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I love the innocence of toddlers. My friend Kelli posted a sweet video of Miss Rylie dancing around the living room in her cast and diaper this week. William perched beside me watching Rylie. He giggled, covered his mouth and said she is so naked! I could not stop smiling! Both are covered in a plaster and fiberglass life vest and yet they see themselves as naked. They are officially cast blind!
We have an influx of new families still very new to this journey. I have been trying very hard to take myself back to the new moments of diagnosis and first cast. It is so cavalier of me to say it will all be fine & don't worry, when 3 years ago I was an absolute basket case. To assure you I am not the robot I may come across now, I wanted to share a little bit of the journal from Cast 1.
Cast Day #1:
Chad and I drove to Chicago the night before Williams first cast. We opted to stay at a hotel near the hospital. It wasn't the best hotel for our family that night, but we were together with our grief and anxiety rather than inflicting it on innocent family members. Once we were checked into our room we chose to visit Bar Louie in Oak park and try to relax. Dinner was nice, we love Bar Louie & its casual atmosphere. When dinner was complete it was time to head back to the hotel. When we returned to the hotel, we gave our little angel his last bath for what would be a 12 week stretch in cast, clothed him in comfortable new jammies and tried to turn off the screaming in our heads as we waited for morning. We ended up not sleeping at all the night before. Some of it was the paper thin walls and the couple reminiscing all night in the room next door, but more of it was the anxious unknown that was to come.
We opted to leave the hotel at around 5:15 a.m. By 5:45 a.m. we were in the parking lot of Shriner's Hosptial for Children, Chicago. A friend had encouraged us to take William's stroller in as he would be awkward to hold after casting. Chad and I wheeled our tiny 10 month old in and were escorted to the same day surgery waiting area. A very sweet nurse came to greet us shortly after arrival. She introduced herself as Gwen and told us she would be with us through the day. William was a so sweet and snuggly. He allowed Gwen to get his vitals and cooed at her in his sweet baby voice. As cast time approached we were greeted by residents, anesthesiology and research assistants. Since William had already had an x-ray taken less than 2 weeks prior we were able to skip that portion of this trip. 8:00 a.m. was now here and it was time to be taken to the surgery holding room. We were allowed to carry William with us as they wheeled his crib into the holding room.
Chad and I put on sterile gowns, booties and hair nets and joined his bed in the holding room. Once in the room, William
was greeted by the Pre-op nurse and fitted for his undershirt. He was once again seen by the anesthesiologist and they gave him liquid versed to ease his separation anxiety. Where was my versed? I think I deserve to forget them taking him from me. I sat in a chair with my husband beside me and sobbed as I rocked and sang William to sleep. The Operating Room staff was so kind and agreed to carry him asleep into the OR. As I passed him over, I knew things were about to change for us, yet still hopeful this would be a change for the best in William's future. Sobs left my chest as I said goodbye for now to my sweet little man.
Waiting has to be the hardest, short of handing your child to someone you don't really know. We sat in the same waiting area that greeted us upon arrival and waited for Dr. Sturm to come tell us William was finished.
45 minutes after William entered the OR, Dr. Sturm and Linda came out to update us on his progress. William was out of
surgery, he was in the PACU, Post Anesthesia Care Unit, and he was doing remarkably. They were able to get his
little spine to 25 degrees in cast, a 42 degree correction from where he started. Dr. Sturm was delighted with
the progress and said they would come and get us when he was ready to come back to the room, he would see us in 12 weeks if we had no complications. It's funny how time stands still when you are waiting for something to happen.
Although only about 10 minutes had passed, it seemed a lifetime. I heard the cry before anyone had to come and
retrieve us. I peeked around the corner to same day surgery and Gwen said "you know it's him momma, come on back." There he was, my beautiful baby boy, mad as hell. Gwen said it's ok, he just wants you mom. She helped put him in my
arms. He was so heavy and awkward to hold. I was afraid every move I made was going to hurt him. Gwen assured us he
was just mad and that he would cry off the anesthesia and get back to his old self in no time.
So many milestones have to be reached before you can leave the hospital. He has to drink, pee and get trimmed/ taped
before we can load up and head out. It took close to an hour and half before he was at a place where Gwen could start
the taping process. William fought her the entire time. She just kept taping and saying is that all you have for me. I am not scared of you William. Come on big guy, show me your fight. She is the QUEEN OF CASTING!
Taping and trimming finally complete, it was time to hit the road to our new lives. The 6 hour trip home was terrifying and liberating at the same time. This was our life and we might as well get used to living it. William slept and cried periodically throughout the journey. We stopped about halfway home and changed his diaper with a fear and care I haven't had since he was a newborn. We quickly buckled him back in his seat and pushed through the last
three hours of our trek. Cody was at my sister's house, so that would be our first stop. Maggie and Peter had dinner ready for us when we finally pulled in at close to 6 p.m. Exhausted we carried him into the house and handed him to his beloved
godmother and aunt. By this time, William was extremely swollen. His face looked as if he had been beaten. My beautiful sister hugged him and kissed him as little tears trickled down her cheeks. This was the first and last time I saw my sister cry over his cast. She is strength in a storm, but even she caved at first look of William post cast. Bedtime was nerve racking. William was still in a pack and play in our room so we placed a pillow under his sweet head and let him rest as peacefully as he could. It was tough to see him struggle with everything from rolling around to breathing. He was getting used to
breathing with 25% of his weight coming from a cast on his chest. My friend had prepared us for this guppy breathing, but it is still horrifying when you hear it for the first time. Once he drifted off to sleep the adrenaline wore off and I gave into the grief. I cried for the baby that was gone, the loss of mobility, our loss of normalcy. I cried out of fear of the unknown and fear of the known. I cried until the tears were gone and sleep took over. William's sleep was restless to say the least. He
was unable to roll and that was hard on both of us. I was convinced this was our new normal so I would help him get into a position where he could fall back to sleep. We alternated Tylenol and Motrin most of the night. I was diligent to make sure he was comfortable. As morning came I was still exhausted and scared. I needed to shower and in a fit of necessity I called my mom who immediately came over. She stepped in and said go shower, we are ok. I let the water take over my world and once again the tears began to pour. When I came out of the shower I was resolved that I would not cry anymore that day. I emerged, a new women willing myself to be strong for whatever the day would bring.
Day one had it's trying times, but I am proud to report, my mom had William standing at his activity table playing with the little pages on the book when I came out of my shower. Throughout the day things started to just start happening.
I would prop him with pillows so he could play with toys. I encouraged him to try to crawl when he could and would help him pull up to the couch so he could cruise. Within the next few days mobility returned and he was starting to get used to what first was a burden for his little body.
We are 12 casts and 2.5 years past this moment. My journals of cast changes now are brief updates. They usually start with he walked himself in and end with he walked himself out. There were hiccups and headaches on the road but no more than raising my child without a cast. It is new and you need to mourn, prepare, and feel however you need to feel. There comes a time when you will tell yourself...enough. That is when it is time to move forward and accept. Not a one of us can say we don't visit the dark space every once in a while on the journey, at that point though, our kids have already figured this thing out. More often than not it is the moments like Rylie and William's, "I'm so naked!" that will remind me they are cast blind. I think it gives us permission to be cast blind too.
Until Next Time, Keep it Curvy~
Catie(Scoliosis Sucks) D.
Filed under: Uncategorized, Wellness
Tags: Chicago Shriners Hospital, First Cast, Infantile Scoliosis, mehta Cast, Serial Derotational Casting


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Vicky Kaushal: 'Zubaan' opening Busaan Festival feels surreal
One-film-old actor Vicky Kaushal is thrilled that his second project "Zubaan", a coming-of-age movie about a Sikh boy, has been chosen as the opening film for the 20th Busan International Film Festival.
Vicky Kaushal
"Zubaan", directed by debutant Mozez Singh, also stars actress Sarah Jane Dias and is co-produced by Guneet Monga of "The Lunchbox" fame.
The 27-year-old actor, who is riding high on the success of his debut film "Masaan", said inclusion in Busan is an honour for him and the team of "Zubaan".
"It feels great. We all are ecstatic. Busan Film Festival is one of the most prestigious festivals in the world. It is an honour for any film to open it and for 'Zubaan' to be that movie, it comes from a very humble and honest approach, it has a new director, it is my second movie," Vicky told PTI.
"I feel so blessed, the kind of year it has been for me.
First 'Masaan' represented India at Cannes... It feels surreal and it is too good to be true."
Vicky said he is happy that a commerical movie has been selected for an international festival.
"Going to another nation and representing your country's cinema and that too taking a commerical film, they found the honesty and power in a commercial film to open the festival."
The film is expected to hit theatres this October days after its world premiere at Busan.
"This news gives a huge mileage to the film, in a very organic, positive and honest way. It feels great when a nation outside respects your film and talent," Vicky said.
The festival will run from October 1-10 in Busan, South Korea.

subset: commoncrawl


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Mark Ford trained and qualified as a solicitor at Clifford Chance, the world's largest law firm. Whilst practising as a lawyer, he specialised in data protection and privacy law and advised clients on compliance issues in over 80 jurisdictions. He wrote Data Protection and Employment (Industrial Society, 2000) and was General Editor of Data Protection Laws of the World (Sweet & Maxwell, 1998) for the work's first ten years. He is now the Director of the firm's Knowledge Centre, a 40-strong legal support unit that he established in the firm's office in Delhi. Mark is also an accomplished musician. He trained as an organist with the international recitalist David Sanger and held an Organ Scholarship at Trinity Hall, Cambridge University.
In 1994, he formed The Purcell Singers, with whom he has performed extensively in London, in venues including St James's Piccadilly, St John's Smith Square and Southwark and St Paul's Cathedrals, as well as the Brighton, Hurstpierpoint and Howden Festivals and in Frankfurt. The choir's first disc of choral works by Georg Schumann was Gramophone Magazine's Critics' Choice in January 2002. Mark and the choir are also active in session work and have recorded in all of the major London studios. Their library of more than 100 film trailer cues has been used in trailers for films such as King Kong, The Da Vinci Code, Spider-man 3 and War of the Worlds. They can also be heard on the original cast album of Mary Poppins.


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Well, it's the signature weapon in the series. Wouldn't have it any other way.
Boxart showcasing the AR it seems. Good. MC knows what's up.
Wish I could stick around and see it be put together, but I run out in about 3 minutes, alas. Great idea for the art reveal. Someone make a thread on the gaming side when it's finished.


