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Master teacher Sianna Sherman created this brief sequence integrating the physical and spiritual to take your practice to one more level.
Yoga's fame is now extensive around the world– and also still increasing by leaps as well as bounds. As an international tourist, I see the rise and autumn of different trends as well as the methods yogis are looking for adjustment as well as improvement in all areas of life. I often hear the comment that westerners only like the physical practice of yoga exercise however not the spiritual. In my encounter, yogis all over are asking for a combination of techniques. They are requesting the whole of the tradition.
Why Did You Begin Practicing Yoga?
Last month, I was teaching in a northern European nation regarding a mainly asana-based yoga exercise culture. In an open conversation regarding 40 yogis, I asked why they started the technique of yoga exercise. The breadth of their solutions represented the wide landscape of the human heart:
- to lift me out of darkness
- to recover me from my consuming disorders
- to help me loosen up from my intense company job
- to heal my injuries from an automobile accident
- to sleep at night
- to clear my mind
- to carry me through my grief when my other half died
- to be as healthy as possible
- to know my true Self
There were lots of profusions of the heart in these solutions. This was a group of outstanding asana specialists seeking the whole garland of practices.
What IS Yoga?
Yoga is an alchemical course, one that metaphorically transforms us from lead right into gold, spinning forth the elixir from within. The alchemy exists within the assimilation of the practices: asana, pranayama, rule, mudra, reflection, as well as a lot more. When life really feels stuck or stagnant, yoga exercise can aid us damage with resistance, heal unresolved wounds, as well as open paths for creative energy to flow.
Use this brief daybreak method to help you shift to a brand-new level, clear your thoughts, and also anchor your objective in your heart for a dynamic and healthy day.
Beyond-the-Breakthrough Sunrise Practice
Chant the Gayatri Mantra
Repeat 3 times with the daybreak
Gayatri is an universal mantra of spiritual illumination as well as invokes light bulb into the thoughts, heart, and also physical body. Gayatri is a goddess in the yoga exercise custom which increases up from a red lotus regarding 5 heads and also ten eyes, so she could see in all instructions plus earth as well as sky. She represents the power of awakening from our slumber and the rajas guna, or the fiery, energetic top quality of nature. Gayatri is a guard of the life force and also blesses with the vigor of the Sun.
Chant 3 rounds at sunrise:
Oṃ Bhūr Bhuvaha Svaha
Tat Savitur varēṇyam
Bhargo devasya dhīmahi
Dhiyo yo naf prachodayāt
Kapalabhati Pranayama
Kapala suggests "head" and also Bhati indicates "shining," "illuminating," or "radiance." Kapalabhati is the "skull-shining breath."
- Energizes
- Uplifts
- Clears sinuses
- Helps dispel slowness from system
- Sit in an unwinded as well as upright position.
- Relax your hands on your legs.
- Breathe a couple of rounds of smooth, deep Ujjayi pranayama.
- Begin a series of short, strong, and also active exhalations via the nose.
- Allow the inhalation to be all-natural and passive.
- The short effective exhalations will pump the low belly.
- Practice for up to one minute.
- Take a few clearing rounds of Ujjayi, after that repeat.
Vira Mudra as well as Uttara Bodhi Mudra
Courage Mudra and also Upward-Reaching Knowledge Mudra
- Invokes courage
- Awakens enlightening forces
- Lifts the energy
- Strengthens willpower
- Increases mental clarity
- Creates both agility and also groundedness
- Works like a lightning pole for motivation and clear sight
- Reach your arms up overhead.
- Extend your forefinger right up, intertwine the middle, ring, and little hands, as well as cross your left thumb over your right thumb.
Also try Enlightenment Meditation
Utkata Konasana regarding Vira Mudra
Goddess Stance regarding Nerve Mudra
- Grounds
- Energizes
- Helps to damage via stagnancy
- Spread your arms wide sideways as well as tip your feet apart as vast as your wrists.
- Turn your feet out and bend your knees over your ankles.
- Reach your arms up overhead as well as bring your hands right into Vira mudra.
Meditation with Svaha Mantra
Svaha is a concept for making providings into your interior fire.
- Visualize a fire at the origin of your heart.
- Relax your recognition into the circulation of your breath.
- Internally chant Svaha as you make providings right into the fire of your heart.
- Offer your fears, limitations, concerns, as well as doubts.
- Offer your thankfulness permanently and also the chance to transform.
- Rest in meditation for at the very least 10 mins to refresh your thoughts as well as rejuvenate in your internal light.
Remember: The morning sunlight consistently appears the darkest night. Deal your restrictions into the heart fire as well as you will experience your very own individual revolutionary and beyond.
Try Much more Practices from the Siren Yoga exercise Project
What Is Goddess Yoga?
The Goddess Every Vinyasa Flow Follower NECESSITY Know
5 Heart Openers Dedicated to Lakshmi
3-Step Meditation to Inspire Intuition
Defeat Fear with Sword Breath


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(Reuters) - Tropical Storm Harvey was weakening as it moved farther inland and flooding rains continued over extreme southeastern Texas and southwestern Louisiana on Wednesday, the National Hurricane Center said.
Harvey was about 35 miles (56 km) north-northwest of Lake Charles, Louisiana, and was packing maximum sustained winds of 40 miles per hour (64 km per hour), the NHC added.
Reporting by Nallur Sethuraman in Bengaluru; Editing by Jonathan Oatis

subset: commoncrawl


text:
The Profish 35 is a compact fishing kayak designed with lure fishing in mind. The shorter 3.5 meter length offers excellent manoeuvrability while hull shape delivers great load capacity and stability. This is a kayak for juniors and adults alike.
The large central storage well offers ample room for tackle and other essential gear. The removable lid provides security and easy access, and also makes an excellent mounting surface for electronics. The flush rod holders are within comfortable reach for larger and smaller paddlers, and the rear well offers ample storage for both fresh and salt water anglers.
Features you will enjoy:
Colors available:
Daybreak (Blue White Yellow)
Kiwi (Black Green)
Lava (Black Red)
Storm (Black White)


text:
From Japan
Times Online, October 11, 2008
A robotic suit that reads brain signals and helps people
with mobility problems became available this week to rent in
Japan for Ą220,000 ($2,100 U.S.) a month — an invention that may
have far-reaching benefits for the disabled and elderly.
HAL — short for hybrid assistive limb — is a computerized suit
with sensors that read brain signals directing limb movement
through the skin.
The 10-kg battery-operated computer system is belted to the
waist. It captures the brain signals and relays them to
mechanical leg braces strapped to the thighs and knees, which
then provide robotic assistance to people as they walk.
Cyberdyne, a new company in Tsukuba, Ibaraki Prefecture, will
mass-produce HAL. Two people demonstrated the suits at the
company's headquarters Tuesday.
A demonstration video also showed a partially paralyzed person
getting up from a chair and walking slowly wearing the HAL suit.
"We are ready to present this to the world," said Yoshiyuki
Sankai, a University of Tsukuba professor who designed HAL.
Sankai, who has worked on robotic suits since 1992 and is also
Cyberdyne's chief executive, said a full device that covers the
entire body is also being designed, though it is unclear when it
will be available commercially.
HAL comes in three sizes — small, medium and large — and also
has a one-leg version for a Ą150,000 monthly rental fee.
Noel Sharkey is a robotics expert not affiliated with the
technology. The professor at the University of Sheffield in
England said HAL will have wide-ranging benefits for the elderly
and others with movement disabilities.
"HAL can only lead to extending the abilities of the elderly and
keep them out of care for longer," Sharkey said in an e-mail.
Cyberdyne said its policy is not to reveal how much it costs to
manufacture the device. It is unclear when HAL will go on sale
to the public or what the price tag will be.
Robotics technology is common in manufacturing sectors, but
product liability concerns restrict its widespread use in
everyday life. Sankai said the HAL technology is devoted to
social welfare purposes only, adding he has refused requests
from military officials to share it.
Some European nations have already expressed interest, and HAL
may soon be on the market there, but U.S. sales are still
undecided, Sankai said.
Researchers at the University of California, Berkeley, and
elsewhere around the world are working on similar robotic suits
that increase mobility.
Daiwa House Industry Co. will lease HAL suits to Japanese care
facilities for the elderly and others for those with
disabilities. It plans to rent 500 units over the next year.
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text:
I finally have a Great Barrier Reef card. I wanted a card from there not only for beeing an Unesco site but also because the cards from there are always really beautiful. This one was sent by Heather "hmassese".
On the cards there's the Green Island, Upalu Cay, the Reef and Michaelmas Cay.
"The Great Barrier Reef is the world's largest reef system composed of over 2,900 individual reefs and 900 islands stretching for over 2,600 kilometres (1,600 mi) over an area of approximately 344,400 square kilometres (133,000 sq mi). The reef is located in the Coral Sea, off the coast of Queensland in north-east Australia.
The Great Barrier Reef can be seen from outer space and is the world's biggest single structure made by living organisms. This reef structure is composed of and built by billions of tiny organisms, known as coral polyps. This reef supports a wide diversity of life, and was selected as a World Heritage Site in 1981. CNN labeled it one of the seven natural wonders of the world. The Queensland National Trust named it a state icon of Queensland.
A large part of the reef is protected by the Great Barrier Reef Marine Park, which helps to limit the impact of human use, such as fishing and tourism. Other environmental pressures on the reef and its ecosystem include runoff, climate change accompanied by mass coral bleaching, and cyclic population outbreaks of the crowns-of-thorns starfish.
The Great Barrier Reef has long been known to and used by the Aboriginal Australian and Torres Strait Islander peoples, and is an important part of local groups' cultures and spirituality. The reef is a very popular destination for tourists, especially in the Whitsunday Islands and Cairns regions. Tourism is an important economic activity for the region, generating AU$ 1 billion per year." - in: wikipedia


