) {
                    System.out.print(cell);
                }
                System.out.println();
            }

            Files.createFile(dest);
            PrintWriter outputFile = new PrintWriter(Files.newOutputStream(dest));
            CSVWriter writer = new CSVWriter(outputFile);
            writer.writeAll(records);

            writer.close();

        } catch (Exception e) {
            e.printStackTrace();
        }
    }

erro log =>
java.nio.file.FileSystemException: C:\zipfile\P003.zip: The process cannot access the file because it is being used by another process.

    at sun.nio.fs.WindowsException.translateToIOException(WindowsException.java:86)
    at sun.nio.fs.WindowsException.rethrowAsIOException(WindowsException.java:97)
    at sun.nio.fs.WindowsException.rethrowAsIOException(WindowsException.java:102)
    at sun.nio.fs.WindowsFileSystemProvider.implDelete(WindowsFileSystemProvider.java:269)
    at sun.nio.fs.AbstractFileSystemProvider.delete(AbstractFileSystemProvider.java:103)
    at java.nio.file.Files.delete(Files.java:1126)
    at com.sun.nio.zipfs.ZipFileSystem.sync(ZipFileSystem.java:1294)
    at com.sun.nio.zipfs.ZipFileSystem.close(ZipFileSystem.java:277)
    at com.shrikant.Main.Main2.modifyTextFileInZip(Main2.java:60)
    at com.shrikant.EditCsvFilesApplication.main(EditCsvFilesApplication.java:21)

I wans able to read the and edit the files using inputStream from Path object and then pass it to CSVReader object. But when I pass outputStream to CSVWriter and try to write data to temp file and can't see any changes inside zip file. And also I am getting a .tmp (zipfstmp3381860066025396844.tmp) file generated every I run my program.
I followed this => https://stackoverflow.com/a/43836969/20783127   to write this program
Don't know what's going wrong, I can anyone explain ?
I am trying read and edit the csv files in zip file using FileSystem class in java, but its not working

A: You did not zip.close(). Better use the try-with-resources syntax, which closes also when an exception is thrown or one returns inside the { }.
try (ZipFile zip = new ZipFile(zipFile)) {
    ...

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Amy Cook, JD
Law and Policy Analyst
Amy Cook is a Law and Policy Analyst with the Policy Surveillance Program at the Center for Public Health Law Research. She has spent her career in public service. Before joining the Policy Surveillance Program, Ms. Cook was a Policy Analyst at the American Board of Internal Medicine. Prior to that, she was a Presidential Management Fellow at the National Institutes of Health (NIH). As a Fellow, she rotated through the Workforce Support and Development Division, Office of the Director; the Division of International Science Policy, Planning and Evaluation at the Fogarty International Center; and the Office of the General Counsel's General Law Division, Procurement, Fiscal, and Information Law Branch, at the Department of Health and Human Services. While in law school, Ms. Cook had internships with the Office of General Counsel at the Thomas Jefferson University Hospital and with the Executive Office for Immigration Review with the United States Department of Justice. Ms. Cook has a bachelor's degree in political science from the University of Pittsburgh and a JD from the Temple University Beasley School of Law. She is licensed to practice law in Pennsylvania.


(4) Notice my Twitter Icon with the X - Notice The "X" in the center of the promotion with the "IamHeroX" and their icon as well.
Additionally, there is reference to DoD and #MicroElectronics.
(5) In summary, #TheProgram is an #extrajudicial #PoliticalTargeting protocol that utilizes tenets of the #Stasi #Zersetzung tactics, Russian #Kompromat, #FBI #COINTELPRO + Operation Phoenix.
(6) utilizing tactics that constitute #terrorism as per #DoD definition.
#PSYOPS is just part of this heinous cancer that now plagues America.
Remember people, we are all tracked - warrantlessly I might add - by our unique #biometric signatures, compliments of a few Fortune 50 companies, #NSA, #CyberCommand and US Mil.
#GPS is not needed to track people anymore.
(9) with this sudden "ghosting." It happened across the board to at least 6 followers within the past two weeks. Very noticeable.
(1) Should destroying a snowman on someone's front lawn, be considered an act of vandalism, trespassing, destruction of "personal" property ....?
Methinks this is a rich legal question.
Who did/does the snow belong to?
(2) If the snow is in your yard (maybe like apples from a tree) - is it your snow?
Is a snowman, snow-woman or SnowXi (can I copyright that word?) a "work of art?"
Can a temporary work of art be vandalized?
As part of the #dehumanization that #DHS #DOJ #FBI #JTTF subject their targets to, they create a lot of deviant & psychopathic fairy-tales, with which to smear their targets.
(2) such detailed stories of perversion, mental illness & deviancy? This is a #Stalinist tactic.
#DHS #FBI #JTTF have labeled millions of Americans as "threats" - and even worse.
(3) #InfraGard #CitizensCorp #JTTF - with their #ThreatFusionCenter partners are running around and destroying the lives of several million Americans with their #Kompromat #targeting charade.
Was a government experiment/program on DNA and genetic traits of Special Genes.
R is the R17 Genome.
R's DNA is the 1st , The Original DNA.
Is a Genetic modified human being.
Q was a Goverment experiment.
DNA was mixed from the Deceased & Living to create R.
R was designed to create a Genetically modified human with R17 Genome .
I want to know the truth !
I want this experiment to end.
The World needs to know the truth.
#Twitter, bot ve aktif olmayan hesapları temizleme işlemini bugün tekrardan gerçekleştirdi. Birçok kişinin takipçi sayısında azalma var.
Aynı zamanda #Twitter zamanında askıya alınan ve takipçimiz olan kişilerin hesaplarını da tamamen ortadan kaldırıyor. Takipçi azalmasının nedeni bunlar. Bu çalışmanın devamı gelecekmiş.
#Twitter profilinizde kaç reel kaç bot hesap var bu site/uygulama üzerinden görebilirsiniz.


text:
Police: Man 'fooling' on escalator falls to death at Providence Place mall
May 29, 2018 at 8:07 AM May 29, 2018 at 5:29 PM
PROVIDENCE, R.I. (AP) — A man who was apparently "fooling around" and riding an escalator rail lost his balance and fell to his death at a Rhode Island mall.
Providence Police Chief Hugh Clements told WPRO-AM the victim fell one floor, from the escalator at the movie theater level to the food court, at the Providence Place Mall just before 11 p.m. Monday.
Clements says the man lost his balance, fell backward, and hit his head on a table. He suffered "massive injuries" and was pronounced dead at the hospital.
The victim was a 45-year-old Pawtucket man. His name wasn't immediately released.
Police say the death appears to be accidental, but they'll review surveillance video.
There have been a handful of deaths at the mall since it opened in 1999, including other people falling from escalators. A man died in 2006 after he jumped between escalators and fell.