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Nirvana's 1993 acoustic performance on MTV's 'Unplugged', recorded for the Nirvana album 'MTV Unplugged In New York' released the following
Beards - not since the dawn of the Brazilian has hair been such a headline subject, but no one can
Zayn Malik changes his hair more than he changes clothes (probably), and his arrival back in the UK seems to
Iman has revealed a touching way she is choosing to honour late husband David Bowie. The supermodel attended Tom Ford's
Gucci has chosen British actress Vanessa Redgrave to star in its Cruise 2017 campaign. The 79-year-old posed for photographer Glen
Summer is officially upon us, which means new footwear is most definitely needed. So it's high time to set your
E3 2015 PS4 Trailers: Shenmue 3, Uncharted 4, The Last Guardian, Final Fantasy VII, Call Of Duty: Black Ops 3
Sony has responded to Microsoft's E3 2015 press conference with a rousing two hours of trailers that included the first
Schoolboy Designs Robotic Trolley To Help Elderly And Disabled People At The Supermarket
A Scottish schoolboy is the brains behind a robotic shopping trolley, which could help thousands of elderly and disabled customers
Kendall Jenner has come under fire for her latest shoot with Spanish Vogue. The reality star-turned-supermodel posed as a ballet
'Rock Band' And 'Guitar Hero' Are Coming Back, Because The World Doesn't Have Enough Plastic Toy Guitars
From about 2005 until 2010 the western world was blighted by the arrival of video games which ostensibly simulated playing


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The award presentation began with a tribute led by Viola Davis, who actually won Best Performance by an Actress in a Supporting Role in any Motion Picture for Fences earlier in the night. Davis, who co-starred in Doubt with Streep, amusingly impersonated the actress, but also quite seriously praised her acting talent. "I can only imagine where you go, Meryl, when you disappear into a character," Davis said.
The actress' latest nomination comes for Best Actress in Motion Picture Musical or Comedy for Florence Foster Jenkins. She's previously won eight times, dating back to 1979. As the Cecil B. DeMille Award recipient, she follows Denzel Washington (2015), George Clooney (2014), Woody Allen (2014), and Jodie Foster (2013).


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We are preparing space at the Yoga Farm for summer 2015 retreats and workshops. If you are a yoga teacher or workshop leader and would like to explore bringing your group out to the Yoga Farm we need to talk!
The Yoga Farm is set on a 65 acre gorgeous farm in Lansing, NY, just outside of Ithaca. We have a finished yoga studio, and a much larger rustic but beautiful barn space with 2 floors, and numerous outdoor environments. The farm is bordered by 2 gorges, and boasts waterfalls, fields, forests, hiking trails and more to provide plenty of room to move about. Nearby lodging – B&B's and hotels can host your out-of-town guests. Workshop leaders can stay on-site in the farm house.
As this is our first season offering retreat space, our rates are unbeatable and we'll work with you on amenities, catering, dates and times that suit you. You can use the space for as little as 2 hours and as much as a week. We will make it work for you so call 607.280.2010 and let's discuss.


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Puttur: Subba Rao (100), a resident of Amala Kavinamoole in Koltige village in the taluk, has passed away at home.
He is survived by wife Saraswati, daughters Sharada and Jayanti, sons Shankaranarayana who is engineer in Bengaluru, Seetaram Amala, Shivaram Amala and Ramachandra Amala, Puttur block Congress general secretary.
Prominent persons, including MLA Shakuntala Shetty, former MLC Anna Vinayachandra, zilla panchayat member Anita Hemanath Shetty and many others visited the bereaved family and offered their condolences.


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The Sonora Police Department reported the following:
9:32 a.m., suspicious circumstances - An attempted break-in was reported on Lyons Bald Mountain Road.
1:27 p.m., theft - A man stole merchandise from a Sanguinetti Road store.
2:31 p.m., theft - A bicycle was stolen near the farmers market parking lot on Theall Street.
6:34 p.m., suspicious circumstances - Two men in their 20s were seen looking into vehicles in a Sanguinetti Road parking lot.
7:20 p.m., traffic accident - A man driving with kids in the car, who was believed to be drinking, drove off after hitting a car in a Greenley Road parking lot.
12:41 a.m., public intoxication - A man was seen lying on the side of the road on Mono Way.
9:14 a.m., property damage - A vehicle was reportedly vandalized at a Sanguinetti Road parking lot.
12:17 p.m., traffic accident - Drivers exchanged information after a car accident on South Washington Street.
2:47 p.m., disorderly conduct - A woman was inside an Old Wards Ferry Road business yelling and throwing around her belongings.
3:08 p.m., vandalism - A man said his car was vandalized on South Barretta Street by a former tenant.
3:40 p.m., theft - Someone stole items from a South Washington Street store.
4:41 p.m., traffic accident - A man said he was in an accident the night before that occurred between downtown Sonora and Tuolumne Road after falling asleep at the wheel, likely between midnight at 3 a.m. He said he received a head injury, but did not know where the vehicle was located.
5 p.m., theft - Someone reportedly stole money from a South Washington Street motel room.
6:58 p.m., theft - A woman on South Washington Street said her son's friend stole video games and cash the day before.
8:35 p.m., suspicious circumstances - A woman on Greenley Road believed her son and ex-husband were tampering with her safe and her computer.
10:30 p.m., reckless driving - Someone was driving fast eastbound on Mono Way and shooting "silly string" at pedestrians out of the vehicle.
8:39 a.m., disorderly conduct - Someone on South Arbona Circle said a loud chainsaw was used in violation of a noise ordinance.
2:15 p.m., civil problem - A man on Elk Drive believed his deceased fiancee's son stole his personal belongings.
5:44 p.m., suspicious circumstances - Someone on South Sonora Avenue said women were stealing from an evicted home. Officers on scene determined they had permission from the homeowner to move their belongings.
7:17 p.m., disorderly conduct - A transient at a West Stockton Road business cursed at a man and his daughter.
10:36 p.m., disorderly conduct - Someone on East Lyons Street said a neighbor pointed an "extremely bright light" at their home.
The Sheriff's Office reported the following:
8:09 a.m., Twain Harte - A minor vehicle accident occurred on Joaquin Gully Road.
8:25 p.m., Sonora area - Vehicles were covered in sand at a Mono Way car dealership.
9:22 p.m., Sonora area - Someone called a resident on Beckwith Road claiming to be with Publishers Clearinghouse, making several requests of the resident.
12 p.m., Tuolumne - Someone broke into a vehicle on Rozier Street, attempted to take the stereo and stole $450 out of a wallet.
2:25 p.m., Soulsbyville - Someone appearing to be from a local nonprofit agency was calling a Soulsbyville Road resident asking for credit card and other information. Further investigation revealed the callers were likely using a program that disguises their number on caller identification.
5:42 p.m., East Sonora - Two people tried to shoplift from a Mono Way business.
5:50 p.m., Groveland - A message was left on a James Circle resident's answering machine claiming there was a federal judgment against her for tax fraud.
5:57 p.m., Groveland - Thefts of solar panels were reported on Rocky Point Drive.
7:37 p.m., Tuolumne - A fire was reported on Apple Blossom Lane.
7:57 p.m., Sonora area - An assault was reported on Brookside Drive.
2:45 a.m., Tuttletown area - A vehicle went down the hill and into trees near the Stevenot Bridge on Highway 49.
9:03 a.m., East Sonora - A vehicle ran out of gas and was stopped in the middle of Mono Way.
11:05 a.m., Jamestown - A man was lying on the side of Chicken Ranch Road.
12:26 p.m., Sonora area - A woman woke up in bed with a man she did not know on Chukar Circle. The woman reported not knowing how she got there, though saying she did not believe she was assaulted.
3:13 p.m., Sonora area - A woman on Highway 49 said people were walking on her property to get to a nearby creek. She asked if she could shoot at them with a paintball gun when they walked by.
4:36 p.m., Jamestown - Someone found marijuana wrapped in condoms in a black plastic bag in a garbage can on Shotgun Creek Drive. Once investigated, it appeared the bag contained tobacco rolled up in sandwich bags and multiple cell phones.
6:20 p.m., Jamestown - A man threatened to burn down a house on Wigwam Road.
6:49 p.m., Beardsley Reservoir- A man pulled a knife on someone at Beardsley Reservoir.
1:05 a.m., Sonora - Someone broke the window in a Washington Street jewelry store.
10:04 a.m., Twain Harte - Someone on Middle Camp Road said there was a suspicious car parked on the road for "several days." Deputies confirmed the owner of the car was working at a home nearby.
1:50 p.m., Jamestown - A woman at a Wigwam Road mobile home park said her husband twisted her arm and took her phone after she forgot it was July and called her daughter to wish her "Happy Thanksgiving."
7:42 p.m., Jamestown - A transient at a business on Victoria Way laid on a table and ignored requests for him to leave.
9:51 p.m., Tuolumne - A man on Gardner Avenue said an unknown person "jumped out" of a car and punched him in the mouth.
10:30 p.m., Sonora area - A man on Serrano Road said one of his "drivers" received counterfeit bills after they delivered cigarettes and lottery tickets.
11:09 p.m., East Sonora - A man on Hillsdale Drive said he woke up to his wife choking him.
1:08 a.m., Columbia - A woman on Green Street said a man caused a disturbance and took a dog and a marijuana plant.
Felony bookings
3:11 p.m., East Sonora - Brian Keith Robert Hernandez-Barclay, 20, of the 10500 block of Blanchard Road, Coulterville, was booked on suspicion of burglary after an arrest on the 13000 block of Mono Way.
7:01 p.m., Sonora area - Kalen Lon Collins, 31, of the 20100 block of Jamestown Road, was booked on suspicion of attempted murder, battery with serious bodily injury and threatening a crime with the intent to terrorize after an arrest at his residence.
11:34 a.m., Jamestown - Johnny Ray Cagle Jr., 31, of Turlock, was booked on suspicion of possessing, manufacturing or selling dangerous weapons and possessing burglary tools after an arrest on Highway 108 and Chicken Ranch Road.
7:30 p.m., Jamestown - Pamla Kay Baker, 47, of the 500 block of South Lenhill Avenue, Oakhurst, was booked on suspicion of bringing alcohol or drugs into a prison after an arrest at Sierra Conservation Center.
7:30 p.m., Jamestown - Desirae Michelle Shuck, 18, of the 500 block of Cassidy Avenue, Yuba City, was booked on suspicion of bringing alcohol or drugs into a prison after an arrest at Sierra Conservation Center.
4:40 a.m., Jamestown - Deborah Sue Nevarez, 55, of the 21900 block of Fallview Lane, Sonora, was booked on suspicion of drug possession, and misdemeanors possession of drug paraphernalia and driving under the influence of drugs or alcohol.
Cited on suspicion of driving while under the influence of alcohol or drugs:
2:44 p.m., Sonora - Tony Guy Wallace, 55, of Jamestown, was booked after an arrest on Mono Way near Highway 108.
3:17 a.m., Sonora area - Daniel James Blust, 30, of the 17100 block of Valley Oak Drive, Sonora, was booked after an arrest on Highway 49 near Reynolds Ferry Road.
7:08 p.m., Sonora - Rick Alan Christiansen, 48, of no listed address, was booked after an arrest on Highway 49.
8:20 p.m., Pinecrest area - Michael Wade Drayer, 41, of the 400 block of Williams Street, Murphys, was booked after an arrest on Beardsley Road.
8:47 p.m., Sugar Pine - Keenin Bodean Vickers, 32, of the 16400 block of West Brookside Drive, Sonora, was booked after an arrest on Highway 108 near Lyons Dam Road.
9:51 a.m., East Sonora - Joseph David McHardy, 28, of the 200 block of Woodlands Drive, Murphys, was booked after an arrest on Highway 108 at Hess Avenue.
2:06 a.m., Jamestown - Matthew Thomas Howard, 21, of the 2200 block of St. Francis Drive, Palo Alto, was booked after an arrest at Highway 120 near Keystone Ranch.
7:02 p.m., Sonora - Matthew Brent Wechsler, 46, of the 200 block of Mono Way, was booked after an arrest on Snell and Green streets.
9:06 p.m., Sonora area - Randall Verne Gordon, 62, of the 16500 block of Creekside Drive was booked after an arrest at his home.
The Sheriff's Office reported the following:
8:47 a.m., Valley Springs - A burglary was reported on Highway 12.
9:05 a.m., Valley Springs - An off-white pillow case was seen near Highway 12 and Highway 26 with something that looked strange in it. Further investigation revealed the contents to be a pillow.
10:15 a.m., Copperopolis - A vehicle break-in was reported on Poker Flat Road.
11:14 a.m., Valley Springs - A burglary was reported on Highway 12.
11:50 a.m., Valley Springs - Someone took water from a fire hydrant on Country View Drive.
1:37 p.m., Valley Springs - A burglary was reported at Hogan Observation Point on Hogan Dam Road.
1:56 p.m., Valley Springs - A suspicious man was walking in and out of traffic on Highway 26 carrying a duffel bag.
2:45 p.m., Copperopolis - Residents on Copper Meadows Drive said an unknown man had their dog and would not return it.
7:36 p.m., Valley Springs - A "suspicious" man and woman walked down Redman Road and Mann Street.
9:48 p.m., Jenny Lind - Two "suspicious" people walked down Highway 26 near Milton Road.
1:31 p.m., Angels Camp - An arrest was made after a welfare check at a mobile home park on South Main Street.
7:25 p.m., San Andreas - An arrest was made after a noise complaint on Sunset Street.
Felony bookings
8 a.m., Copperopolis - Troy Trustyn Taliaferro, 23, of the 200 block of Millie Court, was booked on suspicion of drug possession, burglary, receiving stolen property, and grand theft, after an arrest at his home.
2:25 p.m., San Andreas - Michael Anthony Wold, 42, of an unlisted address in Valley Springs, was booked on suspicion of drug possession and receiving stolen property after an arrest on East Saint Charles and Main streets.
2:25 p.m., San Andreas - Emmet Cleveland James, 62, of the first block of Laurel Street, Valley Springs, was booked on suspicion of drug possession and receiving stolen property after an arrest on East St. Charles and Main streets.
3 p.m., Angels Camp - James Rick Moore, 46, of the 300 block of South Main Street, was booked on suspicion of violating terms of alternate sentencing after an arrest at his home.
6 p.m., San Andreas - Rachel Elizabeth Bean, 20, of the 3200 block of Rose Avenue, Ceres, was booked on suspicion of assault and misdemeanor cruelty to a child after an arrest on the 500 block of Angels Road.
Cited on suspicion of driving while under the influence of alcohol or drugs:
9:20 p.m., Angels Camp - Carol Irene Wilson, 55, of the 1900 block of Country Club Boulevard, Stockton, was booked after an arrest on the first block of Main Street.
12:40 a.m., Angels Camp - Kevin Scott Geary, 29, of the 16600 block of South Creekside Drive, Sonora, was booked after an arrest on Highway 49 at Monte Verde Street.
2:30 p.m., Angels Camp - Amii Ann Nagle, 18, of the 500 block of Williams Street, Murphys, was booked after an arrest on Murphys Grade Road and Gardner Lane.