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Our Dot Day celebrations spanned several days and then it took me a bit longer to pull everything together but here's a look at the students' creativity.
After brainstorming what a dot could become, the children were given a "swirly gold frame" from Fablevision's Dot Day Handbook and a dot sticker to make their own creation.
First Grade:
First graders decorated their dot by using the sheet from the ColAR website. ColAR Mix is an augmented reality coloring book app that brings images to life! Dot Day Fun! Watch Your Dot Come to Life describes what special things happen to a dot when viewed through this app. Talk about oohs and ahhs from students! What I found amusing was watching the teachers getting just as involved as the children!
Second Grade:
Second grade took this activity a step further. After discussing that, yes, even young children can make a difference in other people's lives, we brainstormed how this could happen. The students wrote their thoughts on their dot then decorated with crayons and markers. Then the fun began with the ColAR Mix app.
Third Grade:
After talking about how Vashti's teacher made a difference in her life and then how Vashti guided a little boy to "make his mark," the third graders were challenged to illustrate how they could make a difference in someone's life. Students used the Microsoft Paint program to create their pictures. Since not all students wrote how they would make a difference, what they were thinking might not be readily observed but all had something in mind. We had a fabulous discussion about how even the little things you do are important! Here is a look at what they drew.
Fourth Grade:
By the time the students came to me, they had either read The Dot or had watched a video of it but that didn't mean they didn't want to hear it again! And, when the students found out that I also had Peter Reynold's Ish and Sky Color, we couldn't even begin the activity until I had read BOTH books! The students decided they just wanted to create. Some created images from a dot while others made some type of "ish" picture. Then I decided to let them experiment with the ColAR Mix app. They weren't as "wowed" as the younger children but they did enjoy doing a walk-around to see what their classmates had created.
Co-Curricular Classes:
The music, art, and Spanish teachers incorporated dots into their lessons too. From dot paintings to dot music to oxcart wheels to mirrors, the students' imaginations flowed!
Another Dot Day has passed but the creativity will continue!


text:
Legal Status
Document Details
Published Document
Agency Holding the Meeting: United States International Trade Commission.
Time and Date: October 22, 2001 at 2:00 p.m.
Place: Room 101, 500 E Street SW., Washington, DC 20436 Telephone: (202) 205-2000.
Status: Open to the public.
Matters to be Considered:
1. Agenda for future meeting: none.
2. Minutes.
3. Ratification List.
4. Inv. Nos. 701-TA-365 and 731-TA-734-735 (Review)(Certain Pasta from Italy and Turkey)—briefing and vote. (The Commission is currently scheduled to transmit its determination and Commissioners' opinions to the Secretary of Commerce on October 29, 2001.)
5. Inv. No. TA-201-73 (Injury Phase)(Steel)—briefing and vote. (The Commission is currently scheduled to transmit its recommendations to the President on December 19, 2001.)
6. Outstanding action jackets: none.
In accordance with Commission policy, subject matter listed above, not disposed of at the scheduled meeting, may be carried over to the agenda of the following meeting.Start Signature
By order of the Commission:
Issued: October 11, 2001.
Donna R. Koehnke,
[FR Doc. 01-26117 Filed 10-12-01; 12:46 pm]