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Nov 01, 2015
May 08, 2016
May 15, 2016
Jun 15, 2016
LSAT-India 2016 - Exam Pattern
Exam Paper Type : Pen & Paper | Exam Duration : 2.5 Hr
|# Questions
|Analytical Reasoning
|35 Minutes
|Logical Reasoning- I
|35 Minutes
|Logical Reasoning-II
|35 Minutes
|Reading Comprehension
|35 Minutes
The LSAT—India breaks critical thinking skills down into three main types: logical reasoning, analytical reasoning, and reading comprehension. Since the first of these types is most predictive of success in law school, there are two sections of logical reasoning questions in the LSAT—India. There is also one section each of analytical reasoning and reading comprehension questions, which contribute to the predictive validity of the test.
- Total Marks - 96
- Total Questions - 96
- Total Duration - 2 Hours 20 Minutes
- Marking System - There is no negative marking or penalty for guessing. Only correct answers contribute to a candidate's score.
LSAT-India 2016 Syllabus
- Critical Reasoning
- Number series
- Letter Series
- Series Missing Numbers
- Odd one out
- Blood Relation
- Calender
- Clock
- Matrix
- Cubes
- Coding Decoding
- Direction and Distance test
- Ranking Tests
- Sitting Arrangement
- Linear Arrangements
- Matrix Arrangements
- Mathematical Puzzles
- Venn Diagram
- Data Sufficiency (statement type)
- Strong & Weak Arguments
- Statements Assumptions
- Statements and Conclusions
- Statements & Inferences
- Logical Sequence of Words
- Analogies
- Verbal Classification (Odd one out)
- Syllogism
- Logical Deduction (based on syllogism)
- Course of Action
- Causes and Effects
LSAT-India Exam Prepartions FAQ
1) What is the duration of the test?
The total duration of the test is 2 hours and 20 minutes without any break.
2) What is the marking scheme for the different sections?
There are no significant differences in the marking scheme for the different sections. All questions on the test count equally. However, the number of questions in a section can vary somewhat, but it is always approximately 24.
3) Is there any negative marking or penalty for guessing in LSAT—India?
There is no negative marking in LSAT—India and candidates should answer every question, even if they need to make guesses.
4) Is it possible to alter the sequence of answering the sections?
No. Sections are taken in the order that they appear in the test book. Candidates are allowed to work only on the section being timed by the invigilators, at any given point of time. Candidates are not permitted to go back to an earlier section or go forward to a later section, even if they finish before the section time is called.
5) In case a candidate completes answering and reviewing a section before the allotted section time completion, can he go to the previous or next section(s) to complete?
No, in LSAT—India, candidates are not allowed to work on any section other than the one being timed by the invigilators. In case a candidate completes answering and reviewing a section before the allotted section time gets over, he/she is not allowed to attempt any other section — previous or next. The balance time cannot be used on any other section.
6) Is there a special need to prepare for the LSAT—India? If yes, then what are preparation tips for the LSAT—India?
Yes, the candidates should become as familiar as possible with the test pattern, which includes the question types and test timing, by taking advantage of the test preparation material available on this website. Since the LSAT—India measures critical thinking skills, not mastery of any specific body of knowledge, cramming facts, mathematical formulae, and other general knowledge is not necessary and will be a waste of time.
7) What is the difference in preparation for the LSAT—India for 5-year and other law programmes?
For all programmes, candidates should become as familiar as possible with the test pattern, which includes the question types and test timing, by taking advantage of the test preparation material available on this website or Handbook available at a nominal cost. Scores are derived separately for these two groups, and scores for one candidate group cannot be compared to those for the other candidate group since they are based on group performance.
8) Which are the good guide books available in the market in addition to the material available on the website?
There are some guide books available in the market for the LSAT—India. However, the LSAT—India is a test of reason, so one doesn't need to cram facts/data. Understanding of the test pattern and gaining familiarity hold the key for a candidate's success. To gain familiarity with the LSAT—India test pattern, candidates are recommended to go through test pattern and official test prep links.
9) How much preparation is needed to get a good score?
Ideally, a candidate should prepare as long as it takes to feel comfortable with the question types and how they are answered. Candidates can use the free preparation material available on this website to gain familiarity and comfort. To achieve a deeper level of preparation, candidates can purchase The "Official LSAT—India Handbook". This inexpensive guide can help in gaining familiarity with every type of LSAT—India question.
10) What all things can a candidate carry to the test centre?
The candidates should carry their IDs in original, their Admit card and writing material (HB pencils, sharpeners and erasers) for the test. Candidates must not carry any article such as cellphones, calculators, pens, arms, etc., as all these articles have to be left outside the test hall at the candidate's own risk.
11) What if a candidate receives a question paper that is in poor condition?
Once the test paper is distributed and the tamper proof seal on the question paper is broken, a candidate should inform the invigilator immediately if the question paper is defective or in poor condition. The candidate will be given a replacement question paper.
12) What if a candidate believes there is an error in one of the test questions or answer choices?
A candidate should inform the invigilator if he or she believes there is an error in a test question or answer choice. Invigilator will note the candidate's concerns and share with LSAT—India team. Regardless of the concerns, the candidate should mark an answer to the question on the answer sheet. The concerns will be investigated and the candidate will not be penalized if the concern is warranted.
13) Are there any breaks during the test?
There are no scheduled breaks in the test. However, candidates can take unscheduled breaks if they want during the test session. An unscheduled break means a break during which the test clock does not stop. If a candidate chooses to take a break, the actual time spent on the break will be deducted from the time allocated to complete the test. No extra time will be given to complete the test. During the break, the candidate must leave the testing room. Upon re-entering the testing room, the candidate must show a valid ID.
14) Will a candidate be allowed to access his/her personal belongings during the unscheduled breaks that he/she takes?
Candidates will not be allowed to access any personal belonging including study material and mobile phone during the breaks they take. Candidates will only be allowed to access food or medicines, if necessary for medical reasons.