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Lady Cathy does not like her house messy. She is a clean freak and her slave did not understand that. She punished him to make him clean up after himself. She used her bare feet to trample on him. With her full weight on him, he was not breathing properly and wished she could stop. But she was not yet done. When she did finally, he swore never to be untidy again.
Pretty blonde BBW Lady Cathy has been walking around in sandals outside and she didn't bother to wash her feet off because she knew that her foot slave would lick all the dirt off. She sits down relaxing in her bra and panties while the slave worships and licks her bare feet polishing them with his tongue. He is persistent until he licks the soles of her bare feet clean.
Big and busty blonde BBW Lady Cathy makes her slave lie down on the hard shower floor while she crushes him with her full weght. She stands over him with her sexy boot on and lowers her massive ass down on his face. Her pussy covers his nose and her ass covers his mouth. He starts to smother and she just pushes down harder and laughs at his humiliation and pain.
Lady Cathy is a bubbly and playful BBW who is so hot with her curvy body and cute face. She loves to dominate skinny slaves and punish them in ever way she can think of. She makes her slave lie in the floor while she pushes her bare feet in his face making him smell and lick her toes. She sits on him with her full weight and pulls his leg to punish him.
Blonde and beautiful BBW Lady Cathy has found yet another way to dominate her slave using her weight. She doesn't even give this slave the satisfaction of having contact with her body when she sits on him with her full weight. She makes him lie down with a stool on top of him while she sits her big beautiful BBW ass down on top of it. His body is crushed under the weight.
Dominating BBW Lady Cathy has a variety of slaves. Today she has one male slave and one female slave and she makes them get on all fours in front of her. She starts humiliating the male slave first by making him lick her shoes. Then she makes the female slave smother the male slave with facesitting. She steps up her domination when she makes the male slave whip the BBW slave.
BBW blonde Lady Cathy has numerous ways to tease you and turn you on. Today she is going to turn you on with her feet. She uses pedal pumping to make you want her even more. She has her heels on when she gets in the car and starts pumping the pedals. She takes her shoes off so you can see her bare feet and painted toes as she presses the pedals.
Lady Cathy is one of the hottest BBW ladies out there and she is also one of the most cruel. She is wearing jeans today so there is no way her slave can breathe under the full weight of her ass. The slave likes it at first because he smells her ass and pussy through her jeans, but her huge jeans ass covers all of his head and he can't even breathe in.
BBW Lady Cathy went shopping all day and her sexy feet got nice and sweaty. It's a good thing she has a footslave to clean off her shoes and feet with his tongue. He lies down under her stool with his face right under her feet. He licks all the dirt off the soles until his tongue is covered in dirty. She takes her shoes and socks off and he licks her sweaty bare feet.
Radiant BBW Lady Cathy has a fun time in the bathroom with her slave. Well, it's fun for her, but it's humiliating for the slave. She grabs his face and buries it in her fat belly. Her blubber wraps around his face and she pushes it out and laughs. She keeps pushing and pins his head against the wall with her sexy belly. She keeps pushing and he starts to smother.
Plump and cruel BBW mistress Lady Cathy doesn't show any more mercy on her female slaves than she does on her male slaves. This one lies helpless on a cushion while Lady Cathy crushes her face with her ass. She dominates her with hard facesitting and forces the female slave's head down into the cushion with her full weight. She starts to smother and Lady Cathy likes it so she won't stop.
BBW Lady Cathy crushes her slave's face with hard facesitting. She stands over him and plops down with her huge ass covering his whole face. He starts to smother but she just pushes her big ass down farther. It feels good when her slaves fight for air under the full weight of her massive ass. It makes her feel so good to dominate like this and she will not stop until she's satisfied.
Hot, but fat blonde BBW Lady Cathy teases you and makes you think you can have sex with her. She never planned on giving you anything. All you get is tease and humiliation. She tells you how small your cock is and she would never have sex with you. She laughs at you and humiliates you with verbal abuse because you will never get to touch her. You are only a slave.
Lady Cathy is a hot blonde BBW who can't get enough of torturing slaves. Today, she can't decide whether she wants a female slave or a male slave. She decides to humiliate both slaves with facesitting and ass worship. She lowers her huge ass down on both of the slaves and sits down hard until they start to smother. She loves to torture people with her ass while they fight for air.
Hot BBW Lady Cathy likes to crush her slave's head between her thighs. She gets her slave in a painful scissor hold and squeezes. Her heavy and powerful legs wrap around his neck and squeeze until he can't breathe. She doesn't think he's feeling enough pain so she bends his arm back. She gets him in a scissor hold in every position. The more painful the better for this BBW.
Lady Cathy is a sexy blonde bbw mistress wearing a black shirt with a pair of blue jeans and black boots as she gets ready to go on her daily pony ride. She makes her slave get on his hands and knees and she gets on top of him and rides him like a pony. She knows that her full weight causes her slave pain but she does not care so she whips him with her crop to make him go faster.
BBW Lady Cathy is wearing a pair of black pants with a white blouse as she gets ready to mistreat her loser slave. She has him lay down on the floor and she puts her couch on top of his chest. He is able to handle the weight of the light couch but then Lady Cathy sits down on top of the couch and she smothers him as the couch crushes his entire body!
Lady Cathy is a polite bbw mistress and many people mistake it for a weakness. But they soon discover that she's tough when she takes them, tramples them, smothers them and makes them feel her weight on them
Lady Cathy entered the room and her slave did not greet her with the respect that she deserves so she decided that he needed a painful punishment to remind him that she was his mistress. She strips down to her pink bra and panties and makes her slave lay down on the floor. She then sits on his face and smothers him with her sexy ass and her full weight.
Lady Cathy wants to do some workout practices. But she doesn't want to lose some weight... she wants to sweat! After some minutes her body is covered with a lot of sweat. Now everything is ready to humiliate the slave! She sits down on his face on he has to suffer under her sweaty butt! To further increase his punishment she takes off her pants - wow, this way she smells even more...!
BBW Lady Cathy is in a good mood today and so she decides to take off her clothes until she wears her underwear only. She looks sexy in her black panties as she face sits the slave making his face her own personal sitting stool. His face gets crushed by her immense ass cheeks. The blonde beauty poses and checks out her nails as she face sits him.
Huge girl Savannah is an obese woman who absolutely squashes the slave with her immense ass. She moves from timidly and controllably sitting on Matt on the couch to sitting on his head and dropping onto him on the couch. Her huge thunder thighs squash his head like a little bug burying the slave under neath her sweaty legs. She almost snaps his neck with all her weight.
BBW Lady Cathy's dirty boots are preventing her from resting because they are all dirty and they need to be cleaned by the slaves tongue. She stretches her boots into his face and he has to clean them with his tongue only. He has to lick the soles very clean and he has to suck the heels too. Then he has to sniff on her socks after he's done.
BBW Lady Cathy punishes the slave by pressing he rbig bbw body on top of him on the work out bench. She presses her huge tits in the slaves face making him suffocate from her big pillows in his face. Than she sits on his chest with her big beautiful ass pressing all of her weight on top of the slave. The slave is crushed by her full weight.
Lady Cathy is never going to give her slave a break, especially if the slave isn't doing what he is told. She is going to have her slave come into a bathroom at a gas station where bathrooms can be very nasty. She will spit in the toilet, and demand her slave stick his head in the nasty toilet bowl and lick her spit up with everything else.
Cathy is a very hot bbw with curves for days. She loves to have one of her many slaves over, so she can put on a pair of sexy panties and sit right on his face. She loves the feeling of smothering her slaves with her big ass. Her slaves all adore the smell of Cathy's ass and pussy. Her slave is enjoying her scent today.
Cathy is a sexy bbw with a really nice big ass. She loves to use her big ass and sit right on the faces of her slaves. The slaves all love her sexy curves, so they obey any command. Cathy has her favorite slave over, so she can sit right on the slave's face with her juicy ass on his face. The slave is in heaven.
Wishes are mean to be granted. There are some things that beautiful BBW femdoms can't resist. A slave has been extremely good and he wants his mistress and her friend to sit on him. He loves their big asses and wants to smell them as close as he can. Lady Cathy calls Lady Cora and they sit all over his body. They put their full combined weight on his face so he can smell them together.
There are no excuses. When Lady Cathy gives an order, it's to be carried out immediately or there will be consequences. She knows her round ass is the cause of extreme delight or pain. Her favorite punishment is one that involves her putting on sexy black lingerie and sitting on her slave's face. She knows they can't help but breathe her scent in. She makes sure she puts her full weight on their face so they can't move or breath anything until she says so.
The big sexy blonde Lady Cathy has a mouthful of spit for you and she wants to spit it right in your face. This is how she treat losers like you in this hot pov spitting clip. The spitting will be extreme too as Lady Cathy will make sure to cover your entire face with her gobs of spit so your face can't even be seen. It's the ultimate bbw humiliation.
Bbw Lady Cathy and her bbw friend Coras have a special surprise planned for their slave today. He not only gets Cathy's big blue jeans ass on his face but Coras too. He gets the full weight of these big sexy blondes on his face and on his crotch. It is an erotic but humiliating experience for the slave who is smothered by these bbw's. They also laugh at him too.
Lady Cathy is a business woman but that doesn't mean she doesn't have a willing slave with her wherever she goes. She has her fat slave bitch with her at the office today ready to be foot dominated. Bbw Cathy rubs her black pumps on the slave bitch's face and than removes her shoes to relax her bare feet on the slave girl's head. The sexy blonde knows hot to dominate
BBW mistress Cathy wants you to worship her sexy big feet while she relaxes on the stairs. She won't be satisfied until her feet are totally spotless clean! So you know what you have to do, don't you...!? You have to put out your tongue and start licking away the sweat and all the dirt! And better do a good job - because she knows some other nasty things you could do...
If one slave doesn't do a good job, mistress Cathy always knows how to punish him. In this clip she makes her slave lay his head on a leather seat while she sits on his face with her sexy bbw ass! She's wearing a bikini and her victim is made to smell and taste her ass all the time...


text:
On Monday Jenna and I got to go on a little day trip for the OKC Mom's Blog. (To see my most recent post on the blog, click here, and then leave a comment because I really was asking for advice!) For our trip we headed an hour south of OKC to the little town of Sulphur.
First stop was the Bedre' Chocolate Factory.
Oh yes there were free samples! It really is some of the most delicious chocolate you will ever try!
Then we got to tour the new Artesian Hotel and Spa.
Yes my daughter managed to get in the hot water with all her clothes on.
Then we went to Bedre' Cafe for lunch
Then we toured the amazing Chickasaw Cultural Center. It's a whole beautiful campus that you can tour for free! The museum does cost money but not much. There is lots to see and do and they are always hosting events and activities. We might have to check some out.
They also have a library that I think might come in handy one day if we ever want to research Ryan and Olivia's background. They have information about all of the 5 civilized tribes there for genealogical research.
They also have a huge state park there that we drove through a bit and spotted two deer!
Sadly at the Bison preservation we did not see any Bison but maybe next time!
Be checking the OKCMB for the rest of the pics soon!