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margaret pearl disharoon GA OK
I have just rec'd marriage lic. info on the marriage of my grandparents, Margaret Pearl Disharoon to George Harris in Cleveland, OH 12/4/1912.The printed app. was hand written by the Rev. A. A. Shaw so this may account for Margaret's name written as DISHRON. On this app Margaret P.s father is given as BETHEL and her mother as JOSEPHINE BAILEY.
On my father's birth records, Margaret states she was born in Canton, GA. On Margaret's brother JOHN EDWARD DISHAROON's social security application his parents are listed as BETHEL QUILLAN and ELVERIA BAILEY and as having been born in Marble Hill GA in 1900.His obit states that 3 sisters and 1 brother preceeded him in death but did not give their names.
I only recently came across the 1880 Dug Rd., Pickens Co., GA census with BETHEL and LUVIRA DISHAROON, both 18, living next door to MARTHA DISHAROON, DIVORCED, along with several children.
In Mary Markley's 10/3/01 message referring to this same census suggested that the ex-husband (and possibly father to Bethel) might be Erwin Disharoon, 59, who later married Abagail, 26.
This info made me think that some of the pieces to this puzzle were perhaps falling into place...
...but who the heck is JOSEPHINE?


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Arizonans enjoying cool temperatures - in May!
Very unusual for the month of May, Arizonans are still enjoying cooler weather. In Phoenix alone, temperatures were 20 degrees cooler than normal.
"It makes me think that it might snow next," said Kim Hermanski.
Hermanski may not be that far off. Up in portions of the High Country, snow has reportedly fallen. Viewer Christina Cantu shared a video of snow in Flagstaff with FOX 10 Phoenix.
Diners outside a sushi restaurant decided to soak up the cool weather, on the patio.
"I like this weather because we never see it," said Justin Hermanski. "It's always so hot, so getting the clouds and the wind and the rain is awesome."
While many are enjoying the cooler weather, some who are from out of town apparently don't understand what's the big deal behind this streak of cooler temperatures.
"It blows me away to see people wearing long pants and long shirts," said Sean Johler, who is from Philadelphia. "I mean, this is so warm from where we are back home."


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Archives of 2013
Instrument building
Few images of musical instruments I've built available in the gallery.
Banjo Uke - instructions here
Lap Steel Guitar - instructions on this book
Categories: Creations, Music
Posted by Matias at 29.12.2013 15.35 (3 years ago) | 0 comments
Ooh! I had the time to modernize the online version of The Rasterbator, which, sadly had been offline for over half a year.
Go here to satisfy your poster-generation needs!
The online version has, I think, quite nice user experience including lots of Ajax and drag and drop file upload. Although it is quite a small detail, I'm quite proud of the human silhouette that indicates the poster size.
Coders would probably be interested that it is based on ASP.NET MVC 4, jQuery (with some plugins) and iTextSharp.


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Would you like to save as your currency permanently or for this visit only?
There is also a sun bathing terrace available to guests which also offers a wonderful view of the surrounding panorama. DC 09/08 The hotel resembles an antique palace from the last Century. The hotel lies only 150m from the historical centre of Tropea and is situated by the Sea. The beaches of Tropea are easily reachable from the hotel, some even by foot.


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Friday night is turning out to be a fine mess indeed. Not only will Sofada be debuting the S/S '09 collection, but the latest boutique addition to E Burnside, Frank James, is having their soft opening, and now this: Seaplane is setting up a pop-up shop on the ground floor of 919 NW 23rd (which you might recognize as the address of the Frumoasa Boutique) for a weekend-long trunk show with KitKitDodge and a smattering of vintage accessories from Lovebirds for KitKit. The pop-up store will be in effect through Sunday, but hit it up on Friday between 7-10 for refreshments and libations, including "Pixie Retreat" raw chocolates, also for sale. Look for KitKitDodge samples, pieces in organic, natural, and vintage fabrics, de- and reconstructed denim pieces, plus recycled cashmere panty sets--and who knows what Lovebirds for KitKit will smuggle out from their vintage treasure stash, but most everything looks lovely.
A look from KitKitDodge
Mint-condition Miu Mius from Lovebirds for KitKit


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How to simulate condensor?
I want to simulate the temperature field of a condensor, but i know in Fluent 6.0 only NO-phase change's heat exchanger could be simulated, such as radiator,etc. so who can tell me how to set boundary conditions in order to simulate a condensor? I want to have a further discussion with you!
|All times are GMT -4. The time now is 06:38.


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A Night of Horror Volume 1 Trailer (2015)
"Ten Filmmakers. One Night of Horror." 26 November 2015 Horror 87 mins
Whether you're a fan of Sydney's iconic genre film festival A Night of Horror, or just a fan of the best and bloodiest in new horror cinema, you can't afford to miss this fright-filled anthology. Zombies, demonic entities, self-surgery, cannibalism and more await in the dark corners of this terrifying offering from some of the most talented filmmakers working in the genre today.
Watch the official A Night of Horror Volume 1 2015 trailer in HD below or find more A Night of Horror Volume 1 videos on Vidimovie.


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Free software covers a wide range of applications. CAD viewers, productivity software, mobile applications often are free to download. Other applications are fully functional, production-grade CAD programs.
Autodesk Inventor View 2015 free download
Autodesk Inventor View 2015 allows your project team to share Inventor design files with the entire design team. The Autodesk viewer is freely distributable and available for both 32 bit and 64 bit versions of Windows. This viewing application allows everyone to view and to print Autodesk Inventor assemblies and drawings.
Download PTC Creo View Mobile for free and visualize 3D models from PTC Creo
PTC Creo View Mobile lets PTC customers visualize 3D models from PTC Creo on an iPad or iPhone – best of all, it's free.
Graphisoft BIMx iOS and Android viewer free download for ArchiCAD drawings
Graphisoft's BIMx viewer for mobile devices supports iOS, Android, and Kindle Fire mobile devices. With Graphisoft's BIMx Hyper-model navigation, it is easy to navigate an integrated 2D and 3D building model.
Graphisoft BIMx desktop viewer free download for ArchiCAD drawings
While the ArchiCAD package includes with the BIMx desktop viewer, non-license holders can download the viewer for free and gain access to a very complete CAD viewer.
TopSolid'Viewer free download lets anyone view TopSolid documents
The TopSolid'Viewer is a free addition to the TopSolid family of design and engineering software. Share your documents with the extended team and external partners using this free viewer.
Hoops Mobile Viewer for iOS and Android
Tech Soft 3D, the developers of widely used HOOPS 3D visualization standard, provide a free HOOPS viewer for mobile devices. iPhone, iPad, and Android devices are all supported by this free viewer.
Download 3DVIA Shape 3D modeling application for free
3DVIA Shape is a free 3D modeling program designed for ease-of-use and yet powerful enough for experts. Dassault Systèmes provides this free 3D software and also has created an online community exchange and meeting place at 3DVIA.com where you can upload, share, and get feedback on your models.
Autodesk Inventor View 2014 free download
Autodesk Inventor View 2014 allows your project team to share Inventor design files with the entire design team. This viewing application allows everyone to view and to print Autodesk Inventor assemblies and drawings.
Free Autodesk 360 viewer application for mobile devices : Apple's iPhone and iPad and Google Android smart phones and tablets
Autodesk 360 is a cloud service providing storage and tools to support your design projects. A wide range of professional tools are available. Some - like this viewer - are completely free, some are available on a pay per use fee, and others come 'free' with your subscription to Autodesk products. This mobile viewer makes the core project team as well as the extended team more productive on the road, in the conference room, or on-site with a free tool for viewing, commenting, and annotating project designs.
Download OpenFOAM for free : the Open Source CFD Toolbox
OpenFOAM is an open source CFD toolkit. It runs on multiple Linux platforms and on Amazon's EC2 cloud platform.
3D CAD modeling : download Creo Elements/Direct Modeling Express 6.0 for free
Are you looking for a free, 3D, direct-modeling, professional CAD program for part and assembly design? Creo Elements/Direct Modeling Express program can be that solution. Download it here, register, and get to work. For free.
FreeCAD : Open Source, free 3D CAD download
FreeCAD is an open source, parametric, multi-platform, customizable, 3D modelling program. Available for Windows, Linux, and Mac OSX.
Autodesk Simulation CFD Viewer : free download
Download the free Autodesk Simulation CFD Viewer for Windows to share and visualize your work.
Sculptris 3D Sculpting Software
Sculptris is a modeling program from the 3D experts at Pixologic. If you are new to digital sculpting, then this free software will get you started as a digital designer. If you are already experienced, then you will find this to be a fast tools for realizing your ideas.
Free 2D CAD : Solid Edge 2D Drafting download
Solid Edge® 2D Drafting software delivers a production-proven set of capabilities for 2D CAD and it is a free application available for download anywhere in the world.
DraftSight : professional CAD software for free
Can professional CAD software be free? The short answer is "yes, it can be free". The makers of CATIA & SolidWorks also create a bevy of tools which can be absolutely free, yet still provide great value like AutoCAD DWG editing compatibility.
123D Catch Free Download
123D Catch creates 3D geometry from photographs. It is an easy-to-use cloud application with a Windows-based interface. You only need to download and install the Windows program and then start using this fun and useful program.


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Lee Nyquist
Candidate District 9
Lee Nyquist graduated from Harvard College in 1974, where he received his undergraduate degree in Government. He went to the Washington College of Law at the American University and obtained his law degree in 1978. He has strong roots in District 9, where he married his wife and has lived for 23 years.
Lee has served as the president of the Manchester Bar Association and the New Hampshire Association for Justice. He brought labor and business representatives together as chair of the New Hampshire Unemployment Compensation Advisory Council. He has served as the town moderator for New Boston over 20 years. Lee is a practicing attorney, currently working for Shaheen & Gordon in Manchester.
Lee lives with his wife, Leslie, in New Boston. Lee has had five children.
Will Represent:
Bedford, Dublin, Fitzwilliam, Greenfield, Hancock, Jaffrey, Lyndeborough, Mont Vernon, New Boston, Peterborough, Richmond, Sharon, Temple, and Troy
Lee Nyquist 2012 Committee
PO Box 3
New Boston, NH 03070
(603) 759-0129
Campaign Website