text:
One of two gladiators became aware of his evil deeds and quit fighting in an arena to become a hunter. He would learn about life and the secret wisdom hidden within the relativity of the universe while providing for the people. The other gladiator did not like this and plotted to destroy this hunter. "Look at how he talks and how he represents himself! He surely finds himself to be better than us!?"
He convinced the evil King to bring the hunter back into the arena to defend his crimes! As the hunter was placed into the arena some of the audience wanted blood and some wanted justice. The evil King deemed both of those moralities as one in the same and beckoned for the games to begin. "Before we begin then, shall I not issue a challenge of my own?". asked the hunter. The evil King became intrigued and beckoned for the hunter to propose it. "It is easy for one such as I to be brought to your eyes? Then for a moment shall I not bring you to see what I have seen? Allow me to bring forth a rabbit out of the dark forest and place it within the forest of light. As I release the rabbit into the forest of light let the gladiator stand behind it and we'll see if the rabbit would still prefer the light or the darkness".
The evil King agreed and the entire arena followed the hunter. As the wager began the rabbit did not budge even thought the gladiator stood behind it and the gladiator had just about enough of the peoples laughing at his failing. "I'll show you who the rabbit fears!" and at that the gladiator stepped forward unafraid of the dark forest. As the gladiator neared the rabbit he heard a horrifying roar come from the dark forest. The rabbit ran off past the gladiator and further into the forest of light as the gladiator stood frozen face to face with a terrifying creature.
All the people who were gathered remained watching to see what would happen next. As the hunter began to leave into the forest of light the evil king quickly asked, "So are you trying to say that you are a terrible creature?" "No, I'm the rabbit who chose the light".


text:
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text:
Anyway, she turns 54 today. Holy crap.
This is her back in the day.
Here she is in 2008, at 49. Frankly, and this may be my own age showing, she looks better now.
Thoughts on Politics, Culture, Books, Sports and Anything Else Your Humble Author Happens to Think Is Interesting


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Author Affiliations
- Maryam Hejazi1,
- Angela R. Manser1,
- Julia Fröbel2,
- Andrea Kündgen2,
- Xiaoyi Zhao1,
- Kathrin Schönberg1,
- Ulrich Germing2,
- Rainer Haas2,
- Norbert Gattermann2 and
- Markus Uhrberg1⇑
- 1Institute for Transplantation Diagnostics and Cell Therapeutics, Medical Faculty, Heinrich-Heine University Düsseldorf, Germany
- 2Department of Hematology, Oncology and Clinical Immunology, Medical Faculty, Heinrich-Heine University Düsseldorf, Germany
- Correspondence: Markus.Uhrberg{at}med.uni-duesseldorf.de
Natural killer cells are well known to mediate anti-leukemic responses in myeloid leukemia but their role in myelodysplastic syndromes is not well understood. Here, in a cohort of newly diagnosed patients (n=75), widespread structural and functional natural killer cell defects were identified. One subgroup of patients (13%) had a selective deficiency of peripheral natural killer cells (count <10/mm3 blood) with normal frequencies of T and natural killer-like T cells. Natural killer cell-deficient patients were predominantly found in high-risk subgroups and deficiency of these cells was significantly associated with poor prognosis. In the second subgroup, comprising the majority of patients (76%), natural killer cells were present but exhibited poor cytotoxicity. The defect was strongly associated with reduced levels of perforin and granzyme B. Notably, natural killer cell function and arming of cytotoxic granules could be fully reconstituted by in vitro stimulation. Further phenotypic analysis of these patients revealed an immature natural killer cell compartment that was biased towards CD56bright cells. The residual CD56dim cells exhibited a significant increase of the unlicensed NKG2A−KIR− subset and a striking reduction in complexity of the repertoire of killer cell immunoglobulin-like receptors. Taken together, these results suggest that the widespread defects in natural killer cell function occurring in patients with myelodysplastic syndromes are mostly due to either unsuccessful or inefficient generation of mature, functionally competent natural killer cells, which might contribute to disease progression through impaired immune surveillance.
Myelodysplastic syndromes (MDS) constitute a heterogeneous group of bone marrow disorders, which are characterized by dysfunctional hematopoietic progenitor cells and a propensity for evolution into acute myeloid leukemia.1 According to the World Health Organization (WHO) classification system, different MDS subgroups are distinguished based on the degree of dysplasia, the frequency of ring sideroblasts, and the number of bone marrow and/or peripheral blasts.2 Although most patients are initially diagnosed with low-grade disease, approximately two-thirds of patients eventually succumb to multi-lineage cytopenia or transformation to leukemia.3 The risk of tumor progression can be estimated by the International Prognostic Scoring System (IPSS), classifying patients into four risk groups (low, intermediate 1 and 2, or high) based on cytogenetic, morphological, and clinical criteria.4 The etiology and pathophysiology of MDS, which is the most common hematopoietic malignancy of the elderly (subjects aged >70 years), remain incompletely defined.
The role of immunological determinants in MDS are poorly understood. It is known that a subgroup of patients responds to immunosuppressive treatment. However, immunosuppression could compromise proper immune surveillance for aberrant hematopoietic progenitor cells and favor expansion of the malignant clone.5 In this regard, the role of natural killer (NK) cells is of increasing interest. NK cells can produce graft-versus-leukemia responses as previously shown in the setting of haploidentical stem cell transplantation for acute myeloid leukemia.6 NK cell function is determined by a balance of stimulatory and inhibitory receptors surveying the organism for signs of viral infections, cellular stress, and malignant transformation. To this end, NK cells express a variety of stimulatory receptors such as NKp30, NKp46, and NKG2D, recognizing, among others, stress-induced and tumor-associated ligands.7 Stimulatory signals are balanced by arrays of inhibitory receptors such as killer cell immunoglobulin-like receptors (KIR) and the NKG2A receptor, which sense expression of various major histocompatibility complex (MHC) class I molecules. This system enables NK cells to identify and eliminate tumor variants escaping MHC-restricted, adaptive immune control by down-regulation of MHC class I, a mechanism referred to as "missing self" recognition.8,9
With regard to MDS, increased NK-cell-mediated cytotoxicity was found in one study, while several other studies reported impaired NK cell function in peripheral blood and bone marrow.10–13 Kiladjian et al. found decreased cytotoxicity, proliferation and increased apoptosis of peripheral NK cells without changes in expression of inhibitory or stimulatory NK cell receptors.11 Impaired cytotoxicity was also seen by Epling-Burnette et al., who attributed this to a lower frequency of NKG2D-expressing NK cells in peripheral blood.12 In a recent study, Carlsten et al. associated decreased cytotoxicity with decreased expression of DNAM-1 and NKG2D in NK cells from bone marrow but not peripheral blood.13 Overall, the underlying mechanisms for defective peripheral NK cell function remain elusive.
In the present study, a thorough phenotypic and functional analysis of NK cells was performed in a cohort of newly diagnosed MDS patients. In the majority of patients, NK cell defects were found and could be attributed either to an overall lack of NK cells, which was strongly associated with high-risk MDS subtypes and poor prognosis or, more frequently, to the presence of NK cells with an immature phenotype, which were characterized by non-armed granules and an immature NK cell receptor repertoire.
Patients and controls
Peripheral blood was obtained from 75 patients with newly diagnosed MDS (age, 41–90 years; mean 71 years) and 30 age-matched healthy control donors (age, 51–90 years; mean 72 years). Informed consent was obtained from all patients and donors according to the Declaration of Helsinki. The study was ethically approved by the local institutional review board. The patients' characteristics and classification of MDS according to WHO criteria are given in Table 1. Peripheral blood mononuclear cells (PBMC) were isolated from patients and healthy donors using density gradient centrifugation with Biocoll Separating Solution (Biochrom, Berlin, Germany) and subsequently frozen and stored in liquid nitrogen for later analysis.
The following fluorescence-labeled monoclonal antibodies were used: CD56-PE, PC5 or APC (N901), CD3-ECD or PC5 (UCHT1), CD158a/h-APC (EB6), CD158b1/b2/j-APC-Alexa Fluor 750 (GL183), CD159a-PE (NKG2A, Z199), NKG2D-PE (ON72), CD62L-PC5 (DREG56) all from Beckman Coulter (CA, USA). CD158e1-FITC (DX9), CD57-FITC (HCD57), granzyme B-FITC (GB11), perforin-PE (dG9), CD107-APC or FITC (H4A3) and interferon-γ FITC (B27) were purchased from Biolegend (CA, USA) and CD56-PE-vio770 (AF12-7H3) from Miltenyi Biotec (Bergisch Gladbach, Germany). Flow cytometric analyses were performed on a FACSCanto I (BD Biosciences, NJ, USA) using FACS Diva 5.0.1 software.
CD107a and interferon-gamma assay
Thawed PBMC were cultured overnight in RPMI 1640 containing 10% fetal bovine serum, 5% human serum type AB (Biochrom), and 1000 U/mL interleukin-2 (Novartis, Basel, Switzerland) in a concentration of 1×106 cells/mL. PBMC and K562 target cells were mixed at an effector/target (E/T) ratio of 10:1 (5×105:5×104) in a volume of 200 μL in a 96-well plate. For analysis of cytotoxic granule mobilization, CD107a monoclonal antibody was added prior to incubation. To determine spontaneous degranulation, a control sample without target cells was included. After incubation for 1 h, 2 μL of 2 mM Monensin (Biolegend) were added and incubated for a further 5 h. Finally, the cells were washed in phosphate-buffered saline (Lonza) and stained with monoclonal antibodies (CD56, CD3). For measurement of intracellular interferon-γ, effector and K562 target cells were co-incubated for 6 h. After the first hour, brefeldin A (Sigma-Aldrich, Missouri, USA) was added at a concentration of 10 μg/mL. Cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences) and stained intracellularly with anti-interferon-γ monoclonal antibody.
Intracellular staining of granzyme B and perforin
PBMC from MDS patients and healthy age-matched donors were stained with fluorescence-labeled CD56 and CD3 surface marker. After washing with phosphate-buffered saline, cells were intracellularly stained, using an intracellular staining kit (BD Biosciences) and granzyme B and perforin monoclonal antibodies. Expression of granzyme B and perforin was then determined in CD56dim NK cells by flow cytometry.
Cytotoxicity assay
K562 target cells were stained with CFDA-SE (Vybrant® CFDA-SE Tracer Kit, Invitrogen, CA, USA). PBMC (cultured overnight with interleukin-2 1000 U/mL) and stained target cells were mixed at a ratio of 10:1 in a volume of 200 μL. The following controls were prepared: unstained K562 and two wells with stained K562, one as a control for CFDA-SE staining and one to determine spontaneous lysis of K562 cells. After incubation for 6 h, cells were harvested and stained with propidium iodide (BD Biosciences). The frequency of K562 cells double positive for CFSE and propidium iodide was determined by flow cytometry. Specific lysis of K562 cells was calculated as: lysis of K562 cells with effector cells – spontaneous lysis of K562 cells.
Statistical analysis
All tests (Fisher exact test, t-test) were performed at the two-sided 0.05 significance level. All statistical analyses were performed using GraphPad Prism software.
Additional methods regarding cell lines, apoptosis assay, KIR genotyping, and NK cell stimulation are detailed in the Online Supplementary Methods.
A subgroup of patients with myelodysplastic syndromes exhibits profound natural killer cell deficiency
In order to characterize the role of NK cells in MDS, we analyzed peripheral blood NK and T cells from 75 patients who were newly diagnosed with MDS and consequently had not received any prior disease-specific treatment (Table 1). NK cell (CD56+CD3−) frequencies were moderately lower in MDS patients than in healthy, age-matched donors as shown by flow cytometric analysis (Figure 1A). Notably, a subgroup of cases (n=15) exhibited unusually low NK cell frequencies <1% (normal range 2–20%). Remarkably, we could not detect any donors with NK cell frequencies <1% in the age-matched control group or in a second, larger group of 116 healthy adult donors of undefined age (Online Supplementary Figure S1). Reduced NK cell frequency was associated with an even more significant reduction in NK cell numbers. The most severe cases were classified and subsequently referred to as "NK cell-deficient" (n=10; NK cell count <10/mm3 blood). In NK cell-deficient patients discrete NK cell populations were typically not detectable by flow cytometry based on expression of CD3, CD56 (Figure 1D) or CD16 (data not shown). Since NK cells are believed to be generated from T/NK common progenitors, we also analyzed T cells (CD3+) and NK-like T cells (CD56+CD3+). Notably, overall frequencies and cell counts of T and NK-like T cells were comparable to those of healthy, age-matched controls (Figure 1B,C). Direct comparison of NK and T-cell counts in MDS samples demonstrated that the observed reduction was NK cell-specific and that the NK cell-deficient group had normal T-cell (Figure 1E) and NK-like T-cell counts (data not shown). In order to rule out that the observed reduction of NK cells in MDS patients was due to a selective loss of NK cells during the thawing/freezing or storage procedures, we repeated the analysis with PBMC freshly drawn from three MDS patients with NK cell deficiency. The NK cell-deficient phenotype seen in the frozen samples was confirmed in all three fresh samples (data not shown). Longitudinal analyses of three cases (first analysis in 2009, second analysis in 2012) revealed a stable NK cell-deficient phenotype over time (data not shown).
Natural killer cell deficiency is associated with high-risk subgroups of myelodysplastic syndromes and poor prognosis
We next investigated whether NK cell deficiency was associated with specific disease subgroups and/or prognosis as defined by the most recent WHO and IPSS classification systems, respectively. Indeed, NK cell-deficient patients were overrepresented in the WHO refractory anemia with excess blasts I/II subgroup (31%), which has the highest risk for progression to acute myeloid leukemia, compared to the subgroups refractory cytopenia with multilineage dysplasia (11.6%) and refractory anemia/refractory anemia with ring sideroblasts (0%) with a lower risk profile (Figure 2A). Moreover, NK cell-deficient patients were strongly associated with IPSS high-risk groups (5% in the low/intermediate 1 risk groups versus 44% in the intermediate 2/high risk groups, P<0.001), (Figure 2B). In general, NK cell counts (Figure 2A–B) and frequencies (Figure 2C-D) were significantly lower in the WHO and IPSS high-risk subgroups, refractory anemia with excess blasts I/II and intermediate 2/high, respectively. Again, T cells exhibited no association with disease subgroups (data not shown).
Functionally deficient natural killer cells from patients with myelodysplastic syndromes exhibit defects in their killing machinery
Whereas it was not possible to reliably assess NK cell function in NK cell-deficient patients, CD107 mobilization could be readily determined in MDS patients without NK cell deficiency. No difference was observed in CD107 mobilization in response to K562 target cells between MDS patients and healthy, age-matched donors (Figure 3A). In contrast, killing of target cells was significantly decreased in MDS patients (Figure 3B). Plotting NK cell frequency against specific lysis of K562 cells revealed that decreased target cell lysis in MDS patients was only weakly correlated with a low frequency of NK cells among PBMC (Online Supplementary Figure S2). Of note, no correlation was found between CD107 mobilization and target cell killing (Figure 3C,D). Besides cytotoxicity, intracellular interferon-γ production was significantly reduced in response to stimulation with K562 cells (Online Supplementary Figure S3).
Since NK cells from MDS patients showed no change in degranulation activity but did have impaired cytotoxicity, we next investigated whether granules were properly armed with cytotoxic molecules. Intracellular staining demonstrated that granzyme B and even more perforin loading of granules was substantially reduced in CD56dim NK cells from MDS patients (Figure 3E). In most cases, both factors were down-regulated simultaneously (Figure 3F). Importantly, the small subgroup of MDS patients with functional NK cells (cytotoxicity >20%) had high levels of granzyme B/perforin expression. A similar correlation between granzyme B/perforin expression levels and cytotoxicity was found when enriched NK cells were used (Online Supplementary Figure S4). However, there was a subgroup of MDS patients (≈15%) who had impaired killing in spite of normal frequencies of granzyme B/perforin-containing NK cells suggesting additional unidentified mechanisms of functional repression.
In vitro stimulation of natural killer cells resolves functional deficiency
In order to determine whether functional deficiency of NK cells can be resolved in vitro, NK cells from five MDS patients with functional deficiency and from three healthy age-matched donors were subjected to an interleukin-2-based stimulation protocol. Importantly, NK cells could be expanded to a similar extent (10–100 fold in 10 days) in MDS patients and controls (Figure 4A). This observation was in contrast to that in a previous study showing lack of NK cell proliferation in MDS patients.11 However, a different stimulation protocol, including irradiated feeder cells, was applied here (Online Supplementary Methods). Notably, the perforin and granzyme B deficiency of MDS NK cells was fully reversible (Figure 4B). Consequently, in vitro stimulation of functionally deficient NK cells restored cytotoxicity against K562 cells to control levels (Figure 4C).
Phenotypic analyses revealed immature differentiation state of natural killer cells in myelodysplastic syndromes
To further elucidate possible mechanisms for the widespread functional NK cell defects in MDS, we next analyzed NK cell maturation state and receptor repertoires. MDS patients with functional defects showed a significant increase in frequency of CD56bright NK cells, representing an immature, less cytotoxic NK cell subset,14 as well as a decrease in CD56dim NK cells (Figure 5A, B). In contrast, patients with normal NK cell function had CD56bright frequencies that were comparable to those of controls. Analysis of cell counts revealed that the CD56bright subset was unchanged in terms of absolute cell numbers (Figure 5C) whereas the more mature CD56dim subset was strongly decreased in functionally deficient patients compared to cases with normal NK cell function and healthy controls (Figure 5D). Notably, the decrease in CD56dim NK cells was significantly associated (P=0.0004) with reduced target cell killing (Figure 5E).
We next investigated whether the selective decrease of mature NK cells in functionally deficient MDS patients could be due to increased apoptosis of CD56dim NK cells. However, we found no differences in spontaneous cell death of CD56dim or CD56bright subsets between patients and age-matched controls (Online Supplementary Figure S5A,B). Additionally, no correlation between apoptosis of NK cells and the NK cell count of MDS patients was observed (Online Supplementary Figure S5C).
CD56dim NK cells can be separated on the basis of KIR/NKG2A expression into four consecutive maturation stages.15 MDS patients had an unusually high frequency of NKG2A−KIR− CD56dim NK cells (Figure 6A). This subset represents an immature differentiation stage that is functionally not educated and is generally hyporesponsive to various stimuli.16 Furthermore, the two KIR-expressing "late" stages (NKG2A+KIR+ and NKG2A−KIR+) were less represented in MDS patients. Both the frequency and absolute numbers of KIR-expressing NK cells, which are essentially a combination of the two "late" stages, were significantly decreased in patients (data not shown).
In a further analysis of NK cell maturation states, clonal KIR expression patterns of CD56dim NK cell were analyzed. During NK cell differentiation, KIR expression is regulated in a sequential mode with KIR2DL2/3 representing the first inhibitory KIR; KIR3DL1 and KIR2DL1 are expressed subsequently.17,18 The analysis revealed a strong decrease of NK cells expressing the "late" KIR2DL1 or KIR3DL1 as their only KIR, whereas the frequencies of the "early" KIR2DL2/3 were increased in MDS patients (Figure 6B). Direct comparison of NK cells selectively expressing one of the three inhibitory KIR ("single KIR" expressing NK cells) for the major HLA class I-encoded ligands (Bw4, C1, Ct2) revealed a strong bias towards KIR2DL2/3 expression (t-test: P<0.0001) and a significant underrepresentation of KIR2DL1 and KIR3DL1 in the KIR repertoires of patients (Figure 6C). Similar effects were seen when cases where stratified according to KIR genotype, i.e. into patients homozygous for group A haplotypes and those having one or two B haplotypes (B/x; Online Supplementary Figure S6). Furthermore, NK cells expressing multiple KIR, a hallmark of experienced NK cells, were significantly decreased in the repertoires of patients (Figure 6D). Further analysis of other markers associated with distinct stages of NK cell differentiation revealed that CD62L, which is associated with naïve NK cells, was significantly increased in MDS patients (Online Supplementary Figure S7). Furthermore, expression of CD16, a marker of mature NK cells, was significantly decreased in MDS patients (Online Supplementary Figure S7). Low expression of CD16 could specifically be attributed to the CD56dim subset of NK cells (Online Supplementary Figure S8). Of note, the stimulatory receptors NKG2D and NKp30 were not differentially expressed in either NK subset. Collectively, these data reveal a profound deficiency of fully matured NK cells in the majority of MDS patients.
Ineffective hematopoiesis is a hallmark of MDS and affects single or multiple myeloid lineages depending on the MDS subtype. It is less clear to what extent lymphopoiesis is also affected by the impaired differentiation capacity of aberrant hematopoietic progenitor cells or a compromised stem cell microenvironment. Several previous studies in MDS found deficient NK cell responses against various autologous or allogeneic tumor targets.11,12,19,20 The present study suggests that NK cell defects can, in the majority of cases, be attributed to inefficient or defective NK cell differentiation, leading either to a predominantly immature NK cell compartment or to an overall lack of NK cells.
Three different groups of MDS patients could be defined on the basis of NK cell number and function: a small group with normal NK cell numbers and function, a large group of patients with normal or moderately decreased NK cell number and impaired function, and an NK cell-deficient group. This last group of patients, in whom NK cells were rare or undetectable (PBMC: 1–10 NK cells/mm3), was preferentially found in the high-risk WHO subgroups of refractory anemia with excess blasts I/II and had a significantly elevated IPSS risk score. These observations are in line with those of a recent study that found strongly reduced NK cell frequencies in a high-risk (presumably pretreated) cohort of MDS patients undergoing hematopoietic stem cell transplantation from unrelated donors.20 Since NK cells are an important component of immunological tumor surveillance, it is tempting to speculate that a lack of NK cells in MDS patients increases the risk of leukemic progression to acute myeloid leukemia. In this regard, NK cells were previously implicated in relapse control following allogeneic hematopoietic stem cell transplantation for myeloid leukemia.21 Moreover, recent studies have associated certain KIR gene linkage groups with decreased relapse and improved outcome in patients undergoing hematopoietic stem cell transplantation for acute myeloid leukemia.22 Future analysis of larger MDS cohorts and a longer follow-up will be necessary to show whether NK cell deficiency is an independent prognostic parameter for progression to leukemia and/or overall clinical outcome.
The majority of MDS patients had normal or moderately decreased NK cell numbers while exhibiting impaired cytotoxicity, confirming several previous reports. In order to understand the underlying mechanisms better, we analyzed NK cell effector functions on the clonal level. Unexpectedly, cell surface mobilization of CD107 was not affected in MDS patients in spite of strongly decreased target cell cytotoxicity. Moreover, intracellular analysis of perforin and granzyme B revealed significantly decreased expression of either one or both molecules. Normal levels of NK cell cytotoxicity were only seen in patients expressing high levels of granzyme B and perforin. These observations strongly suggest that the lack of armed granules is causally involved in the decreased cytotoxicity of NK cells in MDS. Notably, a small subset (15% of cases) had decreased cytotoxicity despite normal levels of granzyme B and perforin (Figure 3F). In these cases, the cause of the defective function has yet to be elucidated.
The above observations illustrate certain limitations of the CD107 assay, which has emerged in recent years as an established surrogate marker for cytotoxic activity of T and NK cells.23,24 In functionally competent NK cells, mobilization of CD107 to the cell surface correlates with release of granule content and subsequent target cell cytotoxicity. However, if cytotoxic granules are not properly armed with perforin or granzyme B, NK cells might still mobilize CD107 to the cell surface but would not be able to kill. Indeed, NK cells with insufficiently armed granules still mobilized CD107 to the cell surface in MDS patients (Online Supplementary Figure S9). These observations might not only be relevant for MDS but also for other hematologic or pathological settings in which NK cell function might be similarly compromised by deficient arming of cytotoxic granules, which would not then be detected by a standard CD107 mobilization assay.
Several lines of evidence suggest that the observed functional defects are related to the immaturity of the NK cell compartment in MDS patients. Firstly, the distribution of CD56bright and CD56dim subsets was biased towards the immature non-cytotoxic CD56bright subset. Whereas CD56bright frequencies increased, the CD56dim subset was strongly diminished. The CD56dim count significantly correlated with lack of cytotoxicity. Secondly, among CD56dim NK cells, MDS patients had a significant increase in KIR−NKG2A− cells. It was previously shown that this CD56dim subset is immature and can be differentiated in vitro into the other three subsets in a sequential fashion starting with NKG2A+KIR− followed by the NKG2A+KIR+ subset and finally NKG2A−KIR+ NK cells.15 Importantly, KIR−NKG2A− cells are not licensed due to their lack of HLA class I-specific inhibitory receptors and thus exhibit poor cytotoxicity.16,25 In contrast, the more mature KIR-expressing stages KIR+NKG2A+ and KIR+NKG2A− are less abundant and consequently overall KIR expression was significantly reduced in MDS patients. Thirdly, analysis of clonal KIR expression demonstrated a bias towards expression of "early" KIR in MDS patients: KIR repertoires were dominated by expression of KIR2DL3, which is the first inhibitory KIR that is expressed during sequential acquisition of KIR.17,18,26 KIR2DL1 and KIR3DL1, which are expressed at later stages of KIR repertoire development, were strongly diminished in patients. Moreover, the frequency of NK cells expressing multiple inhibitory KIR, which represent a more advanced stage of NK cell development, was also diminished.27
Notably, the above-described phenotypic characteristics resemble those of neonatal NK cells. Like NK cells in MDS, neonatal NK cells have an elevated frequency of CD56bright NK cells, increased expression of NKG2A in the CD56dim subset, and a KIR repertoire biased towards KIR2DL3.28 A further parallel is that effector functions of neonatal NK cells are initially reduced compared to those of adult NK cells but quickly catch up upon stimulation with interleukin-2, similar to what is observed for NK cells in MDS (Figure 4). It is also revealing that the phenotypic changes described here, such as increased frequency of the CD56bright subset, are actually the opposite of the changes observed in the NK cell compartment during healthy aging.29 Thus, although MDS is a disease of the elderly, NK cell repertoires are clearly phenotypically and functionally immature.
The mechanisms leading to the lack of mature, functionally competent NK cells in MDS can only be speculated. Notably, similar analyses of NK cells in patients with acute or chronic myeloid leukemia revealed no such changes of the CD56bright and CD56dim subsets, suggesting that the underlying mechanisms are specific to MDS.30,31 We did not detect a significant increase in spontaneous apoptosis of CD56dim and CD56bright NK cells in MDS patients, making it unlikely that the observed lack of mature NK cells is due to selective cell death of the CD56dim NK cell subset (Online Supplementary Figure S5). One possibility that was not thoroughly addressed in this study is that the malignant clone contributes to altered NK cell differentiation of NK cell progenitors. Indeed, clonal markers were detected in variable fractions of NK cells in some studies,11,32 but not in others.12 Our preliminary analysis confirmed participation of the MDS clone in the NK cell compartment of del5q patients (data not shown). So far it is unknown if and how clonal alterations can influence the efficiency of NK cell differentiation and/or NK cell function. Given the fact that functional NK cell defects and developmental immaturity were found across all MDS subtypes and including many different genetic aberrations, it appears unlikely that clonal involvement is the sole cause.
An alternative, but not mutually exclusive, possibility is that NK cell differentiation is disturbed due to a lack of stromal support in the stem cell microenvironment of MDS patients. In this regard, it was previously shown that efficient generation of mature, KIR-expressing NK cells requires the presence of suitable supportive stromal cells.17 Moreover, it was recently shown that mesenchymal stem cells from MDS patients have a substantially diminished capacity to support myeloid differentiation across all disease subtypes and that hematopoietic stem and progenitor cells from MDS patients can regain their differentiation potential when cultured on mesenchymal stem cells from healthy controls.33
In conclusion, we could associate defective NK cell responses with NK cell maturation defects in the majority of MDS patients. Functional NK cell defects were reversible in vitro by interleukin-2-induced stimulation. In this context, clinical treatment of MDS patients with lenalidomide or other derivatives of thalidomide was previously shown to improve NK cell function and proliferation via increased T cell-based interleukin-2 production.34 It would be interesting to determine whether immunomodulatory drug treatment is also effective in NK cell-deficient patients. The majority of these patients have a high-risk profile with increased risk of progression to leukemia and might particularly profit from improved NK cell-based immune surveillance. Finally, it should also be considered whether MDS patients could benefit from adoptive NK cell-based immunotherapy. This might be a valuable option for patients with a poor prognosis in order to improve immunosurveillance for leukemic cells as well as for patients with secondary immunodeficiency, e.g. experiencing recurrent viral infections.
We would like to thank all individuals who voluntarily donated blood for this study. We also thank Dr. Jürgen Rack for providing samples from healthy age-matched donors. This work was supported by funds from the Deutsche José Carreras Leukämie-Stiftung e.V. (to NG and MU), by the Deutsche Krebshilfe (to UG, NG, RH, and MU) and the Deutsche Forschungsgemeinschaft (research grant UH 91/7-1 to MU).
The online version of this article has a Supplementary Appendix.
Authorship and Disclosures
Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.
- Received October 13, 2014.
- Accepted February 9, 2015.
- Copyright© Ferrata Storti Foundation


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Hope everyone had a great week!
Here are the songs for this weekend:
Your Grace Is Enough
For Who You Are
From the Inside Out
At The Cross
I Need You
I'm looking forward to a great weekend of worship and preaching before Matthew and I head off to Mexico next week to work with our team on the ground there. Be praying for us as we go make friends in Oaxaca, Mexico.
Friday, August 8, 2008
Friday Morning
Posted by Anonymous at 6:21 AM
Labels: worship leading
2 comments:
- Linda said...
It's Linda Carter here. Just wanted to let you know your song "I Need You" was sang and played by the band beautifully !!
Since Church at 11:30 I've been singing it to myself ALL day. You know how when a song gets stuck in your mind?
Well anyway GOOD JOB BAND !!
- August 10, 2008 at 8:17 PM
Miss you bunches! Can't wait for you to get home. I know you are having a great time and making a difference in Oaxaca. Love ya!
- August 12, 2008 at 8:25 PM
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BREAKING: New 'Corrupt' Money Scheme Uncovered in DNC and Hillary Campaign
More corruption has been uncovered in the DNC and Hillary campaign!
A new legal complaint filed with the Federal Election Commission shows that the Hillary campaign and the DNC used state chapters as strawmen to circumvent campaign donation limits and laundered the money back to her campaign.
From Fox News
The Committee to Defend the President, a political action committee, filed its complaint with the FEC on Monday with the allegations that the Hillary Victory Fund (HVF) solicited cash from big-name donors, including Calvin Klein and "Family Guy" creator Seth MacFarlane — money that was allegedly sent through state chapters and back to the DNC before ending up with the Clinton campaign.
Officials with the committee said their filing was spurred by their own analysis of FEC reports, where they said they discovered the HVF either never transferred the money to state chapters and back to the DNC, or did so without the state chapters having actual control.
"What we have found, people need to see," Ted Harvey, chairman of the CDP which emerged from the now-defunct Stop Hillary PAC, told Fox News. "I think it's important that the American public has an understanding of how corrupt this campaign system was and that they were doing anything they could to secure the nomination in her favor."
In its complaint, the CDP alleges that about $84 million was funneled illegally from the DNC through state party chapters and back into the war chest of the Clinton campaign.
"Apparently the DNC's response to the [Supreme Court] ruling was, 'Hold my Beer.'"
"Based on publicly available FEC records, repeatedly throughout the 2016 presidential campaign, HVF would purportedly transfer funds to its constituent political committees, which included between 34 and 40 state parties," reads a passage from a copy of the complaint reviewed by Fox News. "On the very same day each of these transfers supposedly occurred, or occasionally the very next day, every single one of those state parties purportedly contributed all of those funds to the DNC."
CDP counsel Dan Backer said the scheme was a flagrant violation of a Supreme Court ruling that determined such moves were illegal — and broke FEC campaign contribution rules which state that an individual can contribute only $2,700 directly to a presidential campaign.
"Apparently the DNC's response to the ruling was, 'Hold my Beer,'" he said, referring to a phrase from an Internet meme. "These donations were specifically earmarked for the DNC and the campaign committee."
Backer continued, "It's not entirely clear if they transferred the money to the state parties to begin with, and if they never had control of the money, it's not a contribution. It's an earmark. If the money is in control of the candidate, then it is a contribution to them."
Backer reiterated a point from the complaint — that the Supreme Court first recognized these practices were illegal when it ruled in a 2014 case involving Shaun McCutcheon, a businessman and electrical engineer from suburban Birmingham, Ala., who created the Conservative Action Fund PAC. McCutcheon filed suit against the FEC challenging federal campaign contribution limits. It was Backer who assisted in filing the suit.
"This is exactly the scenario that they said would occur," Backer says. "They were so brazen about it yet so sloppy."
The new complaint filed against the FEC said previous reports showed a series of transactions in which the HVF disbursed contributions to its state party committee members — and they would receive the funds on the same day. The HVF would also allegedly disburse funds to up to 40 state parties at the same time, and those parties would send the money back within 24 hours.
"It appears virtually every single disbursement from HVF to a state party resulted in an immediate transfer of the same amount of funds from the state party to the DNC," reads another passage from the complaint. "Over 99% of funds transferred through HVF to state parties wound up at the DNC."
Officials for the DNC did not return requests for comment when contacted by Fox News.
The allegations seem to echo claims by former DNC chair Donna Brazile while promoting her book earlier this year.