text: Home > Tech Biz Google gains entry to home and prized team with $3.2B Nest deal Reuters Posted at Jan 14 2014 05:39 PM | Updated as of Jan 16 2014 08:43 PM Share Save Facebook Twitter GPlus LinkedIn SAN FRANCISCO- Google Inc took its biggest step to go deeper into consumers' homes, announcing a $3.2 billion deal to buy smart thermostat and smoke alarm-maker Nest Labs Inc, scooping up a promising line of products and a prized design team led by the "godfather" of the iPod. Nest will continue to operate as its own distinct brand after the all-cash deal closes, Google said on Monday. The deal is the second largest in Google's history after the $12.5 billion acquisition of mobile phone maker Motorola in 2012. Like the Motorola deal, which marked Google's first major foray into hardware, the Nest acquisition gives Google a stepping stone into an important new market at a time when consumer appliances and Internet services are increasingly merging. "Nest Labs appears to be focused on thermostats and smoke alarms, but it's not far-fetched to see Google expanding this technology into other devices over time," said Shyam Patil, an analyst at Wedbush. "Home automation is one of the bigger opportunities when you talk about the Internet of everything and connecting everything. This acquisition furthers their strategy around that," he said. Shares of Google were up 0.5 percent at $1,128.49 in extended trading on Monday. Nest gained a large following with its first thermostat - a round, brushed-metal device with a convex glass screen that displays temperature and changes hue to match the color of the wall it attaches to. It also tracks usage and employs that data to automatically set heating and cooling temperatures. With the acquisition, Google gets Tony Fadell, a well-connected and well-respected Silicon Valley entrepreneur credited with creating Apple Inc's iconic iPod music player, along with co-founder Matt Rogers and a host of talented engineers and designers. According to a search on professional network LinkedIn, roughly 100 of Nest's 300 employees have worked at Apple in the past. Google, the world's largest online search engine, is increasingly expanding into new markets, with efforts ranging from a high-speed Internet access business to advanced research on self-driving cars and robotics. But while Google's engineering expertise has generated major advances in technology, the company has at times struggled to create hardware products that resonate with consumers as much as Apple's products do. The consumer experience of Nest's products "is Apple-like and it gives Google that," said Pat Moorhead, an analyst at Moor Insights and Strategy. "What Google wants to do is be the backbone for your home, how you consume energy, how you consume content like music through your entire house," said Moorhead. Some commenters on Twitter expressed concerns about the privacy implications of the deal for Google, which collects scads of personal data about its users' online habits. "Not content with your personal data, Google now wants your home data by buying Nest," read a Tweet by Irene Ng. "So basically Google will know when homeowner is away, when they've had a fire & what the power bill is?" Tweeted Brian Makas, who appended the hashtag #creepy to his comment. In an interview with Reuters, Nest's Fadell said the company spent a lot of time discussing privacy issues with Google during talks that led to the deal. "The reality of the situation is inside of Google they take privacy so incredibly seriously you have no idea," Fadell said, noting that Nest's terms of service would not change after the deal. Google said the deal is expected to close in the next few months pending regulatory approval. EARLY BETS Google has tried to gain a foothold in the smart home market before, launching the PowerMeter service in 2009. The service let consumers use the Web to monitor their home electricity consumption, but Google shut it down in 2011, noting that it hadn't caught on as much as Google hoped. It was that same year that Nest's Fadell met with Google co-founder Sergey Brin at a TED conference, showing him a prototype of the thermostat. Google's venture capital arm, Google Ventures, made an investment in Nest not long after that. Kleiner Perkins Caufield & Byers, the venture-capital firm that started backing Nest in 2010, made a return of 20 times on the $20 million it has put in over the years, for a return of around $400 million, a person familiar with the situation said. Shasta Ventures, which invested at the same time as Kleiner, stands to make a similar rate of return on its investment, a person familiar with the matter said. Fadell said the deal with Google was the culmination of "countless" discussions that began in the summer of 2013. "It took us months to get comfortable that they are going to bring to the table the things we need for scale and to realize our decade-long vision and that they really truly respected what we did," he said. While Fadell's expertise in mobile products could be a boon to Google and its money-losing Motorola smartphone division, he stressed that his focus was on home automation products. "That was one thing I was very clear about. I said 'Larry, I have already built all kind of mobile products, I have done all those things. I am not here to build those,'" Fadell said, referring to Google CEO Larry Page. "I am here to build out this vision. Not to go and build the other things I have already built in the past," said Fadell. Share Save Facebook Share on Twitter GPlus LinkedIn Read More: Google Inc Nest Labs /entertainment/11/20/17/balikan-unang-pagbisita-ni-franco-sa-ggv/entertainment/11/19/17/i-can-see-your-voice-erik-santos-fooled-by-singer-like-looks/trending/11/19/17/american-trio-airspoken-share-their-favorite-opm-songs/life/11/19/17/panoorin-love-story-nina-nerissa-at-johnny-na-may-conjoined-twin/entertainment/11/19/17/tom-doromal-ng-hashtags-nagsalita-na-sa-pagkalunod-ni-franco


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T regulatory (Treg) cells are implicated in maternal immune tolerance of the conceptus at implantation; however, the antigenic and regulatory signals controlling Treg cells in early pregnancy are undefined. To examine the role of male seminal fluid in tolerance induction, the effect of exposure to seminal fluid at mating on responsiveness to paternal alloantigens was examined using paternal tumor cell grafts and by delayed-type hypersensitivity (DTH) challenge on Day 3.5 postcoitum. Exposure to seminal fluid inhibited rejection of paternal tumor cells, independently of fertilization and embryo development, while seminal fluid from major histocompatability complex (MHC)-dissimilar males was less effective. Similarly, mating with intact males suppressed the DTH response to paternal alloantigens in an MHC-specific fashion. Excision of the seminal vesicle glands diminished the tolerance-inducing activity of seminal fluid. Mating with intact males caused an increase in CD4+CD25+ cells expressing FOXP3 in the para-aortic lymph nodes draining the uterus, beyond the estrus-associated peak in cycling mice. The increase in CD4+CD25+ cells was abrogated when males were vasectomized or seminal vesicles were excised. Collectively, these data provide evidence that exposure to seminal fluid at mating promotes a state of functional tolerance to paternal alloantigens that may facilitate maternal acceptance of the conceptus at implantation, and the effects of seminal fluid are likely to be mediated by expansion of the Treg cell pool. Both seminal plasma and sperm components of the seminal fluid are necessary to confer full tolerance and elicit the Treg cell response, potentially through provision of immune-deviating cytokines and antigens, respectively.
Survival of the semiallogeneic fetus in pregnancy depends on adaptations in both the innate and adaptive immune compartments [1, 2]. A transient state of peripheral immune tolerance is one key mechanism, since the maternal immune system is not ignorant of paternal major histocompatability complex (MHC) and other conceptus antigens [3–5]. This tolerance is mediated, at least in part, by regulatory T (Treg) cells [6], a unique subpopulation of T cells that are potent suppressors of the generation and effector function of type 1 (cell-mediated) immune responses. Treg cells comprise 5%–10% of CD4+ T cells in rodents [7, 8], and are identified on the basis of their constitutive expression of the interleukin 2 receptor, CD25 [9], and the transcription factor, FOXP3 [10].
Treg cells generated in peripheral tissues require antigen-driven activation and proliferation to mediate their full suppressive function [11, 12]. The events associated with activation and expansion of the Treg cell pool during early pregnancy are not defined, and the nature and origin of the eliciting antigens are unclear [12]. In mice, there is some evidence that paternal alloantigens associated with the gestational tissues serve in this role, acting together with antigen-nonspecific hormonal factors to facilitate expansion of Treg cell populations [13]. Several studies in T-cell receptor-transgenic systems specific for paternal H-Y antigen [4], paternal MHC class I [5], or employing chicken egg ovalbumin (OVA) as a model paternal antigen [14] infer that T-cell activation and proliferation occurs in response to antigens shed by fetal or placental trophoblast cells, and cross-presentation by maternal dendritic cells may be involved [14]. However, this is not reconciled with studies showing that mice expressing transgenic T-cell receptors reactive with paternal MHC exhibit functional tolerance and show evidence of T cell anergy even prior to embryo implantation [3]. The increase in Treg cells that occurs in early pregnancy can partly be attributed to elevated estrogen associated with ovulation [15], but this is not a complete explanation, since there is evidence of antigen specificity in the Treg cell response even at this early time point [16, 17].
Female reproductive tissues are first exposed to paternal antigens during transmission of seminal fluid to the female reproductive tract at mating [18]. This raises the questions of whether the female immune response can recognize and respond to male antigens in seminal fluid, and whether mating constitutes an opportunity for initial activation of the maternal immune response. The kinetics of expansion in CD4+CD25+ Treg cell populations within days after mating [6], and the necessity for Treg cell function prior to embryo implantation [19], suggests that expansion of the Treg cell pool occurs independently of the products of conception, and is, instead, consistent with a role for seminal fluid.
This study aimed to determine whether seminal fluid has a role in activating Treg cell populations and eliciting functional immune tolerance to male alloantigens at the time of embryo implantation. A paternal tumor graft model and a delayed-type hypersensitivity (DTH) assay were utilized to investigate the significance of the seminal plasma and sperm components of seminal fluid, and male partner MHC status, in eliciting a functional tolerogenic response. The effect of seminal fluid on populations of CD4+CD25+ Treg cells in lymph nodes (LNs) draining the uterus and systemically was examined by flow cytometry. Our findings show that both the sperm and seminal plasma components of seminal fluid act in concert to induce a state of functional tolerance acting to suppress the type-1 immune response to male alloantigens, and this is associated with expansion of the CD4+CD25+ Treg cell pool beyond the estrogen-regulated increase at ovulation. We conclude that male seminal fluid contributes to the events through which maternal immune tolerance is established at the outset of pregnancy.
BALB.K (H-2k) and C57BL/B10.BR (B10; H-2k) mice were purchased from the Animal Resource Centre (Perth, Australia). BALB/c (H-2d), C57BL/6 (B6; H-2b), and (CBA × C57BL/6) F1 (CBB6F1; H-2b/k) mice were purchased from the University of Adelaide Central Animal House. All mice were housed under specific pathogen-free conditions at the University of Adelaide Medical School Animal House on a 12L:12D cycle, and were administered food and water ad libitum. All experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, with approval from the University of Adelaide Animal Ethics Committee.
Some females underwent surgery 2–4 wk prior to mating to ligate the oviductal-uterine junction after flurothane anesthesia. Vasectomized and seminal vesicle-excised (SVX) male mice were prepared surgically, as previously described [20], and were allowed 2 wk to recover, then proven capable of mating by caging with females and detection of vaginal plugs and sperm deposition, respectively, prior to experimental use. For matings, one to three adult (8–12 wk) intact or uterine-ligated female mice were caged with intact, vasectomized, or SVX stud males and checked daily for vaginal plugs. For female mice caged with SVX males, vaginal smears were taken each morning and examined for the presence of sperm. The day of detection of a vaginal plug or sperm-positive smear was designated Day 0.5 postcoitum (pc). Mated females were removed from males and housed in groups of one to three females per cage.
Tumor challenge experiments utilized JR-5 fibrosarcoma cells or Lewis lung tumor (LLT) cells, both provided by Drs. Lindsay Dent and John Finlay-Jones (Flinders University, Adelaide, SA, Australia). JR-5 tumors were originally induced by subcutaneous inoculation of 3-methylcholanthrene into male BALB/c mice [21], and LLT cells arose spontaneously as a carcinoma of the lung in a B6 mouse [22]. Tumors were propagated by serial passage in BALB/c and B6 mice, respectively, with culture for one to three passages in vitro prior to experimental use. For in vitro culture, both JR-5 and LLT cells were cultured in RPMI-1640 containing 10% fetal calf serum (FCS) and antibiotics (RPMI-FCS) at 37°C in 5% CO2, and were harvested using EDTA (20 mM in PBS for 10 min at 37°C). Expression of MHC class I antigens was confirmed by flow cytometric analysis in cultured cells, or cells recovered from solid tumors excised from mice and mechanically disaggregated with the aid of fine scissors and a manually operated glass homogenizer.
For tumor challenge experiments, adult, virgin intact or uterine-ligated BALB.K or B10 mice at estrus or at Day 3.5 pc after mating with intact, vasectomized, or SVX BALB/c, B6, or CBA × B6 F1 males were administered 1 × 105 JR-5 cells or 5 × 105 LLT cells in 50 μl PBS by s.c. injection into the right ventral flank. Mice were killed by cervical dislocation 13 days later (Day 16.5 pc), and tumor growth was quantified by making two measurements each of the length and width of tumors using digital calipers (Mitutoyo Corp., Kawasaki, Japan), and calculating the average diameter as the mean of these four readings. The incidence of pregnancy was noted, and late gestation pregnancy parameters were measured by excision of the intact uterus and counting total, viable, and resorbing implantation sites.
Adult virgin BALB.K mice were evaluated to determine the stage of estrous cycle by phase-contrast examination of vaginal smears prepared at 0900–1000 h. Four sequential stages of the cycle were identifiable on the basis of the cellular composition of the smear: proestrus (>50% intact, live epithelial cells); estrus (100% cornified epithelial cells); metestrus (~50% leukocytes and ~50% cornified epithelial cells); or diestrus (>70% leukocytes ± cornified or intact epithelial cells). Mice in diestrus were primed by administration of 1 × 107 BALB/c spleen cells s.c. into both the left and right dorsal flank at 1200–1400 h (Day 0). Prior to injection, spleen cells were incubated with mitomycin C (0.5 mg/ml; Sigma-Aldrich) for 20 min at 37°C, then washed three times in Hanks Balanced Salt Solution (HBSS). On the afternoon of the next day, immunized females were placed with either BALB/c males or BALB.K males. Mice that mated on the first, second, or third night (identified by detection of a vaginal plug on the mornings of Days 2–4) were included in the experiment. On Day 3.5 pc (Days 6–8 after priming), or on Day 7 after priming (unmated virgin controls), all mice were challenged by injection of the left footpad with 1 × 107 mitomycin C-treated BALB/c spleen cells in 25 μl HBSS. The right footpad was injected with 25 μl HBSS. Immediately prior to challenge and 24 h after challenge, footpad thickness was measured as the average of five replicate readings made using a constant pressure caliper (Mitutoyo Corp.). The DTH response was calculated as the difference in microns between the left and right footpad thickness values.
Adult virgin B6 mice at defined stages of the estrous cycle (as described above) or Day 3.5 pc after mating with intact, vasectomized, or SVX BALB/c males, were administered 2% Avertin anesthesia (2% 2.2.2-tribromomethanol; Sigma-Aldrich), and blood was collected by cardiac puncture and diluted 1:2 in PBS containing heparin (Sigma-Aldrich). Peripheral blood mononuclear cells were collected after centrifugation over Lympholyte (Cedarlane, Hornby, Canada) according to the manufacturer's instructions. Single-cell suspensions were prepared from excised para-aortic (ileac) LNs, mesenteric LNs, and the spleen by mechanical dispersion between glass microscope slides. Cells were washed in RPMI-FCS and resuspended in 0.1% FCS/PBS (fluorescence-activated cell sorting [FACS] buffer) at 107 cells/ml. Aliquots of 106 cells were incubated with anti-Fc-γIIR antibody (BD Pharmingen, BD Biosciences, San Diego, CA) to block nonspecific binding (15 min/4°C) prior to incubation with PE anti-CD3 (145–2C11), allophycocyanin anti-CD4 (L3T4), and biotin anti-CD25 (7D5) (all from BD Pharmingen) for 30 min at 4°C. Cells were washed in FACS buffer and incubated with PE-Cy5 streptavidin (BD Pharmingen) for 30 min at 4°C. After further washing in FACS buffer, the cells were resuspended in 300 μl FACS buffer and analyzed using a FACS Canto with FACS Diva software (both from BD Biosciences). CD4+CD25+ cells were quantified as a percent of total CD4+ cells after gating on the CD3+ T-lymphocyte population and excluding debris and dead cells. The absolute number of CD4+CD25+ cells in para-aortic LN was calculated as follows: absolute CD4+CD25+ cell number = total cell number × % CD4+ cells × CD4+CD25+ cells (% CD4+ cells), where total cell number is mean values from identical treatment groups in our previous study [23].
To evaluate FOXP3 expression, para-aortic LN cells prepared as above were incubated with Fc Block (BD Pharmingen) to block CD16/CD32 receptors, and then incubated with fluorescein isothiocyanate (FITC) anti-CD3 (17A2), PE anti-CD4 (RM4–5) (both from BD Pharmingen), and PE-Cy7 anti CD25 (PC61; eBiosciences). Cells were subsequently fixed and permeabilized using a FOXP3 staining buffer set (eBiosciences) according to the manufacturer's instructions. Following further treatment with Fc Block, cells were incubated with allophycocyanin anti-FOXP3 antibody (FJK-16s; eBiosciences), then analyzed using a BD FACS Canto with FACS Diva software. FOXP3+ cells were quantified as a percent of total CD4+CD25+ or CD4+CD25− cells after gating on the CD3+ T-lymphocyte population and excluding debris and dead cells. Epitope mapping shows FJK-16s reactivity with amino acids 75–125 of the mouse FOXP3 protein (manufacturer's product notes), and specificity for FOXP3 is further demonstrated by the absence of FJK-16s staining in mice with a null mutation in the Foxp3 gene [24].
For quantification of MHC class I expression, JR-5 or LLT cells were incubated with monoclonal antibody anti-H-2Kb (HB176; American Type Culture Collection, Manassas, VA), followed by FITC goat anti-rat (Dako, Glostrup, Denmark), or FITC anti-H-2Kd (SF1–1.1; BD Pharmingen), and analyzed using a FACScan with CellQuest software (Becton Dickinson) using forward and side scatter to exclude debris and dead cells.
All data were analyzed using SPSS version 13 (SPSS, Chicago, IL). Categorical data (proportion of mice with tumors) were compared by chi-square test. ANOVA and independent samples t-test were used to compare differences between treatment groups in tumor size, footpad thickness, and cell numbers. The relationship between tumor size and number of viable implantation sites was assessed using Pearson two-tailed bivariate correlation analysis. Statistical significance in differences between groups or correlation was concluded when P < 0.05.