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Rita's Ramblings: a basically house-bound woman in Fargo, North Dakota blogging for friends, family, and anybody who finds it interesting. I talk about art or craft projects, my grandson (Ian), the weather, movies, books, health, and whatever happens to be going on in my life. Welcome!
Friday, January 23, 2009
5 comments:
Okay, that's hilarious! Animals can be such goofballs. My parents live in a small condo with a big dog ... a 9-year-old doberman. They have to make sure the bathroom and closet doors are firmly closed because if not, Lola goes in to investigate. The rooms are so small that when she turns around to leave, she shuts herself inside. Every now and then, someone will notice she's missing and we have to go on a Lola hunt. What's really funny is she always looks so sheepish when you finally rescue her. LOLReplyDelete
OMG, thank heavens you hadn't gone out when she decided to that, Rita...she would have suffocated for sure, the poor darling. I'm so glad that she's okay but I can't help but laugh at the pickle she found herself in. Cody has a habit of jumping inside my wardrobe when I have the doors open while I change in the morning. One morning, I didn't see him go in so I closed the doors......not long after, we heard his muffled bark and I realised what had happened. lolReplyDelete


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Standardised OTC swaps to launch within weeks
Within weeks, fixed-income market participants will be able to trade a new, exchange-traded version of the over-the-counter interest rate swap, with eight tenors, standard coupons and quarterly maturity dates like those used in the futures market. Some of those involved in the work see it as a way of defending the OTC market against the threat posed by swap futures.
Buy-side firms including Pimco, BlackRock, Goldman Sachs Asset Management and Eaton Vance have been meeting during the past 12 mont
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This time it's not just about the puppets.
"Emmet Otter's Jug-Band Christmas," the world preem tuner based on the 1979 Jim Henson HBO special, will be Goodspeed Musical's inaugural holiday show this December.
The show will play Dec. 7-Jan. 4 at the Goodspeed Opera House, marking the first time the theater will have four productions in a season at the East Haddam, Conn., venue.
TV production of "Emmet Otter" was an all-puppet affair, but the stage version will feature 19 actors in the principal roles and an orchestra of eight.
Goodspeed Musicals will self-produce the offering, which features additional new music by the TV show's original composer Paul Williams. Production will be helmed and choreographed by Christopher Gattelli (choreographer for Broadway's "South Pacific" and Off Broadway's "Altar Boyz").
Book is by Gattelli and Tim McDonald. The Jim Henson Co., under son Brian and daughter Lisa as co-CEOs, is creating the costumes and puppets for the production.
The show, set in Frog Town Hollow at Christmas, centers on two otters down on their luck, according to theater rep Dan McMahon.
The TV tuner was based on the children's book of the same name by Russell and Lillian Hoban.
In spring, "Happy Days," the musical based on the TV sitcom, played prior to its national tour. A rare revival of "Half a Sixpence" is now in previews. The fall show will be "Big River."


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Read this tip to make your life smarter, better, faster and wiser. LifeTips is the place to go when you need to know about Digital Photography and other Digital Photography topics.
I'm a long-time Fuji camera fan. My second SLR was a Fuji (before they were known as "Fujifilm"), and I absolutely loved it. And I'm still impressed with some of their ZLR's.
But Fujifilm's 3-D camera? Sorry... been there; done that. You see, I used to work for Qualex, Kodak's former photofinishing arm. And I remember the 3-D film camera mess.
Fuji's 3-D W1 is similar to the film-based 3-D cameras that were promoted a few years back on one of the big TV shopping channels. Two lenses create separate images that are combined on one print. (Or on one viewer, in the case of the digital version.)
3-D movies were quite a fad in the 1950's. They're a fad now. But it's a gimmick. And 3-D photos are a gimmick, too. The cost-to-benefit ratio just doesn't work out.
Yes, PC Mag is gushing over the "new" Fujifilm 3-D W1... but they haven't "been there; done that." I've seen the novelty wear off before... and I believe the novelty will wear off again.
Fuji is capable of producing terrific cameras... and some of their cameras are genuine bargains. The features far outweigh the cost. (I just recommended a Fuji model to a college marine program, because it absolutely crushed the competition for the price.) Fuifilm's ZLR's are some of the great bargains of the photo world.
But 3-D? Sorry... everyone's entitled to make a mistake. And I think this is Fujifillm's... in spite of what the folks at PC Mag may think. At $600 - more or less - buyer's remose will quickly set in.
Why would you spend $600 for a 3-D camera, when Fujifilm's Finepix S1500 is available for under $175? This 10 MP camera has a zoom that extends from 33 mm to 396 mm. No, it's not 3-D. But it will produce excellent results for less than a third of the price... and you won't need a special viewer to appreciate those results, either. And it's priced far less than the competition, to boot.
So let the neophytes "ooh" and "aah" about the 3-D pictures. Just be careful you're not sucked in by their enthusiasm. 3-D has come and gone before. it may stick around a little longer with animated movies this time. But photos? I doubt it.
|Guru Spotlight
|Mary White


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HI Experts,
I have 14 projects Starting with code 2A,2B,2C,2D,2E,2F,2G,2H AND SO ON. For each project business area is different, Hence while creating the project the business area should fetch form the system to avaid manual errors. Our business area starting with 2001,2002.2003,2004, 2004 and so on. I know substitution is correct, I will be able to enter one to one, but I need how to enter multiple projects to multiple business areas at time? Can any body help me?
Hi Hemu,
If you are using different project profle for different category you can default the business area via OPSA under organisation tab.
If only one project profile then you have to use substitution via OPSN and assign the substitution to the project profile via OPSA.
If you want it into as a single substitution step it can be a substitution exit reading from a mapping Z-table.
Dear Hemu
What do you mean by "but I need how to enter multiple projects to multiple business areas at time?".. Ideally and standard is you create only one project per project profile/Mask at a time using the project structure. Two project coding masks/profile cant combine each other in a single profile. The business area is derived as a default value via project profile for a particular project. The very basic ides of creating multiple project profiles is to have different organizational and controlling parameters as business deem fit.
Kindly justify yout requirement to go against the standard practice in project management.
Hi, Thanks for reply,
Here I have only one project profile i.e.2000. Under, we create multiple projects definitions starting with
2A,2B,2C,2D,2E AND SO ON. From project profile, only one business area will default (if business area is common).
Issue is that for each project business area is different.
Requirement is
When I create project starting with 2A,2B,2C,2D,2E, system should pick the business area 2001,2002.2003,2004, 2004 respectively by default
Could you please answer ?
Thanks in advance
Thanks for your reply Paulo, Please elaborate you answer.