Initially, we investigated whether seminal fluid exposure at mating can induce functional tolerance to paternal alloantigens. A model system was established employing congenic mice in the BALB series, which differ at only the MHC gene locus. Intact or uterine-ligated BALB.K (H-2k) females were mated with intact BALB/c (H-2d) or BALB.K males and then challenged with BALB/c JR-5 fibrosarcoma cell grafts on Day 3.5 pc. JR-5 cells were selected from a panel of candidate BALB/c tumor cells on the basis of their formation of solid tumors with linear growth kinetics over a 2-wk period, poor metastatic potential, and stable expression of MHC class I (data not shown). JR-5 cells consistently developed a single solid tumor when injected s.c. into virgin BALB/c mice (28/28, 100% tumor growth), but were rejected from virgin BALB.K mice (24/24, 0% tumor growth). Pregnancy did not alter the incidence of tumor development, since JR-5 cells grew in BALB/c females mated with BALB/c males and injected with JR-5 cells on Day 3.5 pc (19/19, 100% tumor growth). However, tumors were smaller in pregnant BALB/c mice compared with virgin BALB/c mice (P = 0.003) (Fig. 1).
In contrast to virgin BALB.K females, tumor cell graft rejection was impaired in the majority of BALB.K females following mating with intact BALB/c males and administration of JR-5 cells on Day 3.5 pc (35/48, 73% tumor growth) (Fig. 1). Tumor growth was independent of whether or not pregnancy ensued and conceptus tissue was present, since 31/48 (65%) of mated BALB.K females administered JR-5 cells did not have implantation sites at autopsy on Day 16.5 pc, indicating that viable pregnancy was not necessary for female tolerance of tumor cells. A similar rate of pregnancy failure was seen in BALB/c females mated with BALB/c males, where 7/19 (37%) mice administered JR-5 cells had no implantation sites on Day 16.5 pc (P = 0.088, chi-square test). Pregnancy loss rates in both groups were substantially greater than in BALB.K or BALB/c mice not given tumor cells (<10%; data not shown), indicating that the presence of tumor cells interferes with either embryo implantation or pregnancy maintenance. There was no correlation between tumor size and number of viable implantation sites in either BALB/c or BALB.K mice, or both data sets combined (R = 0.064).
In another group of BALB.K females, the uterus was ligated at the oviductal junction 2 wk prior to mating in order to prevent conception and embryo formation, while allowing seminal fluid access to the uterine cavity. When uterine-ligated BALB.K females were mated with BALB/c males and challenged with JR-5 cells on Day 3.5 pc, tumor cell rejection was inhibited (27/38, 71% tumor growth). The proportion of mated uterine-ligated BALB.K mice that developed tumors, as well as the size of tumors, were similar to mated intact BALB.K females (Fig. 1). This shows that tolerance to paternal tumor challenge is induced by seminal fluid at mating, and that neither conception nor the presence of embryos is necessary.
The effect of male partner strain on seminal fluid induction of tolerance to tumor cell challenge was examined next. Compared with BALB.K females mated with BALB/c males, tolerance to JR-5 tumor cell grafts occurred less frequently in BALB.K females mated with CBB6F1 (H-2b/k) males (11/26, 42%; P = 0.031), or BALB.K females mated with BALB.K males (4/16, 25%; P = 0.003) (Fig. 1). This indicates that development of tolerance after seminal fluid exposure is maximized when the male partner is of the same genotype as the tumor cell graft, and implicates male MHC antigens in tolerance activation.
A second congenic model was evaluated in order to ensure that the tolerance-inducing effect of seminal fluid was not limited to BALB.K mice. Intact or uterine-ligated B10 (H-2d) females were mated with intact B6 (H-2k) or B10 males, and challenged with B6 LLT cells on Day 3.5 pc. LLT cells formed solid tumors with linear growth kinetics over a 2-wk period, and showed poor metastatic potential and stable expression of MHC class I (data not shown). While virgin B10 mice all rejected LLT tumor cells (0/9, 0% tumor growth), rejection was inhibited in the majority of B10 females after mating with intact B6 males (6/10, 60% tumor growth) (Fig. 2). In B10 uterine-ligated females mated with B6 males, tumor cell graft rejection was also inhibited (10/10, 100% tumor growth; P < 0.001 compared with virgin B10.BR mice) (Fig. 2). Compared with B10 females mated with B6 males, tolerance to LLT challenge occurred less frequently in both intact B10 females mated with B10 males (1/10, 10%; P = 0.028), and in uterine-ligated B10 females mated with B10 males (1/10, 10%; P < 0.001) (Fig. 2). This indicates that, in B10 mice, as in BALB.K mice, exposure to seminal fluid is sufficient to induce tolerance to tumor cell grafts in a partner MHC-specific manner.
The requirement for the sperm and seminal plasma components of seminal fluid to induce tolerance to tumor cell grafts was examined next, utilizing both vasectomized males, and males from which the seminal vesicle glands were surgically excised (SVX males) to remove the majority of the seminal plasma from the ejaculate. Compared with BALB.K females mated with intact BALB/c males, tolerance to JR-5 challenge occurred less frequently in BALB.K females mated with SVX BALB/c males (8/21, 38%; P = 0.026), but was unchanged in BALB.K females mated with vasectomized BALB/c males (18/28, 64%) (Fig. 1). This shows that tolerance to tumor cell grafts after mating can occur without transmission of sperm, but that factors of seminal vesicle origin in the seminal plasma are essential for induction of tolerance.
Female tolerance of paternal tumor cell grafts after insemination suggests hyporesponsiveness in the type 1 immune response. In another experimental approach to examine the effect of seminal fluid on the female type 1 immune response to male partner MHC antigens, a DTH assay was utilized. Adult virgin female BALB.K mice were immunized s.c. with 1 × 107 BALB/c spleen cells, then, 2–4 days later, mated with intact, vasectomized, or SVX BALB/c males, or intact BALB.K males. On Day 3.5 pc (6–8 days after priming), all mice were challenged in the footpad with BALB/c spleen cells. Mating with BALB/c males reduced footpad swelling at challenge, indicating a diminished DTH reaction compared with control unmated mice (P = 0.020) (Fig. 3). In contrast, no reduction in DTH response was observed when immunized BALB.K females were mated with BALB.K males, or with vasectomized or SVX BALB/c males.
These data show that natural insemination can inhibit the type I immune response to paternal MHC antigens. The effect is dependent on mating with a male of the same MHC haplotype as the immunizing MHC antigen, and appears to require both the seminal plasma and sperm fractions of the ejaculate.
To investigate whether the immune tolerance induced by seminal fluid exposure is potentially mediated by Treg cells, and the requirement for seminal plasma and sperm components of seminal fluid in eliciting any Treg cell response, CD4+CD25+ cells were analyzed by flow cytometry in para-aortic LN, mesenteric LN, peripheral blood, and spleens recovered from B6 mice on Day 3.5 pc after mating with intact, vasectomized, or SVX BALB/c males. For comparison, CD4+CD25+ cells were also analyzed in tissues recovered from virgin mice at defined phases of the estrous cycle (Fig. 4, A and B).
Clear effects of the phase of estrous cycle on CD4+CD25+ cells in LNs, spleen, and blood were observed. In the para-aortic LNs, which drain the uterus, the CD4+CD25+ fraction (expressed as percent CD4+ cells) was significantly higher at the estrous stage (the time of ovulation and receptivity to mating) than at either the diestrous or proestrous stages of the cycle (P < 0.001 and P = 0.001, respectively) (Fig. 5A). In mesenteric LN, mice had fewer CD4+CD24+ cells at diestrus than at estrus (P = 0.006) (Fig. 5C). Similar fluctuations were seen in spleen and blood, but the variation between mice was higher in these compartments, and statistically significant differences were not observed (Fig. 5, B and D).
Mating with intact males elicited a further 44% increase in CD4+CD25+ cells (expressed as percent CD4+ cells) by Day 3.5 pc in para-aortic LNs compared with estrous mice (P = 0.013) (Fig. 5A). In contrast, there was no increase in females mated with vasectomized or SVX males, where CD4+CD25+ cells were fewer than in females mated with intact males (both P < 0.001) (Fig. 5A), and, in the case of females mated with vasectomized males, less than in estrous females (P = 0.002). This shows that seminal plasma and sperm components of the seminal fluid are required to elicit the postmating increase in CD4+CD25+ cells in para-aortic LN.
In the mesenteric LN, no significant increase was seen in mice mated with intact males compared with estrous mice, but fewer CD4+CD24+ cells were present after mating with vasectomized or SVX males compared with intact males (P < 0.001 and P = 0.001, respectively), and, in both cases, values were less than in estrous females (P < 0.001 and P = 0.009, respectively) (Fig. 5C). A similar effect was evident in the spleen, where fewer CD4+CD24+ cells were present after mating with vasectomized or SVX males compared with intact males (P = 0.051 and P = 0.043, respectively) (Fig. 5B). No significant effect of mating or seminal fluid composition on CD4+CD24+ cell number in the blood was apparent (Fig. 5D).
The Treg surface marker CD25 is also expressed by activated CD4+ effector cells, and T-cell activation is a characteristic of early pregnancy [23]. To confirm that the increase in CD4+CD25+ cells in the para-aortic LN reflects an increase in Treg cells, a different antibody cocktail was utilized to detect the Treg-specific marker FOXP3 in CD4+CD25+ cells from para-aortic LN of estrous mice and Day 3.5 pc females after mating with intact BALB/c males (Fig. 4, C and D). Consistent with previous reports [10], FOXP3 expression was largely restricted to CD4+CD25+ cells, and rarely detected in CD4+CD25− cells or CD4− cells. In both groups of mice (n = 5–6 mice/group), the percentage of CD4+CD25+ cells expressing FOXP3 was comparable (median [range] % FOXP3+ at estrus = 90.2% [83.5%–93.9%] and at Day 3.5 pc = 91.0% [83.2%–92.3%]) (Fig. 4, D and E). Small proportions (≤3%) of CD4+CD25− cells in both estrous and mated mice expressed the FOXP3 marker (Fig. 4, G and H). This indicates that the majority of CD4+CD25+ cells are indeed Treg cells. Since the proportion of FOXP3+ Treg cells among the expanded para-aortic LN CD4+CD25+ pool was unchanged by mating, the increases in CD4+CD25+ cells at Day 3.5 pc reflect an increase in the Treg cell population, rather than activation and proliferation of other FOXP3− CD4+ cells.
Hypertrophy of the para-aortic LN is characteristic of the immune response to early pregnancy, and depends on exposure to factors in the seminal plasma fraction of the seminal fluid [23]. To estimate the scale of effect of seminal fluid on the total size of the CD4+CD25+ cell pool, the absolute number of CD4+CD25+ cells in the para-aortic LN was calculated from flow cytometry data, factoring in the total number of cells recovered from LNs [23] (Fig. 6A) and the proportion of total cells expressing CD4 (Fig. 6B). Exposure to seminal fluid by mating with intact males increased the absolute number of CD4+CD25+ cells by 2.7-fold (P < 0.001), while absolute CD4+CD25+ cell numbers were unchanged after mating to vasectomized or SVX males compared with estrous values, and significantly less compared with the intact mated group (both P < 0.001) (Fig. 6C).
Preventing development of maternal type 1 immunity to conceptus transplantation antigens is essential for pregnancy. Maternal T cells recognize and can respond to paternal MHC antigens, indicating the necessity for mechanisms to limit their effector function [1, 3]. Treg cells are implicated in this protective role [6]; however, the factors regulating Treg cell abundance and function in pregnancy have not been elucidated. The current experiments demonstrate that generation of paternal antigen-specific tolerance in early pregnancy is initiated at the time of conception, when the female is first exposed to paternal alloantigens in the context of seminal fluid. Seminal fluid transmission activates a state of functional tolerance to paternal alloantigens, and this is associated with expansion of CD4+CD25+ cell populations confirmed to be Treg cells by virtue of their expression of the signature Treg cell marker, FOXP3. Both the seminal plasma and the sperm components of seminal fluid appear to be necessary for maximum expansion of the Treg cell pool and for full expression of functional tolerance, but conceptus tissue is not required for this initial phase of the response. Additionally, the data suggest that there is a degree of male partner specificity in the female reaction to seminal fluid.
The current findings concur with published studies showing that seminal fluid influences the female immune response. Insemination causes hypertrophy of LNs draining the uterus [25, 26]. This is associated with expression of lymphocyte activation markers and cytokine expression, and is completely dependent upon factors present in seminal plasma [23]. Mated females or females administered whole seminal fluid to the uterine cavity show partner-specific hyporesponsiveness in type 1 immunity to male MHC antigens, demonstrated by prolonged survival of male skin grafts [25, 27]. Consistent with this, we reported preliminary observations that seminal fluid inhibits rejection of paternal tumor cells, even when conception is prevented by surgical ligation of the uterine-oviductal junction [28]. The current study extends these observations by showing the following in two mouse strains: seminal fluid exposure in the absence of conception is sufficient to activate functional tolerance in females; consistent tolerance induction requires exposure to seminal plasma of the same male strain as the paternal challenge; two functional measures of type 1 immunity—tumor graft rejection and DTH response—are inhibited by seminal fluid exposure; and Treg cells are potentially involved in suppressing these type 1 responses.
Challenge with paternal skin grafts and tumor cell grafts have been previously utilized to demonstrate functional tolerance to paternal MHC and other antigens in pregnancy [3, 25, 29], but these assays can be variable in their specificity and sensitivity, depending on graft tissue expression of alloantigens and other tissue-restricted antigens. These factors presumably contributed to the quantitative differences in the results for the two strain combinations used in the tumor challenge experiments reported herein. Compelling evidence linking suppression of type 1 immunity with Treg-cell activity in pregnancy has now emerged [6, 30], but the antigen specificity and dependence of Treg cells is difficult to study without reliance on in vivo functional assays, since reagents to distinguish gestational tissue-reactive Treg cells among the systemic Treg pool are not available.
Previous studies have alluded to the likely importance of gestational tissues as the source of antigens and immune-deviating signals for activating tolerance mechanisms [3], including Treg cells [16]. However, the kinetics of expansion in CD4+CD25+ Treg cell populations within days of mating [6], and the necessity for Treg cell function prior to embryo implantation [19], argues against their dependence on fetal alloantigens. Furthermore, since the preimplantation embryo comprises, at most, a few hundred cells encapsulated in the zona pellucida, antigen associated with the embryo before implantation would be insufficient and inaccessible.
A role for seminal fluid in activating the female Treg response provides an explanation for why functional tolerance and Treg cell expansion are evident even before the time of embryo implantation. Since T cells generally take several days to generate a robust response after stimulation, a Treg response initiated at the time of conception would allow protective suppression to manifest by the time the embryo implants 4 days later. Several of the same antigens are present in seminal fluid and later expressed from paternal genes by the conceptus [31], including classical class Ia and nonclassical class Ib MHC antigens and minor antigens, such as H-Y [32, 33]. Therefore, Treg cells activated during the preimplantation period would exhibit protective suppression upon reencounter with MHC and other antigens expressed by fetal cells and placental trophoblast cells following embryo implantation [34, 35].
The male partner specificity evident in both the tumor challenge and DTH assays suggests that antigen specificity is conferred by alloantigen in seminal fluid. Both somatic cell- and spermatozoa-associated MHC class I, as well as soluble MHC class I, is present in seminal fluid [32, 36–38]. The growth of tumors after challenge in some BALB.K mice after mating with males expressing third-party MHC suggests that antigens other than alloantigens might also be involved, and H-Y antigen or other unique differentiation antigens on the surface of spermatozoa could fulfill this role [39]. These antigens are present on non-sperm cells, such as desquamated epithelial cells in the seminal plasma fraction, as well as expressed at lower levels by sperm, and this could explain why seminal plasma in the absence of sperm was sufficient to allow acceptance of BALB/c tumor cell challenges in BALB.K mice after mating with vasectomized males. A greater dependence on seminal plasma than on sperm for Treg cell generation is also evident in the qualitatively greater reduction in absolute Treg cell numbers seen after seminal plasma-deficient mating compared with sperm-deficient mating.
Previous reports have concluded that both antigen-dependent and antigen-independent mechanisms contribute to controlling expansion of the Treg cell pool in early pregnancy, suggesting that Treg cells might arise through both activation and proliferation, and conversion of phenotypically distinct precursor cells. Ovariectomy and steroid hormone replacement experiments demonstrate that estrogen is responsible for eliciting an increase in CD4+CD25+ Treg cell numbers and Foxp3 mRNA expression in the spleen, and in vitro experiments showed direct induction of Foxp3 in CD4+CD25− cells by 17-β-estradiol [15]. Fluctuating estrogen levels likely explain the elevated Foxp3 mRNA expression, indicating accumulation of Treg cells in the uterus at estrus, potentially in response to estrogen-induced expression of chemokines that target the CCR5 chemokine receptor controlling Treg cell recruitment [17]. Our data, showing significantly increased numbers of CD4+CD25+ cells in para-aortic LNs and mesenteric LNs at the estrous phase of the cycle, further support a role for estrogen, and suggest that these LNs, as well as the spleen, may contribute to generating an elevated circulating pool available for periodic Treg-cell recruitment into the uterus. Whether these cells are generated within peripheral lymphoid tissues or recruited via the circulation from the spleen, or perhaps the thymus, remains to be determined.
Similar to previous reports [6, 17, 40], our study shows a relative increase in Treg cell numbers in LNs during the first 4 days of pregnancy and, importantly, demonstrates that this increase is significant even relative to nonpregnant mice timed for the estrous stage of the cycle. Thus, while cycle-associated fluctuations partly explain the elevated CD4+CD25+ cell number in early pregnancy, the estrogen-regulated increase at the time of ovulation in not sufficient to explain the surge in Treg cell numbers during early pregnancy. This is consistent with other studies concluding a role for alloantigen-driven expansion in Treg cell numbers once pregnancy is initiated [16, 17]. Pregnancy-associated Treg cells appear to arise in peripheral tissues as opposed to the thymus, with several studies implicating the uterus draining para-aortic LNs as the predominant site of Treg pool expansion [6, 16].
Our data clearly identify seminal fluid as the missing signal of early pregnancy acting to stimulate Treg cell pool expansion in readiness for embryo implantation. Female exposure to both the sperm and seminal plasma components of seminal fluid appears to be necessary. Mating events deficient in either sperm or seminal plasma failed to sustain the high levels of Treg cells seen in LNs and spleen after mating with an intact male, and cell numbers instead were comparable to those seen in nonovulatory phases of the cycle. Since ovarian hormone levels are functionally comparable at Day 3.5 pc, regardless of the presence of sperm and seminal plasma in the ejaculate [41], these results discount the possibility that the ovarian hormone parameters of early pregnancy are sufficient to sustain high Treg cell numbers until the time of embryo implantation.
Collectively, these experiments suggest that the seminal fluid-driven expansion in Treg cell numbers is the mechanism underpinning the capacity of mated females to tolerate paternal tumor cell grafts. While this seems a likely proposal, we were not able to confirm a causal relationship by demonstrating any consistent reduction in tolerance of JR-5 cells after depletion of Treg cells by passive administration of anti-CD25 antibody to mated mice (data not shown). Thus, mechanisms in addition to Treg cells may contribute to suppression of type 1 immune responses after mating.
Like other antigen-specific immune responses, activation and proliferation of Treg cells requires an active process of antigen uptake, processing, and presentation of antigenic peptides by antigen-presenting cells (APCs). The female reproductive tract is well equipped to mount tolerogenic immune responses to seminal fluid antigens, since APCs are abundant in the endometrial tissue at the time of mating [42–44]. Indeed, seminal fluid has an active role in APC recruitment, with coitus eliciting a local inflammatory response in the female reproductive tissues [45]. Soluble factors in the plasma fraction of seminal fluid induce expression of proinflammatory cytokines and chemokines in epithelial cells lining the cervix and uterus, causing recruitment of macrophages, dendritic cells, and granulocytes, which accumulate in endometrial and decidual tissue [42–44]. These dendritic cells express markers indicative of a tolerogenic phenotype [46], which would be maintained by the type 2 cytokine-dominated environment of the decidual tissue [47].
Seminal plasma contains potent immune-regulatory molecules, including prostaglandins E (PGE) and transforming growth factor-β (TGFB) [48, 49], both of which have been linked with generation of immune suppression mediated by regulatory cells [7, 8]. Naïve CD4+CD25− T cells differentiate into a suppressor T-cell phenotype and express FOXP3 when TGFB is present [50]. TGFB is also implicated in proliferation of mature Treg cells through modifying the function and signaling capabilities of dendritic cells [51]. PGE2 may synergize with TGFB in this role, since in vitro experiments indicate that PGE2 can enhance the inhibitory capacity of human CD4+CD25+ Treg cells and induce a regulatory phenotype in CD4+CD25− T cells [52].
We have utilized a T-cell receptor-transgenic model with OVA as a model paternal antigen to provide direct evidence that male antigens delivered in seminal fluid can activate an antigen-specific T-cell response in female mice. OVA delivered in the ejaculate was found to activate and elicit proliferation of adoptively transferred CD4+ and CD8+ OVA-specific T cells specifically within para-aortic LNs draining the uterus, and female dendritic cells were required to activate the response [53]. This demonstrates the feasibility of uptake of seminal fluid antigens by uterine dendritic cells and cross-presentation to T cells in para-aortic LNs after mating.
The extent to which Treg cells arise from differentiation of naïve CD4+ T cells, or, alternatively, from proliferation of existing Treg cells, and the role of antigen in either pathway, remains to be determined. The effect of successive exposures to paternal antigens through multiple matings, or previous pregnancy, warrants examination to investigate whether these events progressively enlarge the paternal antigen-reactive Treg pool. Another unanswered question is the physiological significance of the seminal fluid-driven expansion in the Treg pool for successful pregnancy. Embryo transfer studies will be required to evaluate whether it is essential or simply a facilitating mechanism to boost suppressive activity prior to the sustained immune-regulating function of placental tissue taking effect. The success of artificial insemination with diluted semen or washed sperm, as well as embryo transfer in mice and other species, clearly shows that successful pregnancy can occur without a natural mating at the outset, although there is mounting evidence that assisted reproductive technologies are associated with suboptimal reproductive outcomes [54], and lack of seminal fluid conditioning of the female immune response may partly account for this [55]. In mice, embryo transfer generally employs recipients mated to vasectomized males, but fetal loss and abnormality is considerably greater when embryos are transferred without recipient exposure to male fluids [56]. When recipient females are mated with seminal vesicle-deficient males, transferred embryos give rise to fetuses with impaired growth trajectories and placental development [57]. In rats, implantation rates and fetal growth are similarly impaired unless females are inseminated prior to embryo transfer [58].
The prospect of Treg cell regulation by seminal fluid has implications for human reproductive health. In women, seminal fluid elicits inflammatory cytokine expression in cervical epithelial cells [59] and, in vivo, causes recruitment of APCs into the ectocervical epithelium (Sharkey and Robertson, unpublished results). Although, to date, there are no studies on the effects of intercourse on Treg cells in women, clinical studies indicate that there may be both acute and cumulative effects on fertility of exposure to seminal constituents, and implicate a partner-specific mode of action. Live birth rates in couples undergoing in vitro fertilization treatments are significantly improved when intercourse occurs around the time of embryo transfer [60, 61], and administration of seminal plasma may increase pregnancy rate in women experiencing recurrent spontaneous abortion [62]. In the condition of pre-eclampsia, which results from a type 1-dominated immune response linked with insufficient Treg cell activity [63, 64], there is a protective benefit of exposure to semen over time [65, 66]. Markedly increased rates of pre-eclampsia are evident in pregnancies with donor oocytes or semen [67], when prior exposure to the donor sperm or conceptus antigens has not occurred. Primary unexplained infertility is also associated with reduced expression of FOXP3 mRNA [68], but whether seminal fluid exposure can alter endometrial Treg cell populations is unknown.
In summary, this study shows that exposure to seminal fluid at mating is necessary and sufficient for conferring maximal immune tolerance at the time of embryo implantation in mice. Seminal fluid appears to act through providing antigen and cytokine signals to further expand populations of Treg cells potentiated by estrogen at the time of ovulation. This finding reinforces a function for seminal fluid beyond simply providing a vehicle to deliver sperm for fertilization, and adds further complexity to its emerging role in signaling physiological changes that prepare the female reproductive tract for embryo implantation [45]. A better understanding of the key antigens and immune-deviating components of seminal fluid and their mode of action in the female will be of utility in designing new therapeutic agents for treating infertility and subfertility in women, and for promoting reproductive competence in animals.
We thank Vicki Mau, Leonie Hicks, Leanne Srpek, and Camilla Dorian for their technical assistance.
1Supported by the National Health and Medical Research Council Australia project and fellowship grants.